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W2198526697.txt	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0145638&type=printable	en		The Effect of Oxytocin on Social and Non-Social Behaviour and Striatal Protein Expression in C57BL/6N Mice	PloS one	2,015	cc-by	9,165	RESEARCH ARTICLE The Effect of Oxytocin on Social and NonSocial Behaviour and Striatal Protein Expression in C57BL/6N Mice Xiaofan Zhang1,2, Qi Li2, Min Zhang1, Sylvia Lam2, Pak Chung Sham2, Bitao Bu1, Siew Eng Chua2, Wei Wang1, Grainne Mary McAlonan3 1 Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), 1095 Jiefang Ave., Wuhan, 430030, P.R. China, 2 Department of Psychiatry, University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region (H.K.S.A.R.), China, 3 Department of Forensic and Neurodevelopmental Sciences, The Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, United Kingdom a11111 * grainne.mcalonan@kcl.ac.uk (GMA); wwang@vip.126.com (WW) Abstract OPEN ACCESS Citation: Zhang X, Li Q, Zhang M, Lam S, Sham PC, Bu B, et al. (2015) The Effect of Oxytocin on Social and Non-Social Behaviour and Striatal Protein Expression in C57BL/6N Mice. PLoS ONE 10(12): e0145638. doi:10.1371/journal.pone.0145638 Editor: Barbara Bardoni, CNRS UMR7275, FRANCE Received: August 14, 2015 Accepted: December 7, 2015 Published: December 30, 2015 Copyright: © 2015 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by research funding from the Hong Kong Universities General Research Fund Award to Dr Grainne McAlonan (HKU 774710) and research funding from National Natural Science Foundation of China to Professor Wang (61327902 and 81030021) and Professor Zhang (81271406). Dr Grainne McAlonan is a member of the EU-AIMS consortium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Oxytocin has been suggested as a promising new treatment for neurodevelopmental disorders. However, important gaps remain in our understanding of its mode of action, in particular, to what extent oxytocin modulates social and non-social behaviours and whether its effects are generalizable across both sexes. Here we investigated the effects of a range of oxytocin doses on social and non-social behaviours in C57BL/6N mice of both sexes. As the striatum modulates social and non-social behaviours, and is implicated in neurodevelopmental disorders, we also conducted a pilot exploration of changes in striatal protein expression elicited by oxytocin. Oxytocin increased prepulse inhibition of startle but attenuated the recognition memory in male C57BL/6N mice. It increased social interaction time and suppressed the amphetamine locomotor response in both sexes. The striatum proteome following oxytocin exposure could be clearly discriminated from saline controls. With the caveat that these results are preliminary, oxytocin appeared to alter individual protein expression in directions similar to conventional anti-psychotics. The proteins affected by oxytocin could be broadly categorized as those that modulate glutamatergic, GABAergic or dopaminergic signalling and those that mediate cytoskeleton dynamics. Our results here encourage further research into the clinical application of this peptide hormone, which may potentially extend treatment options across a spectrum of neurodevelopmental conditions. Introduction Oxytocin has a recognised role in lactation and parturition. However, it also has central binding sites in the limbic system and basal ganglia [1] and is now appreciated to be involved in the regulation of a wide variety of social and non-social behaviours [2]. As a consequence, oxytocin has been proposed to have utility in neurodevelopmental disorders of social processing and PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 1 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice Competing Interests: Grainne McAlonan currently serves as an Academic Editor or Guest Editor for this journal. cognition. Emerging evidence suggests oxytocin can serve as an antipsychotic that modulates glutamatergic signalling [3]. Indeed, several studies have reported that oxytocin ameliorated symptoms of schizophrenia [4] and exerted a generally positive impact on social behaviour, cognition and memory in humans [5]. Preclinical studies are broadly in agreement. Social interaction deficits in rats caused by chronic phencyclidine administration were reversed by oxytocin [6] and rats treated with antipsychotics had elevated oxytocin secretion, suggesting that endogenous oxytocin contributes to the antipsychotic action of conventional antipsychotic drugs [3]. However, there remain a number of gaps in our understanding of oxytocin’s therapeutic potential. First, pre-clinical studies often examine a limited range of behavioural tasks, and it is unclear whether oxytocin modulates both social and non-social behaviours at the doses used. Second, many studies have a male bias but, since brain oxytocin and oxytocin receptor distributions are sex specific [7], it is not known if results generalize across both sexes. Third, the protein substrates in the neural networks targeted by oxytocin remain relatively obscure. Therefore, in this study we sought to clarify the effects of a range of oxytocin doses on social and non-social behaviours in female and male C57BL/6N mice. We hypothesized that peripherally injected oxytocin would improve performance in social and non-social tasks and alter protein expression in a similar direction to that reported for anti-psychotic medication. We also predicted that oxytocin would alter protein expression in brain. We selected the striatum as a region-of -interest because it is a major neural substrate of oxytocin [8]; it is strongly implicated in neurodevelopmental disorders [9], linked to both social and non-social behaviour traits in these conditions [10]. For example, neuroanatomical differences relative to typically developing controls are consistently reported in autism and schizophrenia [11–14]. In line with this, preclinical studies have linked the striatum to behaviors relevant to these disorders including sensorimotor gating, social interaction and amphetamine sensitivity [15–21]. In addition, the striatum is considered to be a key target for the anti-psychotic action of medications used in schizophrenia [22–27]. Therefore, we predicted that oxytocin would alter striatal protein expression a similar direction to that reported in preclinical and clinical studies of conventional anti-psychotic treatment. Materials and Methods Animals One hundred and sixteen adult C57BL/6N mice (8 weeks old, female = 58, male = 58) were used in this study of oxytocin. All mice were obtained from and housed in the Laboratory Animal Unit (LAU) at the University of Hong Kong. The experimental protocol had been approved by the Committee on the Use of Live Animals for Teaching and Research, the University of Hong Kong (CULATR case no: 2189–10, 2624–12). The behaviour holding room was maintained at 21°C but, unlike the general areas of the LAU, had a reversed day-night cycle (light on: 7PM-7AM). Mice were therefore acclimatized for a minimum of one week before testing. All behavioural tests were conducted in the dark phase of the light-dark cycle. Behavioural testing Prepulse inhibition test (PPI). The whole-body startle responses of mice were measured in mouse startle chambers supplied by SR-LAB (San Diego Instruments, San Diego, CA, USA). Mice were placed in the startle chamber immediately after either oxytocin or saline injection and left undisturbed for 10min before the PPI test began [28]. The PPI paradigm conducted followed the standard protocol in our lab [29]. As pharmacological treatments can alter baseline reactivity to startle stimuli, and confound interpretation of the PPI [30], for this paradigm PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 2 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice we employed a design incorporating 3 prepulse and 3 pulse conditions [31]. This allowed us to exclude conditions where altered baseline reactivity might confound interpretation of PPI. All startle stimuli and background stimuli (NS) comprised white noise. The background stimulus was 65dB. The three intensities of a 40ms pulse were 100dB, 110dB and 120dB; the three 20ms prepulse intensities were 6dB, 12dB or 18dB above background. The session began with six consecutive pulse-alone trials. The first block (first six trials) had two of each pulse-alone condition to habituate and stabilize the startle response. These trials were not included in the statistical analysis. Thereafter the test session included 10 blocks of 16 trials in pseudorandom order. Each block was composed of three prepulse-alone trials (+6, +12 or +18dB), three pulsealone trials (100, 110 or 120dB), 9 combinations of prepulse-pulse trials (3 prepulse options×3 pulse options), and one no-stimulus (NS) trial. The test session ended with a final block of six pulse-alone trials as in the first block. The whole session lasted approximately 45 minutes. PPI data were collected by SR-LAB. PPI was taken as the reduction in pulse-induced startle reaction on prepulse_pulse trials relative to the reaction in the pulse_alone trials. The proportional (percentage) reduction of the startle amplitude (%PPI) based on the non-transformed startle response was calculated by the formula [(Rpulse_alone—Rprepulse_pulse)/ Rpulse_ alone] × 100%, where “R” was the individual mean response of the startle reaction. PPI data were analysed by repeated measures ANOVA, with pulse intensity (100 110 or 120 dB) and prepulse intensity (+6, +12 or +18 dB) as within-subject factors, and drug dose (saline and three doses of oxytocin), sex and drug exposure (single or repeated dose), as between-subject factors. Data are presented as (Mean ± SEM) and were analysed with IBM SPSS 22.0 software. P values less than 0.05 were considered to indicate statistical significance. Novel object recognition test. Object recognition in mice followed a standard protocol [32]. Mice were placed into an open field (40 cm×40 cm) test box, in which two identical solid impermeable objects (A1, A2) were positioned at one end of the field, each 10cm from the adjacent walls. Mice were allowed to explore the area for 3mins (Exploration Session-1). One hour later, they were exposed to the same open field and objects, which had been thoroughly cleaned with 70% ethanol, for 3 mins (Exploration Session-2). Either oxytocin or saline was administered immediately after the second exploration session. After 24 hours, the mice were again exposed to the test apparatus this time containing one of the original objects (A2) and a novel object (B) (different size, shape and colour, Fig 1). The time spent exploring each of the objects (Exploration Session-3) was assessed over 3 min. A discrimination index was calculated as the (exploration of novel object-exploration of familiar object)/ (total exploration time), i.e. (B-A2)/ (B+A2). All the mice had received one previous dose of drug injection respectively before the novel object recognition test. Recognition memory was assessed by the discrimination index. The novelty discrimination index was analysed using a two-way ANOVA, SPSS22, with sex and drug dose as between-subjects factors. Social interaction test The social interaction test was carried out in a neutral cage with standard bedding (30 cm×30 cm×30 cm) following methods previously published [33]. On the first three days before the test, each mouse, including mice of the same strain, age and sex to be used as ‘strangers’, were habituated to the cage in one 10min session each day. On day 4, the test day, either oxytocin or saline was administered 30min before the test. Pairs of an unfamiliar, ‘stranger’ mouse and experimental mouse were placed into the neutral cage for 10 min. The time spent in social interaction (active contact such as sniffing, following and grooming the partner) was measured over 10 min. PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 3 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice Fig 1. Novel object recognition test. Blue circles: A1, A2; original objects. Red triangle: B; novel object. Oxytocin doses: 10 μg/kg, 100 μg/kg and 1000 μg/kg. In the first day of the test, the subject explored the two objects of same colour and same shape twice in 1 hour interval. Oxytocin was administrated in the second test day, then the subject explored one of the same objects previously presented and a new object of different colour and shape. doi:10.1371/journal.pone.0145638.g001 The effects of oxytocin dose and exposure were analysed using a Univariate General Linear Model (GLM, SPSS 22), in which the drug dose and exposure (single or repeated) were the between-subject factors. Open field and amphetamine test The open field exploration test was tested following methods previously published [34]. It was performed in 40×40 cm2 rectangular white arenas. At the beginning of the session, the mouse was gently placed in the centre of the arena, and allowed to explore for 1hour while drug free. The mouse was then briefly removed from the arena, and drug administered quickly via the subcutaneous (s.c.) route. The arena was cleaned with 70% ethanol; the mouse was placed back in the arena and allowed to explore for another 30min. The mouse was again briefly removed from the arena and injected with 2.5 mg/kg d-amphetamine (calculated as the salt, sigmaAldrich, Switzerland) dissolved in a 0.9% NaCl solution via the intraperitoneal route. The volume of injection was 5 ml/kg. The arena was cleaned again. The mouse was placed back in the arena and allowed to explore for another 1hour (Fig 2). Order of behavioural testing Equal numbers of male and female mice received a single subcutaneous dose of 10 μg/kg, 100 μg/kg or 1000 μg/kg of oxytocin (Sigma Chemicals, St Louis, Mo., USA) in an injection volume of 5 ml/kg prior to testing. All mice were given one week of ‘rest’ between each test. Each mouse received the same dose of oxytocin or saline for all tests. To control for the potential effect of previous oxytocin exposure and/or behavioural test order, half the mice followed test sequence I; half followed test sequence II (Fig 3). Twenty mice (10 females; 10 males) received Fig 2. Open field and Amphetamine test. Oxytocin doses: 10 μg/kg, 100 μg/kg and 1000 μg/kg. The subject explored the arena for 1 hour while drug free. Then the subject explored the same arena for another 30min after drug (saline or oxytocin) injection. Finally the subject explored the same arena again for 1hour after amphetamine injection. doi:10.1371/journal.pone.0145638.g002 PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 4 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice Fig 3. Behavioural tests. The sequence of behavioural testing. Mice were dived into two groups of equal number and sex. Half the mice followed test sequence I; half followed test sequence II. PPI: Prepulse inhibition of startle. doi:10.1371/journal.pone.0145638.g003 saline injections only. There were 32 mice (16 females; 16 males) in each oxytocin dose group: 10 μg/kg, 100 μg/kg or 1000 μg/kg. Where appropriate, whether this was the first exposure to oxytocin or a repeat exposure was entered as a fixed factor in the analyses. The open-field and amphetamine challenge test were carried out last; therefore, all animals had previously received 3 doses of oxytocin or saline. In this test, to clarify whether pre-exposure to oxytocin was sufficient to modify low-dose (2.5 mg/kg) amphetamine response, or whether an additional dose of oxytocin was necessary, half the cohort received saline immediately before the amphetamine challenge; half received a single dose of oxytocin (10 μg/kg, 100 μg/kg or 1000 μg/kg) Proteomics and Western blot Twelve mice (female = 6, male = 6) were naïve to behavioural testing; half were exposed to 1000 μg/kg oxytocin, half to saline vehicle injection, once a week for 3 weeks were selected for proteomics and western blot analysis. The mice were sacrificed by cervical dislocation. The brain was removed and placed immediately on ice, and the striatum dissected free of the remainder of the brain tissue. The striatal tissue from each mouse was homogenized in 0.5ml of ice-cold 0.32 M sucrose buffered with 5 mM HEPES, pH7.5. Proteomics analyses Striatum was homogenized in 2D lysis buffer (7M Urea, 2M Thiourea, 2% CHAPS, 10mmol DTT, 0.5% IPG buffer) for 30 seconds, then centrifuged at 15,000×g for 15 minutes to remove cell debris. Supernatant for 2-DE was collected and quantified with protein assay kit (Pierce1 660nm Protein Assay, Thermo Scientific). The 2D procedure followed the protocol from Bio-Rad Laboratories (Bio-Rad Laboratories, Inc.), with minor modifications. Rehydration and first dimension were carried out using BioRad isoelectric focusing (IEF) separation (Bio-Rad IEF system). The 350ul rehydration buffer containing 180ug protein was loaded on 18cm, pH 4–7, IPG DryStrips (Bio-rad). IEF conditions were: 50V, 18h, 1000V, 1h; 3000V, 3h, 8000V, 6h. After the IEF run was complete, the IPG strips were equilibrated for 2×20min in the equilibration buffer containing 50mM Tris, 6M urea, 30% glycerol, 2% SDS. The first equilibration was performed in the equilibration buffer with 1.0%w/v DTT followed by a second equilibration with 2.5% w/v iodoacetamide. PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 5 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice The strips were subsequently subjected to a secondary dimensional separation by 12% SDS-PAGE (Bio-Rad vertical system). The SDS-PAGE was performed at 10mA/gel for 30min, and then 45mA/gel, until the dye front reached the bottom of gels. After fixing in 50% ethanol, 12% acetate solution and staining with silver nitrate, gels were scanned at 300dpi resolution and the images were analysed with Image Master Platinum™ software (GE Health care). Protein spots with p < 0.05 and > 1.3-fold change were considered significant. Spots of interest were picked from sliver-stained gels, followed by in-gel trypsin digestion and peptide extraction [35] for protein identification by tandem mass spectrometry analysis using ABI 4800 MALDI TOF/TOF™ MS Analyzer (Applied Biosystems, Foster City, CA, USA). The combined peptide mass fingerprinting (PMF) and MS/MS peptide fragmention data were submitted to the NCBInr database and SwissProt database using the software MASCOT version 2.2 (Matrix Science). For all significant protein identifications, both protein and total ion scores were above or equal to C.I. 99%. Western blot analyses The striatum was homogenized at 4°C in lysis buffer (1:5,wt/vol) containing 1mM EDTA and 20mM phenylmethylsulphonyl fluoride. The proteins from the resultant supernatant were determined (Bio-Rad Protein Assay) for Sodium Dodecyle-Sulphate Polyacrylamide gel electrophoresis (SDS-PAGE). Protein (20ug/lane) was subjected to electrophoresis on a 10% (wt/ vol) polyacrylamide gel in SDS, and gels subsequently processed for electroblotting to polyvinylidene difluoride (PVDF) membranes. The blotted PVDF membranes were saturated with 5% (wt/vol) of skimmed milk in Tris Buffer Saline, PH 7.4 and 0.1% (vol/vol) of Tween 20 for 1h at room temperature. The membranes were sequentially incubated with primary antibodies to anti-Calcineurin B (rabbit polyclonal IgG antibody, 1:500 dilution, Cat# ab136526, Abcam); anti-GAD 67 (rabbit polyclonal IgG antibody, 1:1000 dilution, Cat# ab52249, Abcam) and anti-GAD 65 (rabbit polyclonal IgG antibody, 1:500 dilution, Cat# ab75750, Abcam) overnight at 4°C following by a peroxidase-labelled anti-mouse/rabbit IgG (1:2000 dilution, Boehringer Mannheim, Germany) for 1h at room temperature. After thorough washing, positive bands were revealed using ECL western blotting detection reagents and autoradiography film (Amersham, Biosciences, UK). The intensities of the bands were quantified using IMAGE QUANT software (Molecular Dynamics, USA). Statistics Behavioural results from each task were analysed using a GLM running in SPSS22 with ‘Dose’ of drug (saline, 10 μg/kg, 100 μg/kg, 1000 μg/kg oxytocin), ‘Sex’ and ‘Repeat exposure’ to oxytocin/saline (where appropriate) as between subject factors. Where there was a significant main effect of Sex, data were analysed in each Sex separately. If there was no effect of repeat exposure, data were combined for further analyses. Significant findings observed using GLM were explored using post-hoc Bonferroni tests were appropriate. In PPI, drug treatment may alter the baseline startle response to pulse stimuli. This impacts upon subsequent analysis of %PPI [30]. Therefore, our PPI design included 3 different pulse conditions so that, should baseline startle reactivity to any pulse condition be significantly altered by oxytocin, that pulse condition could be excluded from further analysis of %PPI. Multivariate analysis using partial least squares-discriminant analysis (PLS-DA, SIMCA 12.0, Umetrics, Umea, Sweden) was performed to examine whether C57BL/6N mice with oxytocin exposure could be discriminated from those with saline exposure on the basis of full proteome profile [36]. Subsequently Analysis of Variance running in the standard in multivariate data analysis (SIMCA-P+ 12.0, Umetrics) was used to identify proteins with significant up or PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 6 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice down regulation in the oxytocin treated group compared to saline treated group. Protein expression differences in Western Blot were analysed using t-tests. Results Behaviour In saline treated animals, the order of testing had no effect on any test. Pulse-alone reactivity (Baseline startle reaction). There was significant effect of sex on baseline pulse reactivity (F (3, 100) = 28.883, p = 0.000); and a significant interaction of sex × dose (F (3, 100) = 4.243, p = 0.007). However, previous oxytocin exposure had no impact on the baseline startle response (p = 0.214), therefore data from mice exposed to a single or repeated dose was combined for further analysis in each sex separately. This analysis indicated that male mice had higher startle responses than females at all doses of oxytocin (p < 0.01 for each dose). Post-hoc analyses in each sex subsequently revealed that oxytocin altered startle responsivity at only one pulse condition in each sex; lowering the startle response at pulse 120 dB stimulus in females (F (3, 54) = 3.330, p = 0.026, Fig 4A); increasing the startle response at pulse 100 dB in male mice (F (3, 54) = 4.157, p = 0.010, Fig 4B). Therefore PPI analyses were conducted in each sex separately and data from pulse 120dB in females and pulse 100dB in males was excluded. PPI. Previous oxytocin exposure had no impact on PPI (p = 0.957 in females; p = 0.393 in males), data from mice exposed to a single or repeated dose was combined for further analysis. Oxytocin attenuated PPI in females (F (3, 54) = 3.546, p = 0.020), especially following 100 μg/ kg (Boneferroni test; p = 0.012, Fig 4C). In males, there was a significant dose x pulse interaction (F (3, 54) = 3.120, p = 0.033); oxytocin improved PPI at pulse 120dB (F (3, 54) = 5.112, p = 0.003), at all doses (post-hoc Bonferroni tests, p< 0.01 Fig 4D). Novel object recognition test. Oxytocin increased time spent exploring objects at first presentation (F (3, 108) = 4.292, p = 0.007), especially at doses of 100 μg/kg and 1000 μg/kg oxytocin (Bonferroni tests: p = 0.017 and p = 0.033 respectively). There was no effect of Sex on this measure. Analysis of the novelty discrimination index revealed a significant sex × dose interaction approached significance (F (3, 108) = 2.628, p = 0.054). Post-hoc analyses in each sex separately revealed oxytocin did not alter this index in females but disrupted recognition memory in males (F (3, 54) = 3.869, p = 0.014) at both 100 μg/kg and 1000 μg/kg doses (Bonferroni, (p = 0.023 and p = 0.019 respectively, Fig 5A). Social interaction test. Oxytocin increased social interaction (F (3, 100) = 10.914, p = 0.000) but there were significant interactions of dose with sex (F (3, 100) = 2.827, p = 0.047); exposure (F (3, 100) = 4.007, p = 0.009); and sex × exposure (F (3, 100) = 3.365, p = 0.022) on this measure. Social interaction was therefore examined in each sex separately. Oxytocin increased social interaction time in females (F (3, 50) = 5.281, p = 0.003) and the dose × previous exposure interaction was significant (F (3, 50) = 4.789, p = 0.005). Single doses of oxytocin increased social interaction time in females (F (3, 25) = 5.942, p = 0.03). This result was driven by the 1000 μg/kg dose (post-hoc Bonferroni p = 0.012; Fig 5B). Previous exposure to oxytocin also increased social interaction time (F (3, 25) = 3.635, p = 0.027), especially at doses of 10 μg/kg and 100 μg/kg oxytocin (Bonferroni tests: p = 0.034 and p = 0.049 respectively, Fig 5C). Oxytocin increased social interaction time in male mice (F (3, 50) = 9.779, p = 0.000) regardless of pre-exposure. Therefore data from groups exposed to a single or repeated dose of oxytocin were combined. Post-hoc Bonferroni test confirmed that 100 μg/kg oxytocin significantly increased social interaction time in male mice (p = 0.019, Fig 5D). PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 7 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice Fig 4. PPI test. (A) Oxytocin attenuated the baseline startle response to 120dB pulse in female mice. (B) Oxytocin increased the baseline startle response to 100dB pulse in male mice. (C) 100 μg/kg oxytocin attenuated PPI in females. (D) All doses of oxytocin improved PPI in males. Error bars refer to ± SEM. doi:10.1371/journal.pone.0145638.g004 Open field and amphetamine challenge test Basal locomotor activity: All mice had been given three doses of oxytocin or saline before the open field and amphetamine challenge (Fig 3); all had completed PPI, novel object and social interaction paradigms. The total distance moved in a one-hour open field test provided a measure of basal exploratory locomotor activity. Following an injection of oxytocin or saline, locomotion was measured for 30 minutes. Finally, each animal received 2.5 mg/kg amphetamine and locomotion measured for 60 minutes. Previous exposure to oxytocin lowered baseline activity in both sexes (F (3, 100) = 3.649, p = 0.015), especially at 100 μg/kg (p = 0.009). There was no effect of an additional dose of oxytocin before testing (Fig 6A and 6B). Amphetamine-induced locomotor activity: Oxytocin significantly attenuated the response to amphetamine (F (3, 100) = 11.743, p < = 0.001). There was no effect of an additional dose of oxytocin before testing. As previous oxytocin exposure altered baseline activity, the data were reanalysed with baseline distance entered as a covariate. This did not alter the pattern of results; thus differences in baseline activity could not explain attenuation of amphetamine-induced hyperlocomotion by oxytocin. There was however a significant time × dose × sex interaction in the amphetamine response (F (33, 1167) = 2.115, p = 0.000), therefore, post-hoc analysis examined each sex separately. PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 8 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice Fig 5. Novel object recognition and social interaction test. (A) 100 μg/kg and 1000 μg/kg oxytocin disrupted recognition memory in males. 1000 μg/kg single exposure (B) and 10 μg/kg and 100 μg/kg repeated exposure to oxytocin (C) increased social interaction in females. (D) 100 μg/kg oxytocin increased social interaction in males. Error bars refer to ± SEM. * post-hoc testing: p<0.05. doi:10.1371/journal.pone.0145638.g005 Oxytocin attenuated the response to amphetamine in females (F (3, 54) = 10.477, p = 0.000), especially at 100 μg/kg (p = 0.033) and 1000 μg/kg doses (p = 0.000, Fig 6C). It also attenuated the response to amphetamine in males (F (3, 54) = 3.846, p = 0.015), particularly at the highest dose (1000μg/kg) (p = 0.008, Fig 6D). As 3 repeated weekly doses of 1000ug/kg oxytocin caused a clear suppression of amphetamine-induced locomotion in both male and female mice, this dose regime was chosen for the 2D proteomics analysis. Striatum proteomics A total of 811 individual protein spots were visualized across 12 gels (saline: n = 3 males, n = 3 females; oxytocin: n = 3 males, n = 3 females). All spots were included in a partial least squaresdiscriminant analysis (PLS-DA), which clearly separated protein expression in mice exposed to oxytocin from saline controls (Fig 7A). Analysis of covariance confirmed significant drug-related differences in the expression of 15 proteins (Table 1). Western blot analysis of striatal tissue from animals used for behavioural testing confirmed that a protein identified in 2D proteomics, calcineurin, was significantly lowered by oxytocin. PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 9 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice Fig 6. Open field and amphetamine test. (A) Distances moved in each 5-min time bin at baseline. (B) Total distance moved in 1 hour at baseline. Previous exposure to oxytocin lowered baseline activity in both sexes. (C) 100 μg/kg and 1000 μg/kg doses of oxytocin significantly suppressed amphetamine activity in females. (D) 1000μg/kg Oxytocin exposure significantly suppressed amphetamine activity in males. Error bars refer to ± SEM. post-hoc testing: p<0.05. doi:10.1371/journal.pone.0145638.g006 2D-gel results were confirmed by Western blot analysis in a selected target protein, calcineurin. In addition, because 2D-gel analysis suggested that oxytocin may act upon GABA-glutamate pathways, we examined the effects of oxytocin on glutamic acid decarboxylase (GAD65 and GAD67) post-hoc. GAD catalyses the conversion of glutamate to GABA, and is a marker for GABAergic neurons. Both isoforms have been reported to be lower in schizophrenia and related neurodevelopmental disorders [37] and dopamine inhibits striatal GAD67 [38]. We expected that an ‘anti-psychotic’ effect of oxytocin would include up-regulation of GAD67 in striatum of C57BL/6N mice. In addition, GAD67 expression (but not GAD65) was significantly elevated by oxytocin (Fig 7B and 7C). Discussion Oxytocin has been proposed to be a potential treatment for neurodevelopmental conditions. In this study, peripherally administered oxytocin improved PPI and social interaction in males and attenuated the response to amphetamine in open field test in both sexes. This adds to an increasing body of evidence that oxytocin promotes pro-social behaviour and also modulates non-social behaviour [2]. There were also global differences (PLS-DA analysis) in protein expression elicited by a 1000 μg/kg dose of oxytocin compared to saline. Oxytocin generally, had effects broadly similar to conventional anti-psychotics (Table 1). The proteins altered by oxytocin could be categorized as those modulating glutamatergic, GABAergic or dopaminergic signalling systems, and cytoskeleton components. Such expression changes may in part explain the behavioural differences observed following oxytocin administration. PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 10 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice Fig 7. Striatum proteomics and western blot studies. (A) Multivariate analysis: partial least squares—discriminative analysis (PLS-DA) carried out using SIMCA-P+12.0 software (Umetrics). The striatal protein profile following oxytocin exposure could be clearly differentiated from saline (controls). The XY axis shows the coordinates of the protein distribution in the partial least squares—discriminative analysis (B) Western blot gel images of target proteins. (C) The relative expression of the target proteins/β-actin, Columns show mean±SEM (n = 9 for saline exposure; n = 8 for oxytocin exposure). p<0.05. doi:10.1371/journal.pone.0145638.g007 PPI Deficits in sensorimotor gating measured using PPI are associated with neurodevelopmental disorders. We selected the C57BL/6N mouse strain for this study because it has lower PPI than many other in-bred strains [39] and has been suggested to model aspects of schizophrenia [40]. Thus, any improvement elicited by oxytocin should be evident above the low baseline PPI expected in these animals. Oxytocin increased PPI in male mice—a direction of effect similar to that reported using conventional anti-psychotics [41]. This result is partly consistent with previous reports that high doses of oxytocin (1000μg/kg) improve PPI in male Brown-Norway rats [42] and reverse PPI deficits induced by NMDA antagonists in rats [28]. However, the sensitivity of rodents to oxytocin is likely to be species and strain specific [43], and to be dependent upon the parameters of the PPI paradigm [44]. PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 11 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice Table 1. Proteins with differences in expression levels following 3 injections of oxytocin 1000 μg/kg or saline, at an interval of 1 week apart. Spot No. Fold difference p value Protein name NCBI no. Previous reported change with antipsychotics 33 1.6651 0.0178 protein DJ-1 gi\|55741460 Up [74] 34 1.7887 0.0132 ubiquitin carboxyl-terminal hydrolase isozyme L3 gi\| 139948802 (No report) 37 1.3999 0.0453 alpha-enolase gi\|34784434 Up [75] 42 1.9003 0.0067 D-3-phosphoglycerate dehydrogenase gi\|986918 Up [76] 51 1.6083 0.0433 septin-6 isoform 1 gi\| 293597553 Up [76] 80 1.8686 0.0174 gamma-enolse gi\|7305027 Up [76] 81 2.1161 0.0329 Rho GDP-dissociation inhibitor 1 gi\|31982030 Up [77] 95 1.8751 0.0372 14-3-3 protein zeta/delta isoform 1 gi\|6756041 Up [77] 100 1.9886 0.0290 dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial gi\|21313536 (No report) 109 2.0968 0.0490 triosephosphate isomerase, partial gi\|1864018 Down [78] 127 2.2133 0.0049 dihydropyrimidinase-related protein 2 gi\|40254595 Up [75] 145 1.9783 0.0026 tubulin alpha-1C chain gi\|6678469 Up [76] 275 1.9778 0.0273 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit, isoform 1 gi\| 148677501 Up [74] 322 -6.1599 0.0031 ornithine aminotransferase, mitochondrial precursor gi\|8393866 (No report) 330 -2.5450 0.0275 calcineurin subunit-B gi\|4506025 Down [78] Fold differences in protein expression in the oxytocin group relative to the saline group are shown in column 2. Column 6 (ﬁnal column) indicates how changes in protein expression elicited by oxytocin compare to those reposted to follow antipsychotic treatment in post-mortem studies of schizophrenia and/or rat models (ﬁnal column). doi:10.1371/journal.pone.0145638.t001 Although oxytocin attenuated PPI in female mice, as can be seen from Fig 4C and 4D, the levels of PPI in female mice were higher than even males treated with oxytocin. Thus oxytocin improved a behaviour ‘impaired’ in males, but had limited impact upon behaviour essentially intact in females. The latter observation fits with clinical evidence that PPI is intact in females with schizophrenia [45]. Our results contrast with the previous observation that neither oxytocin knockout [3] nor oxytocin receptor knockout [46] disrupts PPI. Thus, oxytocin is not ‘necessary’ for PPI, but does alter it. One mechanism for this action may be through the glutamate-GABA system which contributes to PPI [47] and is thought to be modulated by oxytocin [48]. Consistent with this, we found that oxytocin down-regulated ornithine aminotransferase, which catalyses the transamination of ornithine to glutamate [49]; and, up-regulated 14-3-3 protein zeta/delta (or KCIP-1), involved in GABAB receptor coupling and dopamine D2 receptor inhibition [50]. In addition, oxytocin increased the level of GAD67 which reflects intraneuronal gamma-aminobutyric acid levels [51]. Thus, we speculate that the net effect of oxytocin may be to promote GABAergic signalling and restrain glutamate and dopamine activity, improving PPI. Novel object recognition In the first step of the novel object paradigm, oxytocin increased time spent exploring the objects, possibly representing an anxiolytic action [52]. However, the novel object component of this paradigm indexes memory for a familiar object and oxytocin disrupted recognition memory in males (Fig 3A) without altering this measure in females. PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 12 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice Again a pro-GABA action may contribute through calcineurin and KCIP-1 regulation of GABAA and GABAB receptors respectively. In line with this, GABAA [53] and GABAB [54] agonists impair recognition memory and calcineurin knockout has also been reported to impair memory in mice [55]. Social interaction Oxytocin increased social interaction time in both females and males, but the dose response was sex-dependent (Fig 5B–5D). Oxytocin had a cumulative effect on social interaction in female mice; whereas previous exposure to oxytocin in males was no different to a single exposure. These facilitatory effects of oxytocin on social interaction are consistent with studies in oxytocin knock-out mice [56]. As discussed above, it is possible that oxytocin triggered changes in GABA signalling contribute. For example, GABAA agonists increase social interaction time in rats, and antagonists do the opposite [57]. No dose of oxytocin used reduced social interaction, ruling out sedative effects. Open field activity and response to amphetamine Amphetamine is an indirect-acting dopamine agonist that increases synaptic dopamine; patients with schizophrenia are particularly vulnerable to amphetamine. Here, oxytocin suppressed amphetamine-induced locomotion in both sexes (even controlling for baseline activity differences). A possible explanation is that oxytocin promotes GABA-medicated inhibition of striatal dopamine release [58], and the increased level of GAD67 following oxytocin exposure points to increased GABAergic inhibitory activity. However, our proteomics results suggest additional pathways for oxytocin to limit dopamine mechanisms. For example, the 14-3-3 protein zeta/delta, up-regulated by oxytocin, is known to inhibit PKC [59] and PKC inhibitors have been shown to block amphetamine-induced dopamine release from rat striatal synaptoneurosome [60]. The attenuation of the amphetamine response occurred in mice previously exposed to oxytocin, with or without an additional acute dose of oxytocin (Fig 6). This indication that intermittent exposure to oxytocin can have ‘lasting’ effects will be important to explore further in light of recent evidence that chronic administration of oxytocin may have undesirable effects, disrupting social bonding in male voles [61]. Effects of oxytocin on other proteins In addition to actions on the glutamate-GABA systems, oxytocin had other wide-ranging effects including on proteins involved in cell structure and plasticity—tubulin-α dihydropyrimidinase-related protein 2 (DRP-2) and D-3-phosphoglycerate dehydrogenase (3-PGDH) [62–64]; and mitochondrial function—upregulating ATP synthase subunit alpha [65]; DJ-1 [66], and enolase (linked to recovery of cerebral metabolism in patients with schizophrenia treated with anti-psychotic medication [67]). Limitations There are some limitations to our current study. To reduce the number of animals used, we tested each mouse in every behavioural paradigm design. The order of testing did not impact upon findings in the animals exposed to saline, and previous exposure to oxytocin did not influence results of PPI in either sex or social interaction in males. However, previous exposure was sufficient to suppress the amphetamine response in both sexes and improve social interaction in females. Unfortunately, our study was not designed to test the effect of an acute PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 13 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice exposure to oxytocin in naïve animals in the amphetamine challenge (without greatly increasing the number of animals used). The half-life of oxytocin is 10-15min [68], but only about 0.1% of oxytocin can cross the blood brain barrier [69]. Therefore the effects studied here are most liekly indirect. The timing of oxytocin administration was challenging to match across tasks. For example, in PPI, as in other drug studies using this paradigm, oxytocin was administered 10 mins before the task, but data collected in the task for a further 45 mins. In the social interaction and amphetamine challenge tests the effects of oxytocin were captured after 30mins. Thus for these tasks the effects of oxytocin from 30mins would have been captured. However, oxytocin was given after the ‘learning phase’ of the object recognition task to establish if it had any effect on the consolidation stage. We observed subtle sex differences in the oxytocin dose response. However, the study was not primarily designed to compare sex differences in response to oxytocin. Rather the study was designed to look at the effects of oxytocin and both male and female animals were included. This is increasingly recognised as important and the NIH now requires the reporting of plans for a balance of male and female animals in preclinical studies unless single sex inclusion is specifically warranted [70]. Unfortunately, the underlying differences in baseline behaviours make interpretation difficult. For example, as previously reported, female C57BL/6N mice had higher levels of PPI than males [71]. This is not thought to be a consequence of the oestrous cycle which does not alter PPI and anxiety-related behaviours in female C57BL mice [72]. Male C57BL/6N mice also had longer social interaction times than female mice (Fig 5B, 5C&5D); possibly meaning they were less anxious than females. However, in addition to these baseline behavioural differences between the sexes, the differential response to oxytocin may also be a consequence of sex differences in expression of oxytocin and its receptors [7] and further work is therefore needed to clarify sex effects of oxytocin. We only examined 2D protein differences in the striatum in response to repeated exposure to 1000 μg/kg oxytocin in naïve animals, and we cannot directly link differences to phenotypic differences. However, confirmatory Western blot studies carried out using tissue from animals involved in the behavioural experiments adds weight to the likelihood that the proteins implicated contribute to the behavioural differences observed. Protein expression differs across brain regions [73] and we do not know if our results generalize across the brain. Nor did we examine protein expression in each sex separately. However, the PLS-DA suggests the distinct protein profile in animals exposed to oxytocin and saline involved both sexes and the effects of oxytocin on social interaction and amphetamine challenge tasks were broadly similar in both sexes. Lastly, the proteomics approach used here was a standard analysis that does not fully sample intracellular organelles. The future separation of subcellular fractions would be useful. Conclusion Oxytocin significantly improves social and non-social behaviours in an experimental animal setting. It elicits a pattern of changes in striatal protein expression similar to that triggered by conventional antipsychotics. We hope that this study encourages further research into the clinical application of this peptide hormone, which may extend treatment options across a spectrum of neurodevelopmental conditions. Acknowledgments The authors would like to thank the staff of the Laboratory animal Unit in The University of Hong Kong for their expert assistance with breeding and husbandry. This study was supported PLOS ONE \| DOI:10.1371/journal.pone.0145638 December 30, 2015 14 / 18 Oxytocin Effects on Behaviour and Protein Expression in C57BL/6N Mice by research funding from the Hong Kong Universities General Research Fund Award to Dr Grainne McAlonan (HKU 774710) and research funding from National Natural Science Foundation of China to Professor Wang (61327902) and Professor Zhang (81271406). Dr Grainne McAlonan is a member of the EU-AIMS consortium. 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https://openalex.org/W1875039158	https://www.mdpi.com/2075-4701/5/4/1997/pdf?version=1446026764	English	null	CFD Modelling of Flow and Solids Distribution in Carbon-in-Leach Tanks	Metals	2,015	cc-by	11,662	Metals 2015, 5, 1997-2020; doi:10.3390/met5041997 Metals 2015, 5, 1997-2020; doi:10.3390/met5041997 metals ISSN 2075-4701 www.mdpi.com/journal/metals/ OPEN ACCESS Keywords: CIL tanks; CFD; hydrodynamics; HA-715 impeller; impeller configuration; design Keywords: CIL tanks; CFD; hydrodynamics; HA-715 impeller; impeller configuration; design 1. Introduction Carbon-in-Leach (CIL) circuit is a process that concentrates gold from 2.5 to 3.5 g/t in ore to 10,000 to 15,000 g/t on carbon. It is a process of continuous leaching of gold from ore to liquid and counter-current adsorption of gold from liquid to carbon particles in a series of tanks. The tanks used in the CIL circuit are continuously stirred tanks and contain high concentration of ores. Efficient operation of CIL tanks requires suspension of the ore particles in the leaching solution and hence, to provide maximum contact between ore and leaching solution. However, problems such as settled solids and presence of dead zones are detrimental to the efficiency, and could be identified and solved by understanding the flow field and solid distribution in the system. Furthermore, reducing the energy consumption to achieve a higher contact is always desired and can be achieved by proper design and optimization while investigating the hydrodynamics. While, some information on the hydrodynamics such as residence time distribution (RTD) can be obtained from experiments using tracer studies; detailed quantitative measurement of the flow field inside the CIL tank is challenging. In such a scenario, computational fluid dynamics (CFD) can prove to be an inexpensive and viable solution. With the availability of improved models for turbulence, interphase drag force, particle-particle interaction models, etc. and advances in computational speeds, resolving the complex multiphase flows phenomenon is possible using CFD. In the present work, models for simulating high solid loading stirred tanks are validated with experimental data. The flow field generated by a pitched blade turbine and a HA-715 impeller is compared. The effect of design parameters such as off-bottom clearance, impeller separation, impeller speed, scale-up, and multiple-impeller configuration is examined by modelling the flow and estimating the suspension quality for each case. Divyamaan Wadnerkar, Vishnu K. Pareek and Ranjeet P. Utikar * Department of Chemical Engineering, Curtin University, Perth, WA 6102, Australia; E-Mails: d.wadnerkar@curtin.edu.au (D.W.); v.pareek@curtin.edu.au (V.K.P.) Department of Chemical Engineering, Curtin University, Perth, WA 6102, Australia; E-Mails: d.wadnerkar@curtin.edu.au (D.W.); v.pareek@curtin.edu.au (V.K.P.) * Author to whom correspondence should be addressed; E-Mail: r.utikar@curtin.edu.au; Tel.: +61-8-9266-9837; Fax: +61-8-9266-2681. * Author to whom correspondence should be addressed; E-Mail: r.utikar@curtin.edu.au; Tel.: +61-8-9266-9837; Fax: +61-8-9266-2681. Academic Editors: Suresh Bhargava, Mark Pownceby and Rahul Ram Received: 29 July 2015 / Accepted: 23 October 2015 / Published: 28 October 2015 Abstract: The Carbon-in-Leach (CIL) circuit plays an important role in the economics of a gold refinery. The circuit uses multiphase stirred tanks in series, in which problems such as dead zones, short-circuiting, and presence of unsuspended solids are detrimental to its efficiency. Therefore, the hydrodynamics of such a system is critical for improving the performance. The hydrodynamics of stirred tanks can be resolved using computational fluid dynamics (CFD). While the flow generated by the impellers in the CIL tanks is complex and modelling it in the presence of high solid concentration is challenging, advances in CFD models, such as turbulence and particle-fluid interactions, have made modelling of such flows feasible. In the present study, the hydrodynamics of CIL tanks was investigated by modelling it using CFD. The models used in the simulations were validated using experimental data at high solid loading of 40 wt. % in a lab scale tank. The models were further used for examining the flow generated by pitched blade turbine and HA-715 Mixtec impellers in lab scale CIL tanks with 50 wt. % solids. The effect of design and operating parameters such as off-bottom clearance, impeller separation, impeller speed, scale-up, and multiple-impeller configuration on flow field and solid concentrations profiles was examined. For a given impeller speed, better solids suspension is observed with dual impeller and triple impeller configurations. The results presented in the paper are useful for understanding the hydrodynamics and influence of design and operating parameters on industrial CIL tanks. Metals 2015, 5 1998 Metals 2015, 5 Keywords: CIL tanks; CFD; hydrodynamics; HA-715 impeller; impeller configuration; design Metals 2015, 5 9.6% v/v), which were significantly smaller than the experimental values of 4.59, 4.37, and 4.23. They attributed the imperfection in the solid suspension prediction to second order effects (particle drag modifications due to liquid turbulence, presence of other particles, particle-particle direct interactions, etc.) that were neglected in the study. Ochieng and Lewis [6], Fradette, et al. [7], Ochieng and Onyango [8], Kasat, et al. [9], Fletcher and Brown [10], Tamburini, et al. [11–13], conducted simulations for volume fractions below 20%, and validated using non-local properties like cloud height, suspension quality, etc. In another similar study, Gohel, et al. [14] used qualitative and quantitative data for cloud height to validate the simulation of high concentration solids suspension in stirred tanks. In these studies, while the parameters, for example cloud height, were accurately predicted, the errors in the predictions of local hydrodynamics were not verified in the absence of data. In the direction of resolving the local hydrodynamics of the stirred tanks, Liu and Barigou [15] used local velocity field and solids concentration data for model validation and found that even though a good agreement in the axial concentration profile was observed, the local concentration predictions could still be very poor and could vary from experiments by several folds. The inaccuracy was attributed to inadequate models for particle sedimentation, lift-off, and particle-particle interaction in their CFD model. They suggested incorporating particle-particle interaction in the solids pressure term given by Gidaspow [16], but did not use it in their models due to convergence problems. The highest solid loading for which the simulation results in stirred tanks are reported is 20% (by volume), and the simulations are not able to predict the local concentration due to limitations of models [4,5]. In the CIL tanks, Dagadu, et al. [17] used a radioactive tracer to investigate the RTD in the CIL tanks and developed a model to predict it. While the results provided an overview of complexity of the system and suggested the presence of stagnant and active volumes, the details such as solid accumulation, dead-zones, uniformity, etc. are still unknown. To address these issues, Dagadu et al. [18,19] investigated the mixing in the CIL tanks by conducting CFD simulations and drawing inferences based on the flow field and eddy viscosity. Metals 2015, 5 In both of these instances, neither the relationship of the flow field or turbulent viscosity with the solid distribution is quantified, nor the solid distribution in the tanks are presented. While validation with experimental data is missing, the scope of both studies is limited to evaluating the ability of the models for predicting the qualitative flow behavior in such systems. Drawing conclusions for critical design and operating parameters requires comprehensive validation of models and controlled investigation of influence of these parameters on the flow field and solid distribution. The current study focuses on the extensive validation of local solid concentration distribution in high solid loading stirred tanks. The shortcomings of previous investigations are addressed by using the appropriate constitutive models for turbulence, drag, particle-particle interactions, etc. The validated model is then used for investigation of critical parameters in the CIL tanks. 2. Literature Review Carbon-in-leach tanks are high solid loading stirred tanks of large diameter (~10–15 m) with solid concentration of up to 50% by weight (~28% by volume). Such a high concentration renders opacity to the system which makes its hydrodynamic investigation challenging even at a small scale. With the advent of radioactive experimental techniques such as Computer Aided Radioactive Particle Tracking (CARPT), Positron Emission Particle Tracking (PEPT), etc., reliable quantitative data can be obtained [1–3]. The data can now be used to validate the computational models for the high solid loading regimes in turbulent flows and further advance the design and optimization of CIL tanks. While simulating high solid concentration (20% by volume) stirred tanks, Altway et al. [4] found major discrepancy while validating the solid concentration profile using data from Yamazaki et al. [2]. Micale et al. [5] used Multiple Reference Frame (MRF) and Sliding Grid (SG) approach to study the clear liquid layer and the suspension height for dense solid-liquid systems. In their simulations, the power numbers were 2.98, 2.74, and 2.68 for N = 5, 6.33 and 8 RPS respectively (particle loading of 1999 Momentum equation:     , , , 12 α ρ . α ρ α . α ρ td q vm lift q q q q q q q q q q q u u u p g F F t          (2) (2) The rotation of impeller can be simulated using sliding grid approach (SG) or Multiple Reference Frame (MRF). While MRF provides reasonably accurate steady state solution, SG is found to be more accurate compared to MRF approach [20,21]. Therefore, SG will be used in the paper to simulate the impeller rotation. Turbulence is not resolved in the RANS simulations and therefore, it needs to be modelled. Standard k-ε is the most commonly used model in the RANS simulations of stirred tanks [4,9,21–25]. However, it finds limitation in modelling anisotropic turbulence in the impeller discharge region and under-predicts turbulent kinetic energy in flow impingement region. RSM model predicts the Reynolds stresses by explicitly solving their governing equations. Hence, it resolves the anisotropic turbulence and results in improved predictions of turbulence in such regions [26]. Large eddy simulation (LES) also resolves the larger anisotropic turbulence scales while requiring simulation of complete domain with fine mesh, which is computationally expensive [27]. For the same scale, the computational requirement using LES can be 100 times higher than that for RSM simulation. Therefore, in this paper, RSM model is used for modelling turbulence. The equations of Reynolds Stress Model (RSM) model for turbulence are given below tions of Reynolds Stress Model (RSM) model for turbulence are given below.            ' ' ' ' ' ' ' ' ' ' 1 2 ε . . 2 / 3 δ 2 / 3 δ 2 / 3 δ ε i j i j q i j s i j i j ij ij ij ij ij k u u u u u u u C u u C u u k t k C P P S                     (3)       ' ' ε 1ε , 2ε ρ ε ρ ε . ρ ε . 3.1. Governing Equations The hydrodynamic simulations are conducted using Eulerian-Eulerian multiphase model. In this model, each phase is treated as an interpenetrating continuum represented by a volume fraction at each 2000 Metals 2015, 5 point of the system. Reynolds averaged mass and momentum balance equations are solved for each phase. The governing equations are: point of the system. Reynolds averaged mass and momentum balance equations are solved for each phase. The governing equations are: Continuity equation: Continuity equation:     α ρ . α ρ 0 q q q q qu t     (1) (1) Momentum equation: Momentum equation: Description Equations Liquid-phase stress tensor   2 α μ α λ μ 3 T q q q q q q q q q u u u I              Solid-phase stress tensor   2 α μ α 3 T s s s s s s s s s u u u I                Solid shear viscosity , , , μ μ μ μ s s col s kin s fr    Collisional viscosity   0.5 , , 4 μ α ρ 1 α 5 π s s col s s s o ss ss s d g e          Kinetic viscosity      , , α ρ 2 μ 1 1 3 1 α 6 3 5 s s s s kin ss ss s o ss ss d e e g e            Frictional viscosity , 2 sinφ μ 2 s s fr D p I  Solids pressure   2 , α ρ 2ρ 1 α s s s s s ss s o ss s p e g     Radial distribution function 1 1/3 , ,max α 1 α s o ss s g                   Diffusion coefficient of granular temperature       2 , , 12 1 η 4η 3 α 15α ρ π 5 16 4 41 33η 41 33η ηα 15π s o ss s s s s s s o ss g d k g                     where ss η 0.5(1 ) e   Collision dissipation energy   2 , 2 3/2 12 1 γ ρ α π ss o ss s s s s s e g d     Description Equations Liquid-phase stress tensor   2 α μ α λ μ 3 T q q q q q q q q q u u u I              Solid-phase stress tensor   2 α μ α 3 T s s s s s s s s s u u u I                Solid shear viscosity , , , μ μ μ μ s s col s kin s fr    Collisional viscosity   0.5 , , 4 μ α ρ 1 α 5 π s s col s s s o ss ss s d g e          Kinetic viscosity      , , α ρ 2 μ 1 1 3 1 α 6 3 5 s s s s kin ss ss s o ss ss d e e g e            Frictional viscosity , 2 sinφ μ 2 s s fr D p I  Solids pressure   2 , α ρ 2ρ 1 α s s s s s ss s o ss s p e g     Radial distribution function 1 1/3 , ,max α 1 α s o ss s g                   Diffusion coefficient of granular temperature       2 , , 12 1 η 4η 3 α 15α ρ π 5 16 4 41 33η 41 33η ηα 15π s o ss s s s s s s o ss g d k g                     where ss η 0.5(1 ) e   Collision dissipation energy   2 , 2 3/2 12 1 γ ρ α π ss o ss s s s s s e g d     Liquid-phase stress tensor Liquid-phase stress tensor Diffusion coefficient of granular temperature Collision dissipation energy Collision dissipation energy The stress-strain tensor in the momentum transfer equation is due to viscosity and Reynolds stresses that include the effect of turbulent fluctuations. Momentum equation: ε ρ ε ε q q q i i k q m k u C u u C G C t k              (4) (3)       ' ' ε 1ε , 2ε ρ ε ρ ε . ρ ε . ε ρ ε ε q q q i i k q m k u C u u C G C t k              (4) (4) where Cs, C1, C2, C1ε, C1ε, and C2ε are constants. And, ' ' ' ' . . ij i k j j k i P u u u u u u     ; 1 2 ii P P  ; 1 ρ α ρ N q i i i  ; 1 1 α ρ α ρ N i i i i q N i i i u u     . 1 ρ α ρ N q i i i  ; 1 1 α ρ α ρ N i i i i q N i i i u u     . Eddy viscosity is computed from Eddy viscosity is computed from Eddy viscosity is computed from Eddy viscosity is computed from 2 , μ μ ρ ε t q q k C  2 , μ μ ρ ε t q q k C  (5) (5) Metals 2015, 5 2001 Evaluation of generation of turbulent kinetic energy is consistent with Boussinesq hypothesis and is computed as Evaluation of generation of turbulent kinetic energy is consistent with Boussinesq hypothesis and is computed as   q T q q q t q k u u u G         : , ,  (6) (6) The equation of conservation of granular temperature is given as: The equation of conservation of granular temperature is given as: The equation of conservation of granular temperature is given as: The equation of conservation of granular temperature is given as: The equation of conservation of granular temperature is given as:         3 α ρ . α ρ . : . Momentum equation: γ φ 2 s s s s s s s s s s s s s ls u p I u k t                      (7) (7) where   q s s u I p     : . is the generation of energy by the solid stress tensor,   s s k     . is the diffusion of energy, γ s  is the collisional dissipation of energy and φls is the energy exchange between fluid and solid phase. Table 1. Constitutive equations. Table 1. Constitutive equations. 3.3. Interphase Drag Force Interphase drag is the resultant force experienced by the particle in the direction of relative motion due to a moving fluid. Since, the solids and liquid phases are treated as inter-penetrating, an inter-phase momentum exchange term is required. The interphase exchange force is calculated using the following expression given by Syamlal et al. [29]:   3 r 3α α ρ Re 4 l s l D l s d p C F u u d V   (9) (9) where,   2 2 r 0.5 0.06Re (0.06Re) 0.12Re(2 ) ( ) V A A B A       ; 5.14 αg A  ; 2.28 0.8αg B  , for α 0.85 g  ; 3.65 αg B  , for α 0.85 g  . The drag coefficient is calculated using an expression that has a form derived by Dalla Valle and Kenning [30]: Kenning [30]: 2 r 4.8 0.63 Re D C V           (10) (10) 3.2. Turbulent Dispersion Force Turbulent fluctuations result in dispersion of phases from high volume fraction regions to low volume fraction regions. The turbulent dispersion force is significant when the size of turbulent eddies is larger than the particle size [9]. The effect of turbulence dispersion force on the hydrodynamics in CIL tanks, is incorporated using the Burns, et al. [28] model. The model equations for turbulence dispersion force are given below: , , α α σ α α t q p q td q TD pq pq p q D F C k             (8) (8) Metals 2015, 5 thermodynamic temperature of gases, the granular temperature is used to model the fluctuating velocity of particles [16]. The constitutive equations for momentum equation are given in Table 1. Momentum equation: Boussinesq’s eddy viscosity hypothesis is used for the closure of momentum transfer equation. The particle-particle interaction is modelled using kinetic theory of granular flow by assuming that its behavior similar to dense gas. Similar to the 2002 4.1. Vessel Geometry In this paper, a flat bottomed cylindrical tank was simulated (see Figure 1). The shaft of the impeller was concentric with the axis of the tank. A Mixtec HA-715 was used as impeller for CIL tanks, but for validation 45° six-bladed pitched blade turbine pumping downwards (PBTD) impeller was employed. For validation, vessel geometry dimensions, material properties, etc. were taken from paper of Guida et al. [3]. The dimensions of tank and impeller are given in Table 2. The fluid and particle properties used in the simulation are also tabulated in the same table. Conditions such as solid concentrations, impeller speed, and the Reynolds number in the tank are tabulated in Table 3. Simulations were carried out at just suspension speed for each case, which was determined by Metals 2015, 5 the center of impeller. The impeller rod outside this zone is considered as a moving wall. Impellers used in all the cases simulated in the study are operated in the down-pumping mode. The top of the tank is open, so it is defined as a wall of zero shear. The solids in the tank are assumed to be settled in the beginning of simulations. Therefore, a solid volume fraction of 0.6 is patched in the stirred tank up to a height such that the volume averaged solid concentration is the same as the uniformly distributed solids specified in Table 3. For modelling the turbulence, a RSM turbulence model is used. The standard model parameters are Cµ: 0.09, Cs: 0.22, C1: 1.8, C2: 0.6, C1ε: 1.44, C2ε: 1.92, σk: 1.0 and σε: 1.3. In the present work, SIMPLE scheme is used for Pressure-Velocity coupling along with the standard pressure interpolation scheme. To avoid any numerical diffusion and unphysical oscillations, a third order Quadratic Upstream Interpolation for Convective Kinetics (QUICK) discretization scheme is used for momentum, volume fraction, turbulent kinetic energy, and turbulent dissipation rates. The convergence of the simulation is verified by monitoring residual values as well as additional parameters namely turbulence dissipation over the volume, turbulence dissipation at the surface right below impeller and torque on the shaft. Once the residuals and additional parameters are constant, a simulation is deemed to be converged. For all the cases presented in the paper, a time-step of 0.001 s is less than the time which the impeller needs to sweep by a single computational cell (one cell time step ~0.003 s). One cell time step is considered appropriate for the CFD simulations in stirred tanks [12]. But using a time-step of 0.001 s resulted in divergence in the initial time-steps. Therefore, the time-step size initially used in the simulations is 0.0001s, which is gradually increased to 0.001 s. For the full scale geometry, the one cell time step increased due to the reduction in the impeller speed. Therefore, the maximum time-step used for full-scale simulation is 0.01 s (one cell time step ~0.025 s). The results of lab scale simulations approached transient steady values after 12 s. Therefore, the data used for results and discussion is time averaged for the last 3 s. Metals 2015, 5 2003 Guida et al. [3] following Zwietering [31] criterion. According to this criterion, no particle should remain stationary on the base of the vessel for longer than 1–2 s. Guida et al. [3] following Zwietering [31] criterion. According to this criterion, no particle should remain stationary on the base of the vessel for longer than 1–2 s. Figure 1. Computational domain, grid distribution, and schematic of the stirred tank. Figure 1. Computational domain, grid distribution, and schematic of the stirred tank. Table 2. Dimensions of domain and properties of materials used in this study. Table 2. Dimensions of domain and properties of materials used in this study Tank (m) PBTD (m) HA-715 T 0.288, 10 D T/2 D T/2, T/3 H T Bl 0.055 Dshaft 0.01152 W T/10 Bw 0.041 - - Ci T/2, T/3, T/4, T/6, T/8 Dshaft 0.01 - - Dhub 0.034 - - Table 3. Conditions in stirred tanks used for simulations. Table 3. Conditions in stirred tanks used for simulations. Name of Case X (wt. %) N = Njs (RPM) ρl (kg/m3) µl (Pa·s) ρp (kg/m3) dp (mm) PBTD-Validation 40 589.8 1150 0.001 2585 3 CIL tanks (Lab Scale) 50 200–700 1000 0.001 2550 0.075 CIL tanks (Full Scale) 50 22.15 1000 0.001 2550 0.075 4.2. Numerical Simulations The stirred tank for validation consists of six PBT blades and four baffles and that of CIL tanks consists of three HA-715 blades and three baffles. In all the cases studied in the paper, simulation of the full tank is conducted. For the case of PBTD, the moving zone with dimensions r = 0.06 m and 0.036 < z < 0.137 is created (where z is the axial distance from the bottom). For the case of HA-715, the moving zone of height T/10 and r = 3T/4 is created, with top and bottom surfaces equidistant from 2004 5.1. Grid Independency and Validation For testing the grid-independency, the experimental values for axial, tangential, and radial velocities at impeller discharge plane for single phase flow were compared with the simulation results using computational grids with 32,000 (coarse), 140,000 (medium) and 900,000 (fine) cells (see Figure 2). The results using mesh with 32,000 cells are accurate at z = 0.2 H plane. The predictions improve slightly while using 140,000 cells, and beyond that the improvement is marginal. Furthermore, the power for the stirred tanks is calculated by integrating the turbulence dissipation rate over the volume. For the coarse, medium, and fine meshes, the values of power number are 1.34, 1.53, and 1.57, respectively, which re-emphasizes that a mesh of 140,000 cells is suitable for simulation and further refinement will only lead to marginal improvement at significant computational cost. Therefore, the mesh with 140,000 cells is used for the rest of the simulations in the study. For the full scale geometry, the computational grid of 140,000, 900,000, and 3,000,000 cells are investigated based on the same parameters. The values of power number obtained for the three cases are 1.27, 1.49, and 1.55, respectively. Mixing time is another parameter that is sensitive to the number of computational cells in the domain. For the full scale tank, the mixing time for the three cases are 75 s, 113 s, and 111 s respectively to achieve 99% homogeneity. It is evident from the analysis that the results of 900,000 cells and 3,000,000 cells are similar. Therefore, the grid of 900,000 cells was selected for the simulation of full scale CIL tanks. The models used in the study are validated by comparing the flow field generated by PBTD and the concentration profiles obtained at 40 wt. % solid loading. The axial, tangential, and radial velocity plots describe the flow generated by the PBTD. The PBTD pumps the fluid downwards leading to highly negative axial velocities at the impeller plane. The jet leaving the impeller flows down to the bottom of the stirred tank, is then redirected towards the periphery and circulates back to the top along the walls. Due to such motion, a flow loop is formed near the impeller. It results in decreasing axial velocity when moving radially outwards in impeller plane that eventually increases due to the upwards flow near the walls. Metals 2015, 5 2005 Metals 2015, 5 Metals 2015, 5 For full scale simulations, the time taken for reaching the transient steady value is 900 s, and the data is time-averaged for 300 s. The numerical solution of the system is obtained by using the commercial CFD solver ANSYS FLUENT 15.0 using 48 cores of Magnus with Cray XC40, Intel Xeon E5-2690V3 “Haswell” processors running at 2.6 GHz system at Pawsey supercomputing center. Each time step takes an average of 20 s of the wall clock time which reduces after approaching convergence when residuals reach at 10−5. The finite volume method solves the partial differential equations in a spatially discretized domain. The discretization should be fine enough to resolve the physics while not incurring excess computational power. For the purpose, a grid independency test is conducted initially on the single phase flow and is presented. The models used in the paper are validated with the experimental results at 40 wt. % solid loading stirred tank published by Guida et al. [3]. The validated model is used for analyzing the flow field generated by HA-715 impeller and assessing its efficacy compared to the PBTD. The influence of different impeller speeds, impeller off-bottom clearance and impeller diameter on the solid suspension, homogeneity and power consumption are examined. The appropriate stirred tank design parameters are used for scale-up and simulations and are conducted to gain an insight on the flow developed. Flow generated by single and multiple impeller systems in the full-scale CIL tanks is also investigated. 5.1. Grid Independency and Validation A 45° inclination of the impeller blade imparts momentum in the tangential direction resulting in moderate values of tangential velocity. Due to the downward flow developed by a PBTD, the magnitude of the radial component of velocity is the lowest. The maximum values of radial, tangential, and axial velocity in the impeller plane are 0.1 Utip, 0.28 Utip, and 0.45 Utip respectively. Both radial and tangential components gain value with the increase in radius as the velocity increases radially along the impeller blades. The maximum value is attained by both of these components close to the impeller tip after which a sudden decline is observed due to no momentum source in the absence of impeller. These characteristics are well represented in the velocity profiles shown in Figure 2. Metals 2015, 5 2006 r/R, [-] 0.0 0.5 1.0 U/Utip, [-] -0.6 0.0 0.6 Experiment 32000 Cells 140000 Cells 900000 Cells (a) Figure 2. Comparison of dimensionless axial (green), radial (black) and tangential (red) velocity at z = 0.2H plane for computational grid with different resolution. Figure 2. Comparison of dimensionless axial (green), radial (black) and tangential (red) velocity at z = 0.2H plane for computational grid with different resolution. The average axial concentration profile is compared with the experimental data of Guida et al. [3] in Figure 3a. In some cases, the average axial concentration does not provide the information for local variation of concentration. Therefore, radial concentration profiles at different heights are also compared with the experiments. As is evident from Figure 3, the results obtained are in agreement with the experimental data. As the impeller speed used is the speed of just suspension, the solid concentration near the bottom of the tank is only double the average concentration. The jet from the PBTD moves downwards and strikes the bottom around the r/R = 0.5. Therefore, the concentration at this location is low. However, the solid is accumulated at the bottom center and the bottom corner of the tanks due to low velocity fields in both of these regions. Near 0.29 Z, the low concentration in the loop of the eye formed by the jet circular loop is also well predicted by the simulations. With increasing height, the axial velocity near the wall and impeller diminish and therefore, the concentration profile also becomes inverted with low concentration near the periphery and impeller rod. Metals 2015, 5 with the observation by Hicks et al. [32] that the cloud height remains nearly constant between solid concentration of 10% and 40%. It is evident that the models used in the simulations are able to predict the local hydrodynamics for high solid loading stirred tank systems accurately and can be used for investigating hydrodynamics in CIL tanks. Cz/Cav, [-] 0.00 0.75 1.50 2.25 3.00 z/H, [-] 0.00 0.25 0.50 0.75 1.00 (a) Experiment Simulation 3 6 3 6 0.0 0.5 0 3 6 0.0 0.5 1.0 r/R, [-] Cr/Cav, [-] z = 0.86 Z z = 0.71 Z z = 0.14 Z z = 0.29 Z z = 0.43 Z z = 0.57 Z (b) Figure 3. (a) Averaged axial concentration profiles and (b) radial concentration profiles plotted at different heights in stirred tanks with 40 wt. % solid loading using PBTD. Simulation 3 6 3 6 0.0 0.5 0 3 6 0.0 0.5 1.0 r/R, [-] Cr/Cav, [-] z = 0.86 Z z = 0.71 Z z = 0.14 Z z = 0.29 Z z = 0.43 Z z = 0.57 Z (b) Cz/Cav, [-] 0.00 0.75 1.50 2.25 3.00 z/H, [-] 0.00 0.25 0.50 0.75 1.00 (a) Experiment z/H, [-] Cz/Cav, [-] Figure 3. (a) Averaged axial concentration profiles and (b) radial concentration profiles plotted at different heights in stirred tanks with 40 wt. % solid loading using PBTD. 5.1. Grid Independency and Validation This shape of concentration profile is maintained till the top of the stirred tank with the magnitude of concentration decreasing with height. The validity of the CFD simulations is further tested by comparing the results of cloud height in stirred tanks. For quantifying the cloud height, Hicks et al. [32] conducted experiments and reported the values at various suspension speeds, D/T, power, torque, and suspension ratios. Bittorf and Kresta [33] developed a model for predicting the cloud height based on the analysis of liquid jet velocity and just suspension speed. Due to inherent complexity due to several variables such as solid concentration, suspension speed, particle diameter, D/T ratio, C/T ratio, experimental errors, etc., error in the predictions lie in the range of ±16%. Therefore, the simulations are conducted using 10.6% solid concentration (by weight) at Njs with D/T of 0.5 and C/T of 0.25. These results were compared with the experimental results of Hicks et al. [32]. The cloud height obtained from CFD simulations is 0.93 compared to the experimental value of 0.94. For the solid concentration of 20% and 40%, the cloud height calculated from CFD simulation is 0.935 and 0.942 respectively. This aligns 2007 Metals 2015, 5 20.79 W, its value for the HA-715 case at 300 RPM is only 3.25 W. Therefore, the power used in HA-715 was increased to 12.89 W by doubling the impeller speed. This change resulted in a flow field that is far more effective than that generated by the PBTD, while using 60% of the power. The efficiency of mixing for both the cases is quantified by determining the mixing time using an 8% solute patched at the top of stirred tank. The value of mixing time for PBTD is 4.2 s, while the same for HA-715 at 660 RPM is 2.9 s. Therefore, for the given power consumption, HA-715 shows better mixing characteristics than PBTD. Figure 4. Flow field contours in a stirred tank generated by PBTD and HA-715. Figure 4. Flow field contours in a stirred tank generated by PBTD and HA-715. 5.2. Flow Field The simulations of lab scale stirred tank with PBTD and HA-715 were conducted to compare the hydrodynamics in both the cases. Only for these set of simulations, four baffles were employed for the HA-715 case, which is equal to that of PBTD cases. The flow field generated by both axial impellers viz. PBTD and HA-715 are shown in Figure 4. The asymmetry seen in the case of HA-715 impellers is due to the use of three impeller blades which cannot simultaneously appear in the mid-baffle plane. Both of these impellers generate strong axial flow with similar flow field profile. Similar to the flow generated by PBDT, the jet originates from the impeller blades for HA-715 impeller and strikes the bottom of the tank. Such a motion is effective for the suspension of ore particles at the bottom of the tank. After hitting the bottom, the fluid changes direction and moves upwards along the wall of the stirred tank. This results in higher axial velocities near the bottom-wall region of the stirred tanks, where both radial and tangential velocities diminish. The value of axial velocity near the wall gradually diminishes with the ascent due to the counter effect of gravity. The height to which axial velocity remains significant is proportional to the magnitude of velocity at the bottom, which exerts force against gravity. Such a behavior is also evident from Figure 4. It can be observed from contours that for the same impeller rotation speed, the flow generated by PBTD is dominant compared to that generated by HA-715. However, the flow generated at a particular impeller rotation speed is not the sole criterion for determining the efficacy of impellers. Rather than the impeller speed, the power consumed is a more reasonable criterion, where the desired flow needs to be generated by supplying a specific amount of power. While the power consumed for the PBTD case is 2008 Metals 2015, 5 lower solid accumulation in this region. In all the cases in which the lower concentration is observed, a circular loop formed by the jet is present. Therefore the presence of circular loop and the magnitude of the jet velocity determines the formation of a low concentration zone near the impeller. This circular loop is not formed in two cases: first, at low impeller speeds and high off-bottom clearance as the velocity diminishes before the jet reaches the bottom of the tank; and second, at low off-bottom clearance, where the jet hits the bottom but cannot get enough distance to form a loop. In both of these cases, a flat profile near the impeller is observed. Cz/Cav 0.60 1.00 1.40 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.6 1.0 z/H 0.00 0.25 0.50 0.75 1.00 C = T/2 C = T/3 C = T/4 C = T/6 C = T/8 200 RPM 300 RPM 400 RPM 500 RPM 600 RPM 700 RPM (a) Cz/Cav 0.60 1.00 1.40 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.6 1.0 z/H 0.00 0.25 0.50 0.75 1.00 C = T/2 C = T/3 C = T/4 C = T/6 C = T/8 200 RPM 300 RPM 400 RPM 500 RPM 600 RPM 700 RPM (b) Figure 5. Axial concentration profiles at different stirrer speeds and off-bottom clearance for impeller to tank diameter ratio of (a) 0.5 and (b) 0.33. Cz/Cav 0.60 1.00 1.40 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.6 1.0 z/H 0.00 0.25 0.50 0.75 1.00 C = T/2 C = T/3 C = T/4 C = T/6 C = T/8 200 RPM 300 RPM 400 RPM 500 RPM 600 RPM 700 RPM ( ) ( ) Cz/Cav 0.60 1.00 1.40 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.60 1.00 z/H 0.3 0.7 Cz/Cav 0.6 1.0 z/H 0.00 0.25 0.50 0.75 1.00 C = T/2 C = T/3 C = T/4 C = T/6 C = T/8 200 RPM 300 RPM 400 RPM 500 RPM 600 RPM 700 RPM (b) Figure 5. 5.3. Concentration Profiles The CIL stirred tanks at 50 wt. % solid loading are simulated at lab scale. The influence of impeller speed, diameter, and off-bottom clearance is analyzed by plotting the concentration profiles for each of the cases in Figure 5. The variation of off-bottom clearance and impeller speed presented in this paper is between 0.125 T and 0.5 T, and 200 and 700, respectively. These variations are studied for impeller diameters of 0.5 T and 0.33 T. At low off-bottom clearance for the impeller diameter of 0.5 T, the concentration profile remains uniform in the bottom half of the tank. It suggests the lower off-bottom clearance contributes to suspension of the settled solids. Therefore, the rise in the solid concentration near the bottom is not visible for C = T/8 case. As the clearance is gradually increased, the velocity of jet hitting the bottom also decreases, and hence the possibility of the presence of settled solids increases. The presence of settled solid at the bottom is dependent on the velocity of jet when it hits the bottom. Also this velocity is governed by the distance between the impeller and the bottom, and the impeller rotation speed. For high off-bottom clearance and low impeller rotation speeds, the jet velocity hitting the bottom is low, and hence a higher solid concentration can be observed near the bottom of the tank. With the increase in the off-bottom clearance, solid concentration assumes a vertically flipped S-shaped profile. This profile is an indication of significant variation in the solid concentration with height. Such a profile means, the concentration of solid is high at the bottom, it lowers around the region of impeller, then gradually increases above the impeller, and then finally diminishes near the top of the stirred tanks. Comparatively lower concentration in the region near the impeller is because the jet carrying solids circulates after hitting the bottom, and forms an eye with very low velocity at the center, resulting in 2009 5.4. Suspension Quality and Power Consumption The suspension quality and power consumption are the two important parameters for determining the efficiency of a stirred tank. A high homogeneity (high suspension quality) achieved at a low power consumption is the objective of design and optimization. Suspension quality in a stirred tank can be defined in two different ways: first, by defining the variation between the minimum and maximum concentration [34] and second, by determining the homogeneity by variation in local concentration compared to the average concentration [35]. Mathematical representation of both these methods is given in equations below: max min max average 100 C C C C     (11) 2 susp 1 average 1 1 1 n i i C H n C             (12) max min max average 100 C C C C     (11) 2 susp 1 average 1 1 1 n i i C H n C             (12) (11) 2 susp 1 average 1 1 1 n i i C H n C             (12) (12) The results obtained from both of these equations are plotted in Figure 6. While the results from the Hsusp equation suggest that the homogeneity increases with the increase in impeller speed and reaches a plateau value, the results of the ΔCmax equation appear quite erratic. Such an observation is a result of error in the Cmin or Cmax values due to local errors during computation. Therefore, a conclusion cannot be drawn from these plots with confidence. Hsusp provides insight with respect to the homogeneity prevailing in the stirred tanks. For the case of Di/T = 0.5, the homogeneity is low at low impeller speeds, which improves with increasing impeller speed and reaches a plateau. However, for an impeller off-bottom clearance of T/3, the homogeneity decreases at very high velocity because of the generation of a dominant flipped S-shaped concentration profile (see Figure 5a). For a clearance higher than T/3 or lower than T/3, the flipped S-shaped profile is not dominant and therefore, the decrease in homogeneity at very high impeller speed is also not observed. The highest deviation in the homogeneity occurs due to the suspended solids at the bottom. Metals 2015, 5 speed is reduced and therefore, the velocity magnitude of the jet is not sufficient to form a circular loop of solids. Therefore, the profiles for all the cases are almost identical, with strong influence of impeller speed. At low impeller speeds, a high accumulation of solids at the bottom and low concentration at the top is observed. At high impeller speeds, the concentration values showed less variation with height. However, the influence of off-bottom clearance on the accumulation of solid near the bottom remained the same as in the case of 0.5 T. A low off-bottom clearance is found beneficial to suspend the settled solids. Metals 2015, 5 Axial concentration profiles at different stirrer speeds and off-bottom clearance for impeller to tank diameter ratio of (a) 0.5 and (b) 0.33. When the impeller diameter is decreased to 0.33 T, the flipped S-shaped concentration profile is not dominant. On the contrary, the concentration is high near the bottom, which decreases to average concentration while reaching the height of the impeller, and then gradually decreases when reaching near the top. Clearly, the conditions for strong circular loop formed in the 0.5 T case is not met in the 0.33 T case. With the decrease in the impeller diameter and similar rotation velocity, the impeller tip 2010 5.4. Suspension Quality and Power Consumption Decreasing the off-bottom clearance allows the high velocity jet to force the solids to suspend. Therefore, at off-bottom clearance of T/8, the homogeneity improves at lower impeller speed compared to other cases. 2011 Metals 2015, 5 Metals 2015, 5 0 200 400 600 800 Cmax, [-] 50 150 250 C= T/2 C = T/3 Figure 6. Suspension quality and power consumption plotted at different stirrer speeds and off-bottom clearance for impeller to tank diameter ratio of (a) 0.5 and (b) 0.33. Impeller Rotation Speed, [RPM] 0 200 400 600 800 Cmax, [-] 50 150 250 Impeller Rotation Speed, [RPM] 0 200 400 600 800 Cmax, [-] 50 150 250 (a) (b) C= T/2 C = T/3 C = T/4 C = T/6 C = T/8 Impeller Rotation Speed, [RPM] 0 200 400 600 800 susp, [-] 0.8 0.9 1.0 Impeller Rotation Speed, [RPM] 0 200 400 600 800 susp, [-] 0.8 0.9 1.0 Impeller Rotation Speed, [RPM] 0 200 400 600 800 Power, [Watt] 0 5 10 Impeller Rotation Speed, [RPM] 0 200 400 600 800 Power, [Watt] 0 50 100 I ll R t ti S d [RPM] 0 200 400 600 800 Cmax, [-] 50 150 250 C = T/4 C = T/6 C = T/8 Impeller Rotation Speed, [RPM] Impeller Rotation Speed, [RPM] Impeller Rotation Speed, [RPM] Impeller Rotation Speed, [RPM] 0 200 400 600 800 susp, [-] 0.8 0.9 1.0 Impeller Rotation Speed, [RPM] Impeller Rotation Speed, [RPM] 0 200 400 600 800 susp, [-] 0.8 0.9 1.0 Impeller Rotation Speed, [RPM] (b) p p , [ ] Impeller Rotation Speed, [RPM] 0 200 400 600 800 Power, [Watt] 0 5 10 (a) Impeller Rotation Speed, [RPM] 0 200 400 600 800 Power, [Watt] 0 50 100 10 (b) (a) Figure 6. Suspension quality and power consumption plotted at different stirrer speeds and off-bottom clearance for impeller to tank diameter ratio of (a) 0.5 and (b) 0.33. The results of cases Di/T = 0.33 are far different from those of Di/T = 0.5. In these cases, the improvement in the homogeneity is not significant with the increase in the impeller rotation speed. Exceptions are the case of off-bottom clearance of T/4, T/6, and T/8, where the homogeneity crosses 0.9 beyond the impeller rotation speed of 600, 500, and 500 RPM respectively. 5.5. Scale-Up 5.5. Scale-Up Different scale-up criteria are available based on attaining homogeneity or complete suspension condition in the stirred tanks [37–40]. Buurman et al. [37] suggested a scaling up rule of 0.666 js N D  for the complete suspension in stirred tanks assuming the turbulence to be isentropic. Barresi and Baldi [40] suggested that 0.666 js N D  is strictly valid in a homogeneous isotropic turbulent field and devised a new rule of 0.666 js N D  by applying the turbulence theory to the solids distribution. Magelli et al. [38] investigated the solids distribution in high aspect ratio stirred tanks under batch and semibatch conditions and found that a constant ND0.93 was more appropriate. Montante et al. [39] analyzed the scale-up criteria for stirred tanks based on the dependency of minimum impeller speed as a function of impeller diameter and the dependency of settling velocity on the λ/dP, and backed the scale-up criterion of a constant ND0.93. Montante et al. [41] further investigated the role of turbulence on the particle settling and suspension in scaled-up vessels, and arrived at the conclusion that intermediate turbulent fluctuation maintains the vertical solid concentration. Therefore, a value intermediate of constant tip speed ( 1 js N D  ) and constant power per unit volume ( 0.666 js N D  ), i.e., 0.93 N D  is appropriate for scaling-up. Based on this criterion, an impeller velocity of 22.15 RPM used for a stirred tank with T = 10 m. The axial concentration profiles in laboratory scale and full scale stirred tanks are shown in Figure 7. The homogeneity in solid concentration observed in the laboratory scale stirred tank at 600 RPM is not extrapolated to the full scale stirred tank at 22.15 RPM. The presence of low concentration on the top can be explained by the very low velocity field present in the top part of the full scale tank (see Figure 8). The velocity imparted by the impeller is observed to be localized in the lower part of the stirred tank and has apparently no influence on the top. Therefore, the velocity vectors between the middle and top of the impeller are nearly invisible showing low velocity zones. The average value of turbulent kinetic energy in the lower half of the tank is 0.033 m2/s2, which reduces to 0.0008 m2/s2 in the lower half of the tank. Metals 2015, 5 2012 Di/T = 0.33 with clearance of T/6 and impeller rotation speed of 600 RPM is appropriate for efficiently suspending high concentration of solids in a stirred tank using an HA-715 impeller. Di/T = 0.33 with clearance of T/6 and impeller rotation speed of 600 RPM is appropriate for efficiently suspending high concentration of solids in a stirred tank using an HA-715 impeller. 5.4. Suspension Quality and Power Consumption This suggests that the higher off-bottom clearance and low impeller speeds result in dead zones in the stirred tank, hence reducing the homogeneity. When the velocities in the lower part of the tank are sufficiently high, the solids suspend and a higher value of homogeneity is achieved. For a particular impeller speed, the power consumption in a stirred tank system is proportional to 5 i D . Therefore, the power consumption for an impeller diameter of Di/T = 0.5 is higher than that of Di/T = 0.33 at a given RPM (see Figure 6). For an impeller rotation speed of 700 RPM, the power consumption for cases of Di/T = 0.33 is less than 9 W. The impellers with Di/T = 0.5 can operate only up to 400 RPM using this power, and beyond 400 RPM, these impellers will require additional power. However, at the same time, homogeneity higher than a value of 0.9 can only be achieved for an impeller rotation speed above 400 RPM using this impeller diameter, while the same can be achieved using an impeller diameter of Di/T = 0.33 and clearance of T/6 or T/8 using 70% of the power requirement at 600 RPM. Therefore, Di/T = 0.33 is more efficient than Di/T = 0.5 for obtaining high suspension quality for a given power consumption. The power consumption also varies with the off-bottom clearance [36]. The closer the impeller is to the bottom of the tank, the higher the turbulence and power dissipation. Although only minor, the power consumption of T/8 off-bottom clearance is more than that of T/6. Therefore out of all the cases discussed, the impeller diameter of Metals 2015, 5 5.5. Scale-Up Without sufficient kinetic energy in the flow field in this zone, the settling of solids is evident. As a result, the solid concentration drops sharply to zero after 8.5 m in the full scale tank. Due to the same low velocity field in the middle to top zone of the stirred tank, the remaining solid stays suspended in the bottom half of the stirred tank. From the above discussion it is clear that the power provided in the lower half of the stirred tank is sufficient to suspend the solids. However, due to the absence of a power source in the upper half, maintaining the homogeneity in the stirred tank is not possible. Therefore, a multiple impeller system is desired to achieve homogeneity. 2013 Metals 2015, 5 Metals 2015, 5 2013 Figure 7. Axial concentration profiles for lab scale (T = 0.288 m) and full scale (T = 10 m) stirred tanks with C = T/6 and Di = T/3. Cz/Cav, [-] 0.00 0.75 1.50 z/H, [-] 0.00 0.25 0.50 0.75 1.00 Lab Scale Full Scale z/H, [-] Cz/Cav, [-] Figure 7. Axial concentration profiles for lab scale (T = 0.288 m) and full scale (T = 10 m) stirred tanks with C = T/6 and Di = T/3. Figure 8. Velocity vectors on baffle-impeller blade plane in a full scale stirred tank. Figure 8. Velocity vectors on baffle-impeller blade plane in a full scale stirred tank. 5.6. Multiple-Impeller Systems As the homogeneity was not achieved with the single impeller and additional power for solid suspension near the top is required, more impellers are required to be installed in the full scale CIL tanks. Therefore, three configurations in multi-impeller CIL systems were investigated, the details of which are given in Table 4. Table 4. Details of impeller configuration in multi-impeller CIL tanks. Variable Twin-CT6 Twin-CT3 Triple-CT4 Off-bottom clearance, Ci T/6 T/3 T/4 Distance between impellers, CiD 2T/3 T/3 T/4 Table 4. Details of impeller configuration in multi-impeller CIL tanks. 2014 Metals 2015, 5 Twin-CT3 case results in the weakening of drawing forces at the interface of low and medium concentration zones. Therefore, compared to Twin-CT6 case, the low concentration zone in Twin-CT3 case has extended to single impeller value of 0.9 T. This value again increases to 0.95 T for Triple-CT4 case, where the distance of impeller from the interface of low and medium concentration zones is reduced. The concentration profiles indicate accumulation of solid particles at the bottom center of the CIL for off-bottom clearance of T/6 and T/4, which is not present in the off-bottom clearance of T/3. A closer scrutiny of the flow near the bottom of the CIL tanks indicate that the secondary loops play a vital role in the suspension of solid particles at the bottom center of the tank. Ibrahim and Nienow [42] made a similar observation while investigating the solid suspension using different impellers and Newtonian fluids with different viscosities. For T/3 clearance, the secondary loop formed near the bottom is strong which results in the values of axial velocity required for solid suspension at the bottom. For all other cases, the secondary loop is not strong and the values of axial velocity also approach zero. This results in accumulation of solids near the bottom center. Twin-CT3 case and Triple-CT6 case present feasible configurations for achieving homogeneity in CIL tanks. Comparison of the suspension quality and power requirement may provide further information on the efficiency of these two cases. In the case of Twin-CT3, the combination of lower intermediate concentration and extended low concentration zone results in a low suspension quality of 0.72, nearly the same of 0.78 for the Triple-CT6 case. The power requirements for single impeller, Twin-CT3 case and Triple-CT4 cases are 6.5 kW, 12 kW, and 18 kW, respectively. Therefore, the increased homogeneity is achieved at the expense of 6 kW of power per additional impeller. Metals 2015, 5 The solid concentration contours on the baffle-impeller plane in the full-scale CIL tanks for single impeller and multi-impeller cases are given in Figure 9. The inhomogeneity in the single and Twin-CT6 cases are evident from the contour plot. A continuous gradient is observed from bottom-to-top in these two cases. This gradient can be categorized into three zones of very low, medium, and high solid concentration. The use of a second impeller in the Twin-CTby6 case forms a jet that draws the low concentration liquid from the top and circulates the high concentration fluid along the periphery of the tank. Such a flow is favorable for increasing the suspension quality near the top of the CIL tanks, as a result of which the extent of the low concentration zone is reduced. The zone of high solid concentration is the largest for the single impeller system (extends to z = 0.6 T), which is reduced by using a second impeller which disperses the high solid concentration present near the middle of the CIL tanks (extends to z = 0.5 T). However, the limited range of the jet formed by this impeller does not significantly affect the high concentration zone which still extends to half of the tank. Nonetheless, the use of a second impeller is partially effective in improving the homogeneity. Figure 9. Concentration profiles in baffle-impeller plane for (a) Single impeller; (b) Twin-CT6; (c) Twin-CT3 and (d) Triple-CT4. Figure 9. Concentration profiles in baffle-impeller plane for (a) Single impeller; (b) Twin-CT6; (c) Twin-CT3 and (d) Triple-CT4. While, the presence of impeller near the gradient at the top was found beneficial, a similar approach near the interface between medium and low concentration zones can be applied. Therefore, Twin-CT3 and Triple-CT4 cases were investigated specifically targeting at the interface of the gradients between the zones. The strategy appears to be beneficial as the interface between the medium and high concentration zone disappears and an intermediate concentration is observed to be prevalent in the majority of tank. The average concentration values of intermediate concentration zones for Twin-CT3 and Triple-CT4 cases are 0.32 and 0.31, respectively. The increased distance from the top in 2015 6. Conclusions CFD was used to simulate the high solid loading carbon-in-leach tanks. The averaged and local concentration profiles in high solid loading systems were validated with the available experimental data. The validated models were used to simulate the lab scale and full scale CIL tanks. The influence of design and operating parameters such as off-bottom clearance, impeller diameter, and impeller rotation speed were investigated. Several impeller configurations in the full scale CIL tanks for attaining homogeneity in the CIL tanks were also assessed. The conclusions deduced from the study are as follows: 1. The Euler-Euler simulation approach with KTGF, Syamlal drag model, RSM turbulence model and turbulent dispersion force model appropriately predict the local hydrodynamics in high solid loading stirred tank systems. 2. For a given power consumption, the flow generated by the HA-715 impeller is more dominant than the PBTD. 2. For a given power consumption, the flow generated by the HA-715 impeller is more dominant than the PBTD. 3. The low off-bottom clearance is favorable in achieving homogeneity at low impeller speed for lab scale CIL tanks. 4. For scale-up, multiple impeller systems are necessary for providing kinetic energy in the upper half of the CIL tanks. 5. While a low off-bottom clearance is suitable for solid suspension, solids can however accumulate at the bottom center in full scale CIL tanks due to weak secondary loops. Metals 2015, 5 2016 6. The dual impeller configuration with T/3 clearance and triple impeller configuration with T/4 clearance minimize the problems encountered in the CIL tanks. Additional impellers require approximately 6 kW of extra power in CIL tanks. 6. The dual impeller configuration with T/3 clearance and triple impeller configuration with T/4 clearance minimize the problems encountered in the CIL tanks. Additional impellers require approximately 6 kW of extra power in CIL tanks. The insight of the prevailing hydrodynamics in the CIL tanks in this study was useful for the conclusions drawn for its design. The models can be further applied to other industrial high solid loading stirred tank systems to investigate the optimal design and operating parameters. Acknowledgments This work was supported by resources provided by the Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia. Author Contributions D.W. and R.P.U. conceived and designed the CFD simulations. D.W. conducted simulation and acquired data. D.W., R.P.U. and V.K.P. analyzed and interpreted the data. D.W. drafted the manuscript and R.P.U. critically revised it. Conflicts of Interest The authors declare no conflict of interest. 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https://openalex.org/W2100261439	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/1471-2164-15-268	English	null	Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes	BMC genomics	2,014	cc-by	14,106	RESEARCH ARTICLE Open Access © 2014 Rudder et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Abstract Background: Recently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies. Results: The 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome. Conclusions: This study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT). Keywords: Ensifer adhaerens, Transformation, Agrobacterium tumefaciens, Genome sequencing Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes Steven Rudder1,2, Fiona Doohan2, Christopher J Creevey3,4, Toni Wendt1,2,5 and Ewen Mullins1* * Correspondence: ewen.mullins@teagasc.ie 1Department of Crop Science, Teagasc Crops Research Centre, Oak Park, Carlow, Ireland Full list of author information is available at the end of the article Background engineering has become the fastest adopted crop technol- ogy in the world with global value of the biotech/GM seed market estimated to be in excess of $13 billion [6]. How- ever, the complexity of the Agrobacterium patent land- scape remains a challenge for non-patent holders [7,8], as the execution of existing patents on crop biotechnology can restrict the widespread application of AMT technol- ogy by non-patent holders [9]. The ability of Agrobacterium tumefaciens to transfer DNA into a plant cell via horizontal gene transfer has been instrumental in progressing the field of plant molecular biology, enabling methods such as T-DNA tagging [1,2], Agrobacterium-mediated transformation (AMT) for deliv- ery of gene expression and silencing vectors [3,4], and the introduction of genes of interest into plant genomes [5]. In effect, these abilities have underpinned the integration of crop biotechnology into mainstream agriculture, driving the development of genetically modified crop varieties equipped with novel traits, which in 2013 were planted across 175 million hectares [6]. Indeed, based on the use of AMT, commodity crop improvement through genetic The possibility of modifying non-Agrobacterium strains to facilitate horizontal gene transfer was first described by Hooykaas et al. (1977), with work by van Veen et al. [10] showing that while Phyllobacterium myrsinacearum (har- bouring the A. tumefaciens tumour inducing (Ti) plasmid) could cause tumorigenesis on plants, Rhizobium meliloti could not. It was not until 2005 though that the potential of non-Agrobacterium species’ to horizontally transfer genes into plant genomes was re-visited through CAM- BIA’s Transbacter™Project. Using the rhizobial species * Correspondence: ewen.mullins@teagasc.ie 1Department of Crop Science, Teagasc Crops Research Centre, Oak Park, Carlow, Ireland Full list of author information is available at the end of the article Page 2 of 17 Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Sinorhizobium meliloti 1021, Rhizobium sp. NGR234 (now Sinorhizobium fredii NGR234) and Mesorhizobium loti MAFF303099, it was demonstrated that non-Agrobac- terium rhizobia could indeed transfer T-DNA into plant cells [7]. However, the transformation frequency of these species was inadequate to provide a viable alternative to A. tumefaciens [11], which prompted the search for al- ternatives from a collection of diverse soil bacteria [11,12]. This initiative unearthed a lesser known rhizo- bial species, Ensifer adhaerens [13], as a rhizosphere inhabiting bacterium with the ability to successfully transform potato, tobacco and Arabidopsis. Background An independent study looking at 14 rhizobia strains, including 1021, noted dif- ferences in gene content in key groups of genes, includ- ing those involved in nodulation, nitrogen fixation, production of exopolysaccharides and Type I to Type VI secretion systems with the authors concluding no simple ‘core symbiome’ exists among rhziobia [46]. In contrast to the number of comparative studies focused on symbi- otic interactions that have been carried out to date, no study has yet focused on the ability of plant transform- ation within the rhizobia. While a draft genome of E. adhaerens CSBa has re- cently been reported to the best of our knowledge only g gy p The genetic and molecular mechanisms supporting the stable integration of A. tumefaciens T-DNA (trans- fer-DNA) into plant genomes have been the focus of in- tense research efforts since the first reports of AMT in the 1980s. A bacterial pathogen that causes ‘crown gall’ disease across a broad range of dicotyledonous and (some) monocotyledonous species [14], A. tumefaciens genetically transforms its host by transferring a single stranded DNA fragment (T-DNA) from its Ti plasmid into the host cell genome [15,16]. The T-DNA is exported from the bacterial cell into the plant cell to- gether with several virulence effector proteins via a Type IV secretion system. By coating the T-DNA on its jour- ney into the plant cell nucleus, this T-DNA structure ap- pears more as a protein complex than a single strand of DNA [17]. For the purposes of genetic transformation, existing bacterial sequences within the left and right border of the T-DNA can be replaced with genes of interest (e.g. sequences coding for herbicide tolerance/ disease resistance/synthesis of therapeutics), which can then be delivered into the targeted host genome using AMT. The reader is directed to a number of excellent reviews for an in-depth explanation and discussion of this process [18-20]. The genome sequences of A. tumefaciens C58 (C58) and S. meliloti 1021 (1021) were completed in 2001 [21-23]. Although these two gram-negative alphaproteo- bacteria are members of the same phylogenetic family (the Rhizobiaceae) and inhabit the rhizosphere, they op- erate very different lifestyles (pathogen vs. symbiont, re- spectively). The primary circular chromosomes of C58 and 1021 have been shown to share large-scale synteny, while only limited stretches of synteny can be found among additional replicons [24]. Background Designated Ensifer adhaerens OV14 (OV14), this strain can deliver sufficient transformation frequencies to present Ensifer- mediated transformation (EMT) as a viable alternative to existing transformation technology platforms [12]. plasmid and genes key to the symbiotic interaction of 1021 with legumes are found on two megaplasmids namely pSymA and pSymB [25,26]. The application of functional genomic studies to dis- sect the processes of AMT have identified a number of genes located on the A. tumefaciens circular and linear chromosomes that are implicated in virulence through the processes of attachment, vir gene regulation, and resisting plant defence responses. Initial reversible at- tachment to plant cells involving beta-1,2-glucan and secondary irreversible attachment involving cellulose fi- brils are early requirements for A. tumefaciens virulence while beta-1,2-glucan in S. meliloti plays an important role in symbiosis [27-30]. While the pAtC58 plasmid is non-essential for virulence of A. tumefaciens, it contains several att genes involved in attachment and pAtC58’s presence has been shown to have a positive effect on vir gene expression [31]. Mutations to a group of chv genes plus ros, aopB and miaA have all been shown to restrict, and in some cases halt virulence [32-37]. The ability of the bacterial cell to protect itself against plant derived reactive oxygen species (ROS) is also required for viru- lence by both plant pathogens and symbionts [38,39]. For example, a catalase (KatA) conferring gene has been shown to be upregulated in response to H2O2 via the peroxide sensor OxyR in both C58 and 1021 [38,40]. The rhizosphere is typically an acidic environment (~pH5.5) enriched by plant exudates including but not limited to sugars, ions, free oxygen and water [41,42]. Transcriptomic profiling of C58 and 1021 in response to a shift to acidic pH, has revealed a shared regulational change in genes involved in membrane composition and motility [42,43]. Separately, a chromosomally located two component sensor gene key to virulence in C58 and to symbiosis in 1021 termed chvG(exoS)/chvI has been cited as a global pH regulator [44]. A recently published study of 48 Sinorhizobium strains concluded that subtle differences in the presence of symbiosis associated genes involved in Nod-factor and polysaccharide biosynthesis, denitrification and Type III, IV, and VI secretion systems leads to varying compatibility among strains in legume- Sinorhizobium interactions [45]. Background It is upon these more unique replicons that genes encoding functions leading to the different lifestyles of these organisms are found. For example, the above-mentioned T-DNA transfer mechanism of A. tumefaciens is located on the large Ti While a draft genome of E. adhaerens CSBa has re- cently been reported, to the best of our knowledge only Page 3 of 17 Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Table 1 Basic genome information for three species; Ensifer adhaerens OV14, Sinorhizobium meliloti 1021, and Agrobacterium tumefaciens C58 OV14 1021 C58 CHR 1 CHR CHR Circular 3956045 bp 3654135 bp 2841580 bp 916 average gene length 939 average gene length 902 average gene length 62.24 GC % 62.7 GC % 59.4 GC % 9 rRNA 3 rRNA 2 rRNA 52 tRNA 51 tRNA 40 tRNA CHR 2 pSymA CHR Linear 2012811 bp 1354226 bp 2075577 bp 916 average gene length 875 average gene length 988 average gene length 61.77 GC % 60.4 GC % 59.3 GC % 3 rRNA 0 rRNA 2 rRNA 4 tRNA 2 tRNA 13 tRNA pOV14b pSymB pAt 1614950 bp 1683333 bp 542868 bp 860 average gene length 949 average gene length 849 average gene length 60.65 GC % 62.4 GC % 57.3 GC % 3 rRNA 0 rRNA 0 rRNA 4 tRNA 1 tRNA 0 tRNA pOV14c pTi 125203 bp 214233 bp 815 average gene length 938 average gene length 58.37 GC % 56.7 GC % 0 rRNA 0 rRNA 0 tRNA 0 tRNA For each entry information is as follows; Replicon id, Replicon size, average gene length, Replicons GC content as percentage, No. rRNA, and No. tRNA. Table 1 Basic genome information for three species; Ensifer adhaerens OV14, Sinorhizobium meliloti 1021, and Agrobacterium tumefaciens C58 DNA sequences for cobalamin biosynthetic (cob) genes are publicly available [47]. In this study the genome of OV14 was sequenced and functionally annotated by com- paring to the already sequenced genomes of C58 and 1021 using the eggNOG database [48,49]. Subsequently, the lit- erature was screened for all genes reported to have a posi- tive effect on A. tumefaciens virulence and then homologs to these genes were sought for in OV14, and also in 1021 for additional comparison. The level of homology between genes was compared and, where relevant, gene copy num- ber was considered. Background In addition, a phylogenetic analysis was completed on a core group of housekeeping genes and Rhizobiales chromosomal-located virulence related genes to clarify the position of OV14 within the large, di- verse Rhizobiaceae family. Results General features of the E. adhaerens OV14 genome versus that of A. tumefaciens C58 and S. meliloti 1021 The OV14 genome is the largest of the three species at 7.71 Mb; 2.04 Mb bigger than the C58 genome and 1.01 Mb larger than the 1021 genome (Table 1). Com- posed of four replicons of sizes 3.96 Mb, 2.01 Mb, 1.61 Mb and 125 kb, the OV14 genome is similar to that of 1021, which is also made up of three large circular replicons minus the small accessory plasmid. In contrast the C58 genome differs dramatically with the circular chromosome being approximately 25% less than the size of OV14’s and 1021’s counterpart and the presence of a linear chromosome being a feature unique to C58. That said, OV14 (Additional file 1: Figure S1) and C58 share a similar sized 120–180 kb mobile plasmid not found in 1021. The GC content of C58’s genome is notably lower (at 58%) compared to that of OV14 (60.75%) and 1021 (62.17%) genomes (Table 1), with a total genome compari- son highlighting a greater level of synteny between OV14 and 1021 (Figure 1). For each entry information is as follows; Replicon id, Replicon size, average gene length, Replicons GC content as percentage, No. rRNA, and No. tRNA. For each entry information is as follows; Replicon id, Replicon size, average gene length, Replicons GC content as percentage, No. rRNA, and No. tRNA. Structurally, chromosome 1 of OV14 shares 54% nu- cleotide homology with the chromosome of 1021, while the circular and linear chromosomes of C58 share 20% and 5% homology, respectively with chromosome 1 of OV14. Chromosome 2 of OV14 shows reduced homology of 20% to pSymB, 2% to pSymA, and 3% to the 1021 chromosome. In regards to C58, 3% and 7% homology to chromosome 2 of OV14 is noted for the circular and lin- ear chromosome, respectively. The third replicon of OV14 shows 7% homology to both the pSymA and pSymB of 1021, and 4% homology to the linear chromosome of C58. Finally the small accessory plasmid pOV14c shows be- tween 1–2% homology to each of the replicons. Homology of pOV14 to C58 replicons are found in pTi and pAt at 22% and 3%, respectively. supervised Orthologous Groups (eggNOG) assignments. The eggNOG database is formatted to functionally cat- egorise genes within twenty-five groups. Results Visualised in Artemis ACT with cut off set at 1000. The replicons within each genome are separated by coloured bars and labelled. Homology between the genomes is displayed via interconnecting lines; red lines representing direct homology with blue lines corresponding to inverted homologous sequence. Figure 1 Comparative synteny plots showing total genome content of Agrobacterium tumefaciens C58 (top bar), Ensifer adhaerens OV14 (middle bar), and Sinorhizobium meliloti 1021 (bottom bar), computed using DoubleACT version2 on tBLASTx setting. Visualised in Artemis ACT with cut off set at 1000. The replicons within each genome are separated by coloured bars and labelled. Homology between the genomes is displayed via interconnecting lines; red lines representing direct homology with blue lines corresponding to inverted homologous sequence. more than that shared between C58 and 1021 at 196 (2.7%) (Figure 2). NOGs for which 31 (66%) were shared by all three species, with no NOGs shared by C58 and 1021. In category [U] (Intracellular trafficking, secretion, and vesicular trans- port) there were a total of 108 NOGs of which 34 (31.5%) were shared by all three species. Category [U] showed the most even distribution of any category. The final category of specific interest is post-translational modification, pro- tein turnover, chaperones [O] with a total of 202 NOGs for which 112 (55.4%) were shared by all three species; OV14 and 1021 with 29 (14.4%), OV14 and C58 and C58 and 1021 with 8 (4%) and 2 (1%), respectively. Individu- ally, OV14 possessed 21 (10.4%), 1021 possessed 17 (8.4%) and C58 possessed 13 (6.4%). Five of the functional categories grouped under the heading of cellular processing and signalling were of most interest to this study (Table 2). In category [V], ‘de- fence mechanisms’, a total of 87 NOGs were recorded with 28 (32.2%) shared across all three species. Within this category OV14 recorded the most individual NOGs at 17 (19.5%) followed by 1021 and C58 with 12 (13.8%) and 11 (12.6%), respectively. For the signal transduction mechanisms [T] category a total of 282 NOGs were found of which 83 (29.4%) were shared by all species, while within this category C58 contained 45 individual NOGs, 10 more than both OV14 and 1021. Also to be noted were the minimal number of NOGs shared by C58 and 1021 (n = 5) compared to OV14 and C58 (n = 30) and OV14 and 1021 (n = 49). Results Within the category cell wall/membrane/envelope biogenesis [M] 119 (38.2%) of a total of 312 were shared by all three species; OV14 and 1021 sharing 59 (19%), which was more than the 16 (5%) shared across OV14 and C58 and the 5 (1.6%) for C58 and 1021. Although C58 shares a lower number of NOGs with the other two species in category [M] it does possess the highest number of species specific NOGs at 49 (15.7%). Cell motility is category [N] representing 47 Results Twenty-one of the 25 eggNOG functional categories have representa- tives in the three genomes in this study (Table 2). Those categories that are not represented are RNA processing and modification [A], nuclear structure [Y], cytoskeleton [Z] and extracellular structures [W]. In total 7261 NOGs were identified in this study with 2454 (33.8%) being shared among the three species (Table 2). OV14 has the most species-specific NOGs at 1048 (14.4%), marginally ahead of C58 with 1010 (13.9%) whilst 1021 has less at 832 (11.5%). Of the NOGs that are shared between two species and not present within the third species; OV14 and 1021 share 1281 (17.6%) almost 3-fold more than that shared by OV14 and C58 at 440 (6.1%), and 6.5-fold supervised Orthologous Groups (eggNOG) assignments. The eggNOG database is formatted to functionally cat- egorise genes within twenty-five groups. Twenty-one of the 25 eggNOG functional categories have representa- tives in the three genomes in this study (Table 2). Those categories that are not represented are RNA processing and modification [A], nuclear structure [Y], cytoskeleton [Z] and extracellular structures [W]. In total 7261 NOGs were identified in this study with 2454 (33.8%) being shared among the three species (Table 2). OV14 has the most species-specific NOGs at 1048 (14.4%), marginally ahead of C58 with 1010 (13.9%) whilst 1021 has less at 832 (11.5%). Of the NOGs that are shared between two species and not present within the third species; OV14 and 1021 share 1281 (17.6%) almost 3-fold more than that shared by OV14 and C58 at 440 (6.1%), and 6.5-fold The three genomes of OV14, 1021 and C58 were com- pared by using evolutionary genealogy of genes: Non- Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Page 4 of 17 Figure 1 Comparative synteny plots showing total genome content of Agrobacterium tumefaciens C58 (top bar), Ensifer adhaerens OV14 (middle bar), and Sinorhizobium meliloti 1021 (bottom bar), computed using DoubleACT version2 on tBLASTx setting. Visualised in Artemis ACT with cut off set at 1000. The replicons within each genome are separated by coloured bars and labelled. Homology between the genomes is displayed via interconnecting lines; red lines representing direct homology with blue lines corresponding to inverted homologous sequence. Figure 1 Comparative synteny plots showing total genome content of Agrobacterium tumefaciens C58 (top bar), Ensifer adhaerens OV14 (middle bar), and Sinorhizobium meliloti 1021 (bottom bar), computed using DoubleACT version2 on tBLASTx setting. Attachment The chromosomal virulence gene A (chvA) is a member of a group of orthologous genes found in the eggNOG database under the code aproNOG01094 (Table 3). En- coding a cyclic beta-1,2-glucan ABC transporter, its function is linked to chvB a member of aproNOG01088, which encodes a cyclic beta 1-2 glucan synthase. To- gether chvA and chvB function to synthesise and trans- port beta-1,2 glucan across the inner membrane, which is required for attachment of the bacterial cell to the plant cell surface. Genes with parallel function named Rudder et al. Attachment BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Page 5 of 17 Table 2 Comparison of eggNOG assignments for Ensifer adhaerens OV14, Agrobacterium tumefaciens C58, and Sinorhizobium meliloti 1021 eggNOG functional category Shared by 3 species OV14 & C58 only OV14 & 1021 only C58 & 1021 only OV14 only C58 only 1021 only Information storage and processing [J] Translation, ribosomal structure and biogenesis 143 4 18 3 10 14 10 [K] Transcription 160 58 135 19 140 116 83 [L] Replication, recombination and repair 99 16 40 7 42 27 37 [B] Chromatin structure and dynamics 1 0 0 0 1 0 0 Cellular processes and signaling [D] Cell cycle control, cell division, chromosome partitioning 22 2 5 0 2 6 5 [V] Defense mechanisms 28 4 12 3 17 11 12 [T] Signal transduction mechanisms 83 30 49 5 35 45 35 [M] Cell wall/membrane/envelope biogenesis 119 16 59 5 34 49 30 [N] Cell motility 31 2 5 0 2 4 3 [U] Intracellular trafficking, secretion, and vesicular transport 34 13 13 10 16 13 9 [O] Posttranslational modification, protein turnover, chaperones 112 8 29 2 21 13 17 Metabolism [C] Energy production and conversion 164 21 82 14 52 55 58 [G] Carbohydrate transport and metabolism 194 38 121 17 76 61 59 [E] Amino acid transport and metabolism 317 51 128 27 120 118 61 [F] Nucleotide transport and metabolism 67 7 14 3 7 7 5 [H] Coenzyme transport and metabolism 97 6 29 3 10 12 9 [I] Lipid transport and metabolism 96 9 48 9 32 36 26 [P] Inorganic ion transport and metabolism 140 23 46 11 35 57 41 [Q] Secondary metabolites biosynthesis, transport and catabolism 56 12 45 6 36 15 31 Poorly characterized [R] General function prediction only 151 39 90 14 97 80 56 [S] Function unknown 340 81 313 38 263 271 245 Table 2 Comparison of eggNOG assignments for Ensifer adhaerens OV14, A Sinorhizobium meliloti 1021 mparison of eggNOG assignments for Ensifer adhaerens OV14, Agrobacterium tumefaciens C58, and m meliloti 1021 Table 2 Comparison of eggNOG assignments for Ensifer adhaerens OV14, Agrobacterium tumefaciens C58, and adhaerens OV14, Agrobacterium tumefaciens C58, a ndvA and ndvB are found in 1021. All three species were found to possess one gene in the aproNOG01094 repre- sentative of chvA/ndvA (Table 3). Attachment Located on chromosome one of OV14 is a gene showing 89% protein sequence iden- tity to ndvA of 1021 and 77% identity to chvA of C58. Two genes downstream of OV14’s chvA/ndvA homolog was a chvB/ndvB homolog (a member of aproNOG01088) show- ing 86% and 68% protein sequence identity to respective sequences in 1021 and C58, respectively. OV14 also has a second chvB/ndvB gene sharing 47% and 50% protein se- quence identity to 1021’s and C58’s chvB/ndvB respectively. A third gene involved in the synthesis of beta 1-2 glucan is pscA/exoC (aproNOG01465) encoding a phosphoglucomu- tase, which recorded 90% and 81% to the respective target in 1021 and C58, respectively. which contains up to 24 genes for which some have been implicated in the early stages of attachment and virulence. The attR gene is part of aproNOG01724 and encodes an acetyltransferase but no copies were found across the OV14 genome, compared to a single copy in 1021 and two attR copies in C58. The genes attB and attD are implicated in bacteria-plant signalling during root colonisation and at the wound site during patho- genesis, with attB part of aproNOG06835 annotated as part of a binding-protein-dependent transport system, which is predicted to transport mannopine in C58 [50]. While 1021 and C58 both possess a single copy, a mem- ber of the aproNOG06835 was not found in OV14. The attD gene of C58 appears to be unique, unassigned to any aproNOG and having no similar sequence in OV14 or 1021. Mutations to genes attC and attG can render Agrobacterium avirulent on tomato and carrot by The OV14 genome was also screened for the pres- ence/absence of genes linked to the C58 pAt att locus, Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Page 6 of 17 Figure 2 Venn diagram illustrating the number of eggNOGs found across the three species. Ensifer adhaerens OV14, Sinorhizobium meliloti 1021, and Agrobacterium tumefaciens C58. Of interest, 1021 did not contain any orthologs to the cel genes of C58 (Table 4). The presence of phosphatidylcholine in prokaryotic membranes is generally confined to species that intim- ately interact with eukaryotic cells [54]. Two pathways present in C58 can lead to phosphatidylcholine produc- tion; the methylation pathway that requires pmtA (apro- NOG06650) and the pcs pathway that requires pcs (aproNOG02893) [54]. In C58 phosphatidylcholine is found in the inner and outer membrane constituting around 23% of total membrane lipids. Attachment The pcs gene of OV14 shares 92% and 85% protein sequence identity with 1021 and C58 respectively, while the pmtA gene of OV14 shares 83% and 67% protein sequence identity with 1021’s and C58’s respecitvely. The pcs pathway is dependent on the uptake of choline from the environ- ment [55]. Screening OV14 for the choline ABC trans- porter genes choXWV (that have been identified in both C58 and 1021), revealed that the choX solute binding protein component (aproNOG00993) was represented by two orthologs in all three species (Table 3). The ABC ATPase choW (aproNOG00971) has one member in each species and the choline permease (aproNOG02245) was noted to have three members in C58, two in OV14 and one in 1021. Figure 2 Venn diagram illustrating the number of eggNOGs found across the three species. Ensifer adhaerens OV14, Sinorhizobium meliloti 1021, and Agrobacterium tumefaciens C58. preventing attachment to the host cell [51]. Both genes are annotated as ABC transporters for which no ortho- logs exist in OV14: an attC ortholog in aproNOG06683 is present in 1021 and C58 (Table 4). The attKLM (renamed blcABC) operon within this locus has been linked to quorum sensing and found to be up regulated in response to salicylic acid (SA) in C58 [52]. All three species contain multiple gene entries (four copies in OV14, three in C58 and five in 1021) in aproNOG00713, which houses the C58 blcA (a NAD-dependent succinyl dehydrogenase). No genes homologous to C58 blcB (aproNOG04363) or blcC (aproNOG02812) were found in OV14. While the role of the remaining genes of the att locus in virulence remains unclear, the genes attE, attF, attG, attH, attO, attT, attV, attY and attZ were not found within the OV14 genome, or that of 1021 either (Table 4). However, they are represented by aproNOGs highlighting their presence among other alphaproteobac- teria. Genes found in the C58 att locus not represented by aproNOGs include attD, attP, attS, attU, attW, and attX. Host cell wall degradation The C58 genome contains two copies of the picA gene (aproNOG09265), which encodes a polygalacturonase to degrade the pectin network in targeted cell walls and aid the secretion of bacterial proteins into the plant cell [56]. The genome of OV14 was equipped with one copy showing 78% identity to picA of C58, while 1021 has no recorded picA homolog (Table 3). A complementary gene involved in pectin degradation is kdgF (aproNOG11632), with all three species possessing a kdgF homolog; OV14 sharing 72% and 47% protein sequence identity with C58 and 1021 respectively. Finally all three species possessed a member of the aproNOG03997, a beta-etherase linked to a lignin degradation protein annotated as ligE in 1021 and C58, with OV14’s ligE homolog sharing 77% and 65% pro- tein sequence identity to 1021 and C58, respectively. Chromosomal regulation of Ti based virulence genes Chromosomal regulation of Ti based virulence genes Key to the regulation of vir genes in Agrobacterium is the chvG/chvI two-component sensor, with a mutation to either chvG or chvI halting virulence [34]. Responsive to acidic pH, the chvG/chvI sensor regulates aopB and katA, two genes involved indirectly in virulence by pro- moting homeostasis in acidic conditions. A homologous system in 1021 is also responsive to acidic pH. This two component sensor encoded by exoS/chvI regulates the production of succinoglycan and is vital for symbiosis with alfalfa [57]. The OV14 genome has genes homologous to The cel locus is comprised of six genes celABCDEG and encodes a synthase for cellulose fibrils implicated in the second stage of attachment referred to as tight binding, which is irreversible [53] and critical for the virulence of Agrobacterium cells [53]. The genome of OV14 had genes orthologous to celABCEG, but not celD, which are thought to be cytoplasmic lipid carriers (Table 4). The aproNOGs representing celABCG genes were found in 23–27 separate alphaproteobacteria while aproNOGs in- cluding celDE were located in only 7 alphaproteobacteria. Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Page 7 of 17 Page 7 of 17 ( ) ( ) ( Table 3 Comparative analysis for the presence/absence of genes identified to have a positive effect on Agrobacterium virulence that are located in the genomic background (not on Ti plasmid) of Ensifer adhaerens OV14, Agrobacterium tumefaciens C58, and Sinorhizobium meliloti 1021 Gene id eggNOG id OV14 gene C58 gene 1021 gene No. species No. Chromosomal regulation of Ti based virulence genes proteins Virulence function Product In C58 Copy number Copy number Copy number In NOG In NOG In C58 chvA aproNOG01094 1 1 1 52 52 Attachment Cyclic beta-1,2-glucan ABC transporter chvB aproNOG01088 2 1 1 39 40 Attachment Cyclic beta 1-2 glucan synthase pscA(exoC) aproNOG01465 1 1 1 65 65 Attachment Phosphoglucomutase pcs aproNOG02893 1 1 1 37 37 Attachment Phosphatidylcholine synthase pmtA aproNOG06650 1 1 1 58 58 Attachment Phospholipid N-methyltransferase choX aproNOG00993 2 2 2 35 41 Attachment Choline SBP choW aproNOG00971 1 1 1 35 35 Attachment Choline ABC ATPase choV aproNOG02245 2 3 1 45 78 Attachment Choline ABC permease chvD aproNOG00260 1 1 1 102 104 vir gene regulation Uracil phosphoribosyltransferase chvE aproNOG03985 1 1 1 29 20 vir gene regulation Multiple sugar-binding periplasmic receptor chvG aproNOG00593 1 1 1 86 89 vir gene regulation DNA-binding/iron metalloprotein/AP endonuclease chvI aproNOG03091 1 1 1 85 87 vir gene regulation Transcriptional regulator chvH aproNOG03687 1 1 1 50 50 vir gene regulation Elongation factor P miaA aproNOG00010 1 1 1 116 116 vir gene regulation tRNA delta (2)- isopentenylpyrophosphate transferase ros aproNOG09171 1 1 1 16 17 vir gene regulation Ros/MucR family transcriptional regulator picA aproNOG09265 1 2 0 13 15 Host cell wall degradation Polygalacturonase kdgF aproNOG11632 1 1 1 15 15 Host cell wall degradation Pectin degradation protein ligE aproNOG03997 1 1 1 31 31 Host cell wall degradation Lignin degradation protein acvB aproNOG05730 1 1 2 28 31 Forms complex with T-strand Acid tolerance and virulence protein aopB aproNOG08879 1 1 1 17 17 Defence Porin-like membrane protien katA aproNOG00015 2 1 1 51 54 Defence Catalase-Peroxidase dps aproNOG08385 0 1 0 19 19 Defence DNA starvation/stationary phase protection protein catE aproNOG02507 0 1 1 40 40 Defence Catalase oxyR aproNOG01190 1 1 0 34 48 Defence Oxidative stress transcription regulator oxyR (1021) aproNOG01330 0 0 1 33 33 Defence Oxidative stress transcription regulator sodB aproNOG00877 2 3 1 115 122 Defence Super oxide dismutase both chvG (exoS) (aproNOG00593) and chvI (apro- NOG03091) situated in an operon as expected for two- component sensors (Table 3). The OV14 exoS (chvG) homolog shared 91% and 79% protein sequence identity to 1021’s exoS and C58’s chvG, whereas the OV14 chvI homolog shared 94% and 91% protein sequence identity to 1021 and C58 chvI homologs, respectively. Chromosomal regulation of Ti based virulence genes The chvD from C58 interacts with virB8 and has a positive effect on virulence, with both virulence and vir gene expression greatly reduced when the function of chvD is disrupted in C58 [32]. A member of aproNOG00260, all three species contained a homolog with the OV14 homolog recording Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Page 8 of 17 Table 4 Comparative analysis for the presence/absence of genes involved in attachment to plant cell that are located in the genomic background (not on Ti plasmid) of Ensifer adhaerens OV14, Agrobacterium tumefaciens C58, and Sinorhizobium meliloti 1021 Gene id eggNOG OV14 gene C58 gene 1021 gene No. species No. Chromosomal regulation of Ti based virulence genes protein Virulence function Product In C58 Copy number Copy number Copy number In NOG In NOG In C58 celD aproNOG11524 0 1 0 7 7 Attachment Cellulose biosynthesis protein celE aproNOG04598 1 1 0 7 7 Attachment Cellulose synthesis protein celG aproNOG12411 1 1 0 23 26 Attachment Cellulose synthesis protein celC aproNOG07630 1 1 0 25 28 Attachment Endoglucanase celB aproNOG09454 1 1 0 23 26 Attachment Cellulose synthase celA aproNOG05761 1 1 0 27 30 Attachment Cellulose synthase attK2 aproNOG02908 0 1 1 17 18 Attachment Semialdehyde dehydrogenase attA1 aproNOG00407 3 2 4 47 73 Attachment ABC transporter, nucleotide binding/ATPase protein [putrescine] attA2 aproNOG05011 0 1 1 15 15 Attachment ABC transporter, membrane spanning protein [mannopine] attB aproNOG06835 0 1 1 16 16 Attachment ABC transporter, membrane spanning protein [mannopine] attC aproNOG06683 0 1 1 12 12 Attachment ABC transporter, substrate binding protein [mannopine] attE aproNOG05033 0 2 0 24 25 Attachment ABC transporter nucleotide binding/ATPase protein attF aproNOG00433 0 3 0 24 26 Attachment ABC transporter, membrane spanning protein attG aproNOG00433 0 3 0 24 26 Attachment ABC transporter, membrane spanning protein attH aproNOG04763 0 2 0 22 23 Attachment Hypothetical protein attJ/blcR aproNOG06067 0 1 0 17 17 Attachment Transcriptional repressor of the blcABC operon attK/blcA aproNOG00713 4 3 5 88 141 Attachment NAD-dependent succinyl-semialdehyde dehydrogenase attL/blcB aproNOG04363 0 1 1 15 19 Attachment Gamma hydroxybutyrate dehydrogenase attM/blcC aproNOG02812 0 2 0 19 23 Attachment Zn-dependent gamma butyryl lactone lactonase attO aproNOG09383 0 1 0 5 5 Attachment Transcriptional regulator, AraC family attR aproNOG01724 0 2 1 24 27 Attachment Transacetylase attT aproNOG12782 0 1 0 3 3 Attachment GNAT family acetyltransferase attV aproNOG09482 0 1 0 12 12 Attachment Mg (2+) transport ATPase attY aproNOG07710 0 1 0 45 48 Attachment Glutathione S-transferase attZ aproNOG08980 0 1 0 21 26 Attachment Transcriptional regulator Table 4 Comparative analysis for the presence/absence of genes involved in attachment to pla in the genomic background (not on Ti plasmid) of Ensifer adhaerens OV14, Agrobacterium tum Sinorhizobium meliloti 1021 Table 4 Comparative analysis for the presence/absence of genes involved in attachment to plant cell that are located in the genomic background (not on Ti plasmid) of Ensifer adhaerens OV14, Agrobacterium tumefaciens C58, and Si hi bi lil i 02 89% and 87% protein sequence identity, corresponding to that of 1021 and C58. Chromosomal regulation of Ti based virulence genes found towards the N terminus of the gene where a puta- tive ligand binding site is positioned. On the C58 circular chromosome, chvE is located adjacent to gguABC compo- nents of an ABC sugar transporter. The same operon ar- rangement is found in all three species chvE-gguABC with all species’ gguABC genes present in the same aproNOGs (A = aproNOG01497, B = aproNOG03238, and C = apro- NOG05875). In regards to the C58 gene chvH (encoding elongation P factor, member of aproNOG03687), viru- lence of A. tumefaciens is decreased in the chvH mutant, which has been linked to reduced levels of virE2 [33]. The Chromosomal virulence gene E (chvE) codes for a multiple sugar binding periplasmic sensor, which inter- acts with the periplasmic domain of virA aiding in the regulation of the vir operon through the virA/G two- component sensor. OV14 possesses chvE as a member of the aproNOG03985, sharing 93% and 77% protein se- quence identity to its 1021 and C58 counterparts. The chvE homologs of 1021 and OV14 are ~100 bp shorter than the version located in C58, with the difference Page 9 of 17 Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 OV14 chvH homolog was found to share 95% and 84% protein sequence identity to 1021’s and C58’s respective copy. protein (aproNOG06937); not found in C58 or 1021. No homologs to the C58 dps (aproNOG08385), which functions to protect DNA from hydroxyl radicals pro- duced during oxidation of Fe (II) by hydrogen peroxide [60] were detected in OV14, or 1021 (Table 3). The A. tumefaciens ros regulator (aproNOG09171) has been shown to repress the virC and virD operons by binding to a ros box within promoter regions of both genes, but the binding activity of virG is able to over- come this repression although the exact mechanism is unclear. The 1021 ros homolog named mucR (apro- NOG09171) is involved in the regulation of both motil- ity and exopolysaccharide production [58]. The OV14 genome has one gene homologous to the ros gene shar- ing 92% and 79% to 1021 (mucR) and C58 counterparts, correspondingly (Table 3). A homolog of the C58 miaA gene was found in OV14, with mutations of the miaA gene in A. tumefaciens reported to marginally decrease virB, virD and virE gene expression [36]. Chromosomal based acvB C58’s acvB (aproNOG05730) contains multiple annota- tions, the most common being an acid induced virulence protein and the virJ-like protein. The acvB protein has been reported to bind to the T-strand in the periplasm increasing transport efficiency to the plant cell compared to an acvB-strain [59]. In this regard, OV14 contained one entry in aproNOG05730 as did C58, while 1021 possessed two. The OV14 acvB homolog shares 53% protein identity to C58 acvB and the two 1021 acvB orthologs SMc00612 and SMc00613 were found to share disrupted homology to the C58 and OV14 acvB genes (Table 3). Chromosomal regulation of Ti based virulence genes The miaA gene (aproNOG00010) encodes a tRNA delta (2)-isopentenyl- pyrophosphate transferase which is involved in protein translation; a homolog was also identified in 1021. C58 and 1021 both have a single catalase gene in apro- NOG02507 that functions in protecting cells from the toxic effects of hydrogen peroxide, annotated as catE in C58 and catC in 1021. No homolog was detected in OV14. Superoxide dismutases help to protect the cell via dismutation of superoxide into oxygen and hydrogen peroxide and three copies of the sodB gene (apro- NOG00877) were found in C58, two copies in OV14 and one copy in 1021 (Table 3). Knockout of all three sodB genes in A. tumefaciens results in avirulence, while only the sodBI mutant shows reduced virulence when targeted individually [61]. Ti based virulence Th f The vir operon found on the C58 Ti plasmid encodes the core machinery for the production and transport of T-DNA from the bacterial cell with the two-component regulator virA/G switching on expression of ancillary vir genes upon detection of plant phenolics. No homologs of this system were found to exist in OV14 (Additional file 1: Figure S2) or 1021. A combination of virB genes and virD4 form the Type IV secretion system of C58. Part of aproNOG03383 (Table 5), the virD4 aproNOG is shared by seventy-two species. The aproNOG03383 has two entries in C58 (Atu4858 and Atu6184), named virD4-like and virD4, respectively. Four virD4-like genes were identified in OV14. Upon inspection only one was found to share a protein sequence identity exceeding 50% with any known alphaproteobacteria gene, sharing 71% protein sequence identity to virD4 (Arad_15020) of A. rhizogenes K84. Protecting against plant defences The virB operon encodes for eleven proteins (numbered 1–11), which form the T-DNA transporting type IV secre- tion system. C58 has three similar Type IV secretion sys- tems, a Ti-plasmid based virB, a Ti plasmid trb operon and a linear chromosome based avh. VirB3, virB4, virB10, and virB11 form part of the same aproNOGs as their avh counterparts, with virB1, virB2, and virB9 found in differ- ent aproNOGs from counterpart’s avhB1, avhB2, avhB11. While avhB5, avhB6, avhB7, and avhB8 are all found in aproNOGs, virB5, virB6, virB7, and virB8 are not associ- ated with any aproNOG. In this analysis, OV14 shares aproNOG02013 (housing avhB3), aproNOG04596 (hous- ing avhB6) and aproNOG02544 (housing avhB11) only. The genes present from OV14 identified as part of apro- NOG02013 showed less than 50% protein sequence identity to the closest matches from Phenylobacterium zucineum HLK1 and Erythrobacter litoralis HTCC2594, with OV14 containing 4 genes for aproNOG04596. The C58 gene katA (aproNOG00015) encodes a catalase- peroxidase implicated in virulence through detoxification of hydrogen peroxide encountered during bacteria-plant interactions [39]. Three catalase genes have been previ- ously identified in 1021; katA, katB and katC [38], with the 1021 katB being a member homologous to and a member of the same aproNOG as the C58 katA. OV14 had two gene members in aproNOG00015; one sharing 89% protein sequence identity with C58’s katA and 61% identity to katB of 1021 and a second more putative gene with 64% similarity to the C58 katB gene. The oxyR per- oxide sensor regulates transcription of katA in C58 with hydrogen peroxide and superoxide anions indirectly/ directly oxidizing oxyR leading to katA activation [40]. Although the oxyR gene of C58 and 1021 are found separately in aproNOG01190 and aproNOG01330 re- spectively, OV14 was found to only contain a homolog of the C58 oxyR. While OV14 does possess a dps family Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Page 10 of 17 Table 5 Comparative analysis for the presence/absence of Ti-based virulence genes in Ensifer adhaerens OV14, Agrobacterium tumefaciens C58, and Sinorhizobium meliloti 1021 Gene id eggNOG OV14 gene C58 gene 1021 gene No. species No. Protecting against plant defences BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Some proteins encoded by the vir regulon are non- essential for transformation but are known to increase transformation efficiency. The respective proteins of virC1 and virC2 may enhance nicking at the right border of T-DNA and virE2 is exported to the plant cell along with the T-strand potentially protecting the ss-DNA from degradation or detection. The virE1 protein binds to virE2 within the bacterial cell blocking interaction with the T-strand until within the plant cell. No homo- logs for either were found in OV14 or 1021. The genes of the virE operon and virC2 were not found in any aproNOG but the virC1 gene was a member of apro- NOG17216 that was identified to be present in 6 alpha- proteobacteria species. The virulence genes virF, virH1 and virH2 have been implicated in the expansion of the host range during Agrobacterium-mediated transform- ation [22], with virF involved in the stripping of virE2 proteins off the T-DNA and virH1 and virH2 involved in the detoxification of anti-bacterial phenolics. With gene virF not part of an aproNOG and virH1 and virH2 of C58 sharing aproNOGs with only 2 other species, Agro- bacterium rhizogenes K84 and Chelativorans sp. BNC1, no homologs were found in OV14 or 1021. sharing 55% and 82% protein sequence homology with the C58 aopB and 1021 ropB1, respectively. Finally a re- cently identified imp type VI (T6SS) secretion system which is up regulated in C58 in response to low pH (5.5) [42] was not found in OV14 nor 1021. The function of this T6SS in C58 has yet to be determined. pH responsive gene networks Key to vir gene regulation is chvI, which functions with chvG (known as exoS in S. meliloti 1021) and is up regu- lated in both C58 and 1021 [42,43]. Nine exo genes (exoF, exoH, exoK, exoL, exoN, exoQ, exoT, exoW, and exoY) involved in the synthesis and metabolism of succi- noglycan are shared and up regulated in both organisms and all nine genes were found to be present in OV14. Two additional exo genes (exoM and exoU) were also noted to be in all three species. The acid inducible mem- brane protein aopB (aproNOG08879), which is involved in C58 virulence was also present as a single copy in OV14 and 1021 (ropB1) (Table 3). All three species are represented in this NOG with the OV14 homolog Phylogenetic positioning of E. adhaerens OV14 in the Rhizobiales By concatenating the full length sequence of 12 house- keeping (16S rRNA, 23S rRNA, atpD, dnaK, exoC, gap, gyrB, infB, nusA, pnp, recA, rpoB, thrC) and 8 rhizobial virulence genes (chvA(ndvA), chvB, chvD, chvG(exoS), miaA, and pcs) OV14 grouped with the Sinorhizobium of the Ensifer/Sinorhizobium group forming a clade which is a sister group to the Rhizobium/Agrobacterium clade. Within the Ensifer/Sinorhizobium group, E. adhaerens formed a sister group to the Sinorhizobium species (Figure 3). BLASTn searches of each replicon of OV14 revealed the highest synteny to S. fredii for the chromosomes and to A. vitis S4 pTiS4 and A. tumefa- ciens pTiC58 for the E. adhaerens pOV14 (Additional file 2). If considering the main chromosome, for which large scale synteny was observed, a gradient of sequence identity can be observed in OV14’s chromosome one; S. fredii strains shows 58% query coverage, S. meliloti strains shows 54% query coverage, S. medicae 49%, Rhi- zobium species including A. rhizogenes from 34–23%, and A. tumefaciens and Mesorhizobium species show 20% query coverage. Upon a BLASTn of the A. tumefa- ciens circular chromosome (Additional file 3) the query coverage to the closet matched rhizobia species was 34% dropping to 27%. Interestingly S. fredii species show higher coverage of the C58 circular chromosome at 26% than A. vitis does at 23%. Finally S. meliloti and E. adhaerens share 22% and 20% coverage with the A. tumefaciens C58 circular chromosome, respectively. Fur- ther support for this is available from the comparative NOG analysis, which reported OV14 as sharing more genes with 1021 than with C58, whilst also showing that OV14 shares more genes with C58 than 1021 and C58 share with each other (Figure 2). Protecting against plant defences protein Virulence function Product In C58 Copy number Copy number Copy number In NOG In NOG In C58 virA aproNOG05576 0 1 0 7 7 vir operon regulation Sensor kinase virG aproNOG04872 0 1 0 9 10 vir operon regulation Regulatory protein virB1 aproNOG10673 0 1 0 21 22 Type IV secretion Type IV secretion system lytic transglycosylase virB2 aproNOG12925 0 1 0 8 10 Type IV secretion Type IV secretion system Pilin subunit protein virB3 aproNOG08388 0 2 1 20 27 Type IV secretion Type IV secretion system Pilin-like protein virB4 aproNOG02013 2 2 1 71 117 Type IV secretion Type IV secretion system ATPase virB5 N/A 0 1 0 N/A N/A Type IV secretion Type IV secretion system protein virB6 N/A 0 1 0 N/A N/A Type IV secretion Type IV secretion system protein virB7 N/A 0 1 0 N/A N/A Type IV secretion Type IV secretion system protein virB8 N/A 0 1 0 N/A N/A Type IV secretion Type IV secretion system protein virB9 aproNOG01070 0 1 1 20 29 Type IV secretion Type IV secretion system protein virB10 aproNOG01880 0 2 1 25 32 Type IV secretion Type IV secretion system ATP energy sensor virB11 aproNOG02544 1 2 1 70 87 Type IV secretion Type IV secretion system ATPase virC1 aproNOG17216 0 1 0 6 6 Generation of the T-strand DNA-binding protein virC2 N/A 0 1 0 N/A N/A Generation of the T-strand Hypothetical protein virD1 aproNOG18795 0 1 0 5 5 T-DNA processing Endonuclease accessory protein virD2 aproNOG06745 0 1 0 13 16 T-DNA processing Endonuclease virD3 aproNOG10158 0 1 0 14 14 T-DNA processing Hypothetical protein virD4 aproNOG03383 1 2 0 72 121 Type IV secretion Coupling protein virD5 N/A 0 1 0 N/A N/A T-DNA processing Hypothetical protein virE0 N/A 0 1 0 N/A N/A Generation of the T-strand Hypothetical protein virE1 N/A 0 1 0 N/A N/A Generation of the T-strand Chaperone protein virE2 N/A 0 1 0 N/A N/A Generation of the T-strand ss-DNA binding protein virE3 N/A 0 1 0 N/A N/A Generation of the T-strand Hypothetical protein virF N/A 0 1 0 N/A N/A Effector Hypothetical protein virH1 aproNOG14518 0 1 0 3 3 Non-essential Hypothetical protein virH2 aproNOG15187 0 1 0 3 3 Non-essential Hypothetical protein virK aproNOG20065 0 1 0 4 4 Non-essential Hypothetical protein Table 5 Comparative analysis for the presence/absence of Ti-based virulence genes in Ensifer a Agrobacterium tumefaciens C58, and Sinorhizobium meliloti 1021 Page 11 of 17 Rudder et al. Type IV secretion systems Compared to the three T4SS found in C58 (based on virB, avhB, and trb), only one T4SS was identified in OV14 (based on trb); equivalent to a single system also present in 1021 (based on avhB). The virB T4SS, which is known to export T-DNA from the bacterial cell into the plant cell is only found on the Ti-plasmid of A. tumefaciens. C58’s trb is known to be responsible for conjugation of the Ti-plasmid between bacterial cells. A homologous trb was found on pOV14c (Additional file 1: Figure S2), with the trb operon sharing the same gene arrangement and located immediately upstream of repABC in both the OV14 and C58 genome. The protein sequence identities of the 11 genes comprising the trb operon ranged from 71% to 90%. Discussion the ability to deliver DNA into plants when equipped with the unitary pCambia5105 vector and strain 5D19 has been found to inhabit alfalfa nodules during a screen for diversity among the S. meliloti population [63]. Of interest, R. etli CE3 has been shown to possess vir gene homologs on a self-transmissible vector indicating a po- tential source of novel vir genes in soil bacteria [64]. g For this study, the genome of OV14 was compared against the genomes of C58 and 1021 using eggNOG as- signments to gain an understanding of their related- ness, with a focus being on an in/ability to transform plant cells. The potential of OV14 and 1021 to transfer T-DNA has only been achieved when equipped with a Ti-plasmid [7,12]. In this study we confirmed that there are no Agrobacterium vir gene homologs present in the OV14 genome. In the cases where homologs appeared in NOGs alongside virB and virD4 it was likely due to parallel functions within type IV secretion systems. For the virB type IV secretion system virB5, virB6, virB7 and virB8 were not found as part of any NOG suggest- ing no similar genes in fully sequenced alphaproteobac- teria to date. However, Sugawara et al. [45] detected five clades of type IV secretion systems within 48 se- quenced Sinorhizobium species. Phylogenetic analysis found A. tumefaciens virB genes to be present in clade I with 1021 virB genes found in clade II along with A. tumefaciens avh genes. Interestingly, seven Sinorhizo- bium strains were found to possess type IV secretion systems in clade I [45]. While many exo genes have been shown to be upregu- lated in C58 in response to acidic pH, the chvG/chvI sensor in C58 has not been shown to control their ex- pression. Exerting control over cell envelope compos- ition is most likely critical to the type of interaction that occurs with the plant cell directly via cell-to-cell contact or indirectly via the ability of the membrane to incorp- orate influential proteins and protein complexes. In- deed, differences in polysaccharide biosynthesis among Sinorhizobium species has been predicted as a host de- termination factor allowing for varying strategies for legume-Sinorhizobium interactions [45]. The C58 aopB has been shown to have a positive effect on virulence and was upregulated by acidic pH while the 1021 aopB homolog ropB1 was not [42,66]. Of interest, a ropB1 R. leguminosarum bv. Discussion While the ability of OV14 to genetically transform plant genomes has previously been demonstrated [12], OV14 has not acquired a known suite of symbiosis or patho- genesis genes but instead appears to effectively utilise supplementary vir or sym genes for virulence and symbi- osis. For example; E. adhaerens strain ATCC 33499 was found to form nitrogen-fixing nodules on Phaseolus vul- garis (bean) and Leucaena leucocephala when equipped with the two symbiotic plasmids from Rhizobium tropici CFN299 [62], strain OV14 used in this study has shown Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Page 12 of 17 Figure 3 Phylogenetic reconstruction based on the concatenated 16S rRNA, 23S rRNA, atpD, chvA (ndvA), chvB, chvD, chvG (exoS), dnaK, exoC, gap, gyrB, infB, miaA, nusA pcs, pnp, recA, rpoB, and thrC gene sequences. Analyses were conducted using the consensus method (majority rule extended) with 100 bootstrap replicates. Bootstrap scores are represented numerically above branches. EAOV14 represents E. adhaerens. Figure 3 Phylogenetic reconstruction based on the concatenated 16S rRNA, 23S rRNA, atpD, chvA (ndvA), chvB, chvD, chvG (exoS), dnaK, exoC, gap, gyrB, infB, miaA, nusA pcs, pnp, recA, rpoB, and thrC gene sequences. Analyses were conducted using the consensus method (majority rule extended) with 100 bootstrap replicates. Bootstrap scores are represented numerically above branches. EAOV14 represents E. adhaerens. however their presence in bacteria such as the animal pathogen Brucella broadens this hypothesis. Transgres- sion into the rhizosphere or an animal host represents a dramatic change in environmental conditions. While nutrient availability may increase, changes in pH and eukaryotic cells defending themselves against invasion becomes a new challenge for the cell to overcome. Tran- scriptomic profiles in response to acidic pH (5.5) in both C58 and 1021 have shown the expression of genes in- volved in succionoglycan biogenesis and the regulation of acid inducible genes [42,65]. Down regulation of genes involved in motility via flagellum is also a shared response. Separately, electron microscopy analysis of OV14 completed by this research group has already con- firmed the presence of functional flagella (Rathore et al. unpublished). The production of flagellum requires a large amount of energy and down regulation may free up energy for rearrangement of the cell envelope. Conse- quently, down regulation of flagellum may make the bacterial cell difficult to detect for the plant cell. Discussion viciae VF39SM mutant was shown to have increased sensitivity to detergents, hydrophobic Homologs to all chromosomal-based genes cited to be essential for T-DNA transfer in C58 were found to be present in OV14 and 1021, as indeed they are also present in several other alphaproteobacteria. Addition- ally all genes shown to be beneficial for Agrobacterium virulence were found in OV14, however not all of these genes were identified in 1021. At first it may appear that these genes are advantageous to life in the rhizosphere, Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Page 13 of 17 Page 13 of 17 in the absence of virJ and has been found to localise to the periplasmic space and associate with the T-stand in Agrobacterium [72]. Homologs to the acvB gene were found in all three species in this study and another 25 alphaproteobacteria upon inspection of aproNOG05730. The homology of the two 1021 copies to the C58 acvB and OV14 homologs was found to be low, with it appearing that the 1021 acvB has split into two genes, which may be why acvB was not initially detected in 1021 [59]. The C58 and OV14 acvB shares 50% hom- ology with virJ a gene found on octopine-type Ti plas- mids which can complement a acvB mutant [73]. While acvB appears to play a role in export maybe as a chaperone to the T-strand [59,73], the OV14 acvB homolog shares 50% identity to its C58 counterpart and could be a key target for future studies focussed on the improvement of EMT. antibiotics, and weak organic acids, with a suggestion that ropB1 plays a role in membrane stability [66]. For OV14, it would be of interest to complete a transcrip- tomic analysis to monitor the expression of these key genes under varying induction conditions. Another cell wall component present in OV14, C58 and 1021 is phosphatidylcholine, a phospholipid and major component of eukaryotic membranes. Phosphat- idylcholine has been shown to be essential for virulence in C58 and for symbiosis and normal growth in 1021 [67]. Interestingly an A. tumefaciens mutant deficient in phosphatidylcholine production was unable to support a type IV secretion system in the bacterial cell wall and subsequently lost its virulence potential [54]. The role of the virB type IV secretion system is known to be critical for T-DNA transfer to plants and is potentially the only system capable of this feat. Discussion Differences in phosphatidyl- choline content in the cell wall of OV14, C58 and 1021 could affect the cells ability to support the complete virB type IV secretion system and greatly affect the transfer of T-DNA. The phosphatidylcholine synthase (pcs) path- way is choline-dependent and requires the uptake of choline into the cell. An Agrobacterium pcs/pmtA double mutant has been shown to be attenuated in ex- pression of the virB operon [54] while a high-affinity choline ABC transport choXWV has been identified in C58 and 1021 [68,69]; OV14 was found to contain ho- mologs to this system. The choV gene encodes the ATPase component of the ABC transporter. The fact that C58 has an additional copy of choV compared to OV14 and two additional copies of choV compared to 1021 suggests C58 may have an increased ability to actively ac- quire choline for phosphatidylcholine synthesis and ultim- ately complete T-DNA transfer into targeted host cells. The ability to defend against oxidative stress leads to increased virulence as the bacterial cell survives plant cell defences and acidic pH allowing expression of viru- lence genes and delivery of T-DNA to plant cell. All three species in this study were found to possess genes involved in protection against reactive oxygen species. One such gene that was absent in OV14 and 1021 was dps, (DNA-binding proteins from starved cells), which in A. tumefaciens protects the cell by acting as an hy- droxyl radical scavenger and could well function with a catalase such as katA to increase the cell’s tolerance to the toxic effect of hydrogen peroxide [60]. In 1982, E. adhaerens was first described as a gram- negative predatory bacteria [13] but more recently, a re- quest to rename E. adhaerens to S. adhaerens initiated a debate as to the appropriate nomenclature [74]. Follow- ing on from this the International Committee on Sys- tematics of Prokaryotes ruled all Sinorhizobium species were to be transferred to Ensifer based on Ensifer being an early synonym of Sinorhizobium [13,75,76]. The current standing appears to be that Ensifer is the correct name for the amended genus, but the Judicial Commission also ac- knowledges the later synonym Sinorhizobium [76]. The work detailed here describes the genome sequencing of OV14 and based on a phylogenetic analysis of 20 house- keeping genes shows OV14 to form a branch separated from the Sinorhizobia. Discussion While the primary chromosome of OV14 shows a high level of synteny with the Sinorhizobia (highest to S. fredii) the remaining replicons share min- imal synteny to any known species and are a potential re- source of novel alpha-proteobacterial genes. One of the most notable differences across the studied genomes was that OV14 and C58 possess cel homologs that are absent in 1021. The cel locus has been impli- cated in the attachment of Agrobacterium to the plant cell but is not required for tumour formation [70]. The presence of cel homologs in OV14 may suggest a role in attachment to plant surfaces or potentially other bacteria, which may explain its discovery within nodules alongside S. meliloti and the recorded ability of E. adhaerens to phagocytose other bacteria. Another observed difference was the lack of a type VI (T6SS) secretion system in OV14 and 1021 compared to C58. The lack of a type VI secretion system in 1021 was also noted by Sugawara et al. [45]. Type VI secretion systems are a relatively new discovery and their application is not well understood. However studies in Vibrio cholerae and Pseudomonas aeruginosa have shown the T6SS system to be involved in aggressive bacteria-to-bacteria cell interactions [71]. Availability of supporting data The following additional data are available with the online version of this paper. Additional file 1 includes Figures S1 and S2. Additional file 2 includes BLAST search scores for OV14 replicons. Additional file 3 in- cludes BLAST search scores for C58 replicons. Phylogenetic analysis FASTA f l f d FASTA files for individual genes were obtained from NCBI and aligned using Clustal Omega. Clustal files were converted to Phylip format using an online tool found at http://insilico.ehu.es/tophylip/. Phylip files were concatenated using Seaview 4. The 40,470 base pair concatenated file was run using raxmlGUI producing a consensus tree with 100 bootstrap replicates. The tree was rooted by treating Brucella suis 1330 and Mesorhi- zobium loti MAFF303099 as the outgroup. eggNOG analysis Gl d Glimmer-predicted coding regions in the OV14 genome were BLASTp searched against an alphaproteobacteria database downloaded from eggnog.embl.de and assigned to NOGs based on similarity with a cut-off of 60 bits used to filter data. A reciprocal blast analysis (genome to EGGNOG and EGGNOG to genome) was also com- pleted to ensure that recorded hits were evident in both directions, regardless of obtained low r values, which may have been due to evolutionary distinctness of the species. For comparative analysis all alphaproteobacterial gene families and their corresponding functional classifi- cations were retrieved from eggNOG. The Functional categories used are based on: A Genomic Perspective on Protein Families [82]. The literature was screened for all genes known and predicted to be involved in T-DNA transfer and genes induced by the rhizosphere/rhizo- plane environment across all NOG categories. Methods OV14 was originally isolated from the rhizosphere of Brassica napus at Oak Park in Carlow, Ireland. The strain was selected for sequencing based on its ability to transform plant cells [12]. Additional file 1: Figure S1. Circular representation of the four replicons of E. adhaerens OV14. Circles, from the inside out, show: (1) GC skew; (2) Coding regions; light blue blocks represent genes with predicted function, red blocks show transposable elements, dark blue and grey blocks show genes of hypothetical and unknown function, respectively. Figure S2. Synteny plots showing total sequence of Ensifer adhaerens OV14 pOV14c (top bar) vs Agrobacterium tumefaciens C58 pTi (bottom bar), computed Genome sequencing h The OV14 genome was sequenced and constructed by BaseClear B. V. Leiden, Netherlands. A hybrid approach using the Illumina HiSeq and PacBio RS platforms was selected. The genome was constructed from 1GB Illu- mina paired-end reads, 500 MB Illumina mate paired end reads, and 100 MB PacBio RS reads. The assembly was built in the following manner. First Illumina raw reads filtered using CASAVA version 1.8.2 and subse- quently trimmed based on the Phred quality scores using the CLC Genomics workbench 1.8.3. Filtering of PacBio CLR reads was performed using the PacBio SMRT ana- lysis suite. The quality-trimmed sequence reads were puzzled into a number of contig sequences with the CLCbio de novo assembler. This set defines the draft as- sembly. Subsequently the contigs were linked and placed into super-scaffolds based on the alignment against the long PacBio CLR reads. Alignment of the contigs was performed with BLASR [79]. From the alignment the orientation, order and distance between the contigs was estimated. As a result contigs were placed in super- scaffolds. This analysis was performed using a modified version of the SSPACE Premium scaffolder version 2.3 Conclusions This study has confirmed the presence of genes in OV14 that are confirmed homologs of chromosomal-based C58 virulence genes. As to how much their sequence di- versity affects their function during T-DNA transfer The chromosomal-based acvB gene has been cited as essential for Agrobacterium to achieve T-DNA transfer Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Page 14 of 17 remains unknown. Whereas the reported transformation efficiency of OV14 was achieved with environmental conditions optimal for A. tumefaciens, it is possible that Ti virulence induction conditions for non-Agrobacterium species may be different to A. tumefaciens and this is therefore an area that requires further attention. Re- engineering these non-Agrobacterium species with im- proved virulence functions offers the opportunity to increase the range of bacterial species that can be used for the genetic transformation of plant cells. Consider- ing the limitations to the host range of A. tumefaciens have already been described [16,18,77], the use of non- pathogenic bacterial species may increase the range of plant species amenable to agronomic enhancement via genetic transformation. [80]. Finally gapped regions within the super-scaffolds were (partially) closed in an automated manner using GapFiller version 1.10 [81]. The method takes advantage of the insert size between the Illumina paired-end reads. The resulting scaffolds define the draft genome and plasmids, with the genome sequence available in the NCBI database under accession numbers CP007236.1, CP007237.1, CP007238.1 and CP007239.1. DNA isolation S O Strain OV14 was grown to midlogarithmic phase in TY medium at 28°C, 200 rpm. DNA was isolated from 20 ml of cells using a modified CTAB (Cetyl thrimethy- lammonium bromide) genomic DNA isolation method [78]. RNase (20 mg/ml) was added to lysis buffer in step 3, and centrifugal spins were extended to 20 mins to allow separation to lysate and supernatant. References 1. AzpirozLeehan R, Feldmann KA: T-DNA insertion mutagenesis in Arabidopsis: going back and forth. Trends Genet 1997, 13(4):152–156. 1. AzpirozLeehan R, Feldmann KA: T-DNA insertion mutagenesis in Arabidopsis: going back and forth. Trends Genet 1997, 13(4):152–156. 26. Finan TM, Weidner S, Wong K, Buhrmester J, Chain P, Vorhölter FJ, Hernandez-Lucas I, Becker A, Cowie A, Gouzy J: The complete sequence of the 1,683-kb pSymB megaplasmid from the N2-fixing endosymbiont Sinorhizobium meliloti. Proc Natl Acad Sci 2001, 98(17):9889–9894. 2. 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Pitzschke A, Hirt H: New insights into an old story: Agrobacterium-induced tumour formation in plants by plant transformation. EMBO J 2010, 29(6):1021–1032. EMT: Ensifer-mediated transformation; AMT: Agrobacterium-mediated transformation; Ti: Tumor inducing; T-DNA: Transfer-DNA; ROS: Reactive oxygen species; SA: Salicylic acid; T6SS: Type VI secretion system. 15. Gelvin SB: The introduction and expression of transgenes in plants. Curr Opin Biotechnol 1998, 9(2):227–232. Curr Opin Biotechnol 1998, 9(2):227–232. Author details 1 22. Goodner B, Hinkle G, Gattung S, Miller N, Blanchard M, Qurollo B, Goldman BS, Cao Y, Askenazi M, Halling C: Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science 2001, 294(5550):2323–2328. 1Department of Crop Science, Teagasc Crops Research Centre, Oak Park, Carlow, Ireland. 2UCD Earth Institute and UCD School of Biology and Environmental Sciences, University College Dublin, Belfield, Dublin 4, Ireland. 3Animal and Bioscience Research Department, Animal and Grassland Research and Innovation Centre, Teagasc, Grange, Dunsany, Co. Meath, Ireland. 4Current address: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, Ceredigion SY23 3FL, UK. 5Current address: Carlsberg Research Centre, Gamle Carlsberg Vej 4-10, 1799 Copenhagen V, Denmark. 1Department of Crop Science, Teagasc Crops Research Centre, Oak Park, Carlow, Ireland. 2UCD Earth Institute and UCD School of Biology and Environmental Sciences, University College Dublin, Belfield, Dublin 4, Ireland. 3Animal and Bioscience Research Department, Animal and Grassland Research and Innovation Centre, Teagasc, Grange, Dunsany, Co. Meath, Ireland. 4Current address: Institute of Biological, Environmental and Rural Sciences Aberystwyth University Aberystwyth Ceredigion SY23 3FL UK 23. Wood DW, Setubal JC, Kaul R, Monks DE, Kitajima JP, Okura VK, Zhou Y, Chen L, Wood GE, Almeida NF: The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 2001, 294(5550):2317–2323. 24. Slater SC, Goldman BS, Goodner B, Setubal JC, Farrand SK, Nester EW, Burr TJ, Banta L, Dickerman AW, Paulsen I: Genome sequences of three Agrobacterium biovars help elucidate the evolution of multichromosome genomes in bacteria. J Bacteriol 2009, 191(8):2501–2511. 5Current address: Carlsberg Research Centre, Gamle Carlsberg Vej 4-10, 1799 Copenhagen V, Denmark. Additional files Additional file 1: Figure S1. Circular representation of the four replicons of E. adhaerens OV14. Circles, from the inside out, show: (1) GC skew; (2) Coding regions; light blue blocks represent genes with predicted function, red blocks show transposable elements, dark blue and grey blocks show genes of hypothetical and unknown function, respectively. Figure S2. Synteny plots showing total sequence of Ensifer adhaerens OV14 pOV14c (top bar) vs Agrobacterium tumefaciens C58 pTi (bottom bar), computed Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Rudder et al. BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Page 15 of 17 9. Nottenburg C, Rodríguez CR: Agrobacterium-mediated gene transfer: a lawyer’s perspective. In Agrobacterium: From Biology to Biotechnology. Edited by Tzfira T, Citovsky V. New York: Springer; 2008:699–735. using DoubleACT version2 on tBLASTx setting with cut off set at 100. Visualised in Artemis ACT. Homology between the genomes is displayed via interconnecting lines; red lines representing direct homology while blue lines represent homologues but inverted sequence. using DoubleACT version2 on tBLASTx setting with cut off set at 100. Visualised in Artemis ACT. Homology between the genomes is displayed via interconnecting lines; red lines representing direct homology while blue lines represent homologues but inverted sequence. 10. Van Veen R, den Dulk-Ras H, Schilperoort R, Hooykaas P: Ti plasmid containing Rhizobium meliloti are non-tumorigenic on plants, despite proper virulence gene induction and T-strand formation. Arch Microbiol 1989, 153(1):85–89. Additional file 2: BLAST of Ensifer adhaerens OV14 replicons.xlsx. Excel file includes tables of BLAST search of individual Ensifer adhaerens OV14 replicons. Additional file 2: BLAST of Ensifer adhaerens OV14 replicons.xlsx. Excel file includes tables of BLAST search of individual Ensifer adhaerens OV14 replicons. 11. 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BMC Genomics 2014, 15:268 http://www.biomedcentral.com/1471-2164/15/268 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W4210744949	https://mhealth.jmir.org/2018/5/e127/PDF	English	null	Designing a Tablet-Based Software App for Mapping Bodily Symptoms: Usability Evaluation and Reproducibility Analysis (Preprint)	null	2,017	cc-by	11,164	Abstract Background: Symptom drawings are widely used as a qualitative and quantitative method of assessing pain symptoms for both clinical and research purposes. As electronic drawings offer many advantages over classical pen-and-paper drawings, the last years have seen a shift toward tablet-based acquisition of symptom drawings. However, software that is used in clinical care requires special attention to usability aspects and design to provide easy access for physically impaired or elderly patients. Objective: The aims of this project were to develop a new tablet-based software app specifically designed to collect patients’ and doctors’ drawings of pain and related bodily symptoms and test it for usability in 2 samples of chronic pain patients (Aim 1) and their treating doctors (Aim 2) as well as for test-retest reliability (Aim 3). Methods: In 2 separate studies, symptom drawings from 103 chronic pain patients and their treating doctors were collected using 2 different versions of the app. Both patients and doctors evaluated usability aspects of the app through questionnaires. Results from study 1 were used to improve certain features of the app, which were then evaluated in study 2. Furthermore, a subgroup of 25 patients in study 2 created 2 consecutive symptom drawings for test-retest reproducibility analysis. Usability of both app versions was compared, and reproducibility was calculated for symptom extent, number of symptom clusters, and the whole symptom pattern. Results: The changes we made to the app and the body outline led to significant improvements in patients’ usability evaluation regarding the identification with the body outline (P=.007) and the evaluation of symptom depth (P=.02), and the overall difficultness of the drawing process (P=.003) improved significantly. Doctors’ usability evaluation of the final app showed good usability with 75.63 (SD 19.51) points on the System Usability Scale, Attrakdiff 2 scores from 0.93 to 1.41, and ISONORM 9241/10 scores from −0.05 to 1.80. Test-retest analysis showed excellent reproducibility for pain extent (intraclass correlation coefficient, ICC=0.92) and good results for the number of symptom clusters (ICC=0.70) and a mean overlap of 0.47 (Jaccard index). Conclusions: We developed a tablet-based symptom drawing app and improved it based on usability assessment in a sample of chronic pain patients and their treating doctors. Increases in usability of the improved app comprised identification with the body outline, symptom depth evaluation, and difficultness of the drawing process. Test-retest reliability of symptom drawings by chronic pain patients showed fair to excellent reproducibility. Corresponding Author: Corresponding Author: Florian Beissner, Dr phil nat Somatosensory and Autonomic Therapy Research Institute for Diagnostic and Interventional Neuroradiology Hannover Medical School Carl-Neuberg-Strasse 1 Hannover, 30625 Germany Phone: 49 511 53508413 Email: beissner.florian@mh-hannover.de Original Paper Original Paper JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 1 (page number not for citation purposes) Abstract Patients’ usability evaluation is an important factor that should not be neglected when designing apps for mobile or eHealth apps. (JMIR Mhealth Uhealth 2018;6(5):e127) doi: 10.2196/mhealth.8409 Original Paper Designing a Tablet-Based Software App for Mapping Bodily Symptoms: Usability Evaluation and Reproducibility Analysis Till-Ansgar Neubert1; Martin Dusch2, Dr med; Matthias Karst2, Dr med; Florian Beissner1, Dr phil nat 1Somatosensory and Autonomic Therapy Research, Institute for Diagnostic and Interventional Neuroradiology, Hannover Medical School, Hannover, Germany 2Section Pain Medicine, Clinic of Anaesthesiology and Intensive Care Medicine, Hannover Medical School, Hannover, Germany these authors contributed equally Neubert et al JMIR MHEALTH AND UHEALTH JMIR MHEALTH AND UHEALTH Neubert et al Designing a Tablet-Based Software App for Mapping Bodily Symptoms: Usability Evaluation and Reproducibility Analysis Till-Ansgar Neubert1; Martin Dusch2, Dr med; Matthias Karst2, Dr med; Florian Beissner1, Dr phil nat 1 Till-Ansgar Neubert1; Martin Dusch2, Dr med; Matthias Karst2, Dr med; Florian Beissner1*, Dr phil nat Introduction Bodily symptoms, such as pain, headache, discomfort, or paresthesias, are among the most common reasons to see a doctor [1,2]. Quantification of these symptoms has been challenging ever since because of their purely subjective nature, often leaving patient self-report as the only available source of information. Common tools to measure bodily symptoms include questionnaires [3], rating scales [4], and symptom drawings (better known as pain drawings [5] or discomfort drawings). In the latter, the patient receives an outline of the human body or parts thereof and marks or shades the location and distribution of his different symptoms. Such drawings can then be used to extract features such as the body area affected by the symptom, the number of sites, or the average intensity. The spatial distribution of symptoms may also carry valuable information for diagnosis, such as patterns of segmental or peripheral innervation or association with the location of internal organs. Participants of both studies were chronic pain patients and their treating doctors from a pain outpatient department. The project was conducted in accordance with the Declaration of Helsinki and had been approved by the Ethical committee of Hannover Medical School (#2987-2015). All participants were informed about the purpose of the project and gave written informed consent. Patients were asked to draw their pain and related symptoms before their appointment with the doctor. All of them were using the app for the first time. Following data entry, each patient filled out a usability questionnaire and continued with the routines of the pain outpatient department, namely, filling out standard pain questionnaires and having the appointment with the clinician. Several groups have developed symptom drawing approaches that were based on tablet computers [6-11]. Such electronic drawings have many advantages over pen-on-paper drawings, the most important being the ability to analyze drawings right after their completion without the need for prior digitization. Of particular interest are tablets with an electronic pen (stylus) as they have 2 main advantages: a much higher precision than drawing with the finger [12,13] and high similarity with pen-on-paper drawings [7]. High reproducibility of electronic drawings has been validated by Barbero et al for chronic low back and neck pain and acute induced pain [6,14]. Doctors were asked to enter the findings of their anamnesis and bodily examination during or shortly after seeing the patient. KEYWORDS pain drawing; symptom drawing; body outline; usability testing; reproducibility; tablet computers; eHealth; app; chronic pain pain drawing; symptom drawing; body outline; usability testing; reproducibility; tablet computers; eHea pain drawing; symptom drawing; body outline; usability testing; reproducibility; tablet computers; eHealth; app; chronic pain designed to evaluate the final app with all improvements that had been made. The final app version was also used to assess reproducibility of our symptom drawing approach in a test-retest design. The reproducibility study is reported according to the guidelines for reporting reliability and agreement studies (see Multimedia Appendix 1) [15]. Pain Patients In both studies, participants were recruited consecutively from patients of the Pain Outpatient Department of Hannover Medical School. Inclusion criteria were chronic pain (pain duration of ≥3 months), age ≥18 years (legal age in Germany), physical ability to draw symptom drawings on a tablet personal computer (PC), and ability to give written informed consent. Due to our consecutive recruiting approach, our sample should reflect the normal composition of patients in outpatient departments similar to ours. The aims of this project were to develop a new tablet-based software app and test it for usability in 2 samples of chronic pain patients (Aim 1) and their treating doctors (Aim 2) as well as for test-retest reliability (Aim 3). This app is specifically designed to collect patients’ and doctors’ drawings of pain and related bodily symptoms. Aiming toward high drawing precision, the app contains 4 different views of the human body, and the drawings were collected on a stylus-based tablet. We screened 70 patients in study 1, of which 52 were included, 15 declined participation, and 3 had to be excluded because they did not fulfill the inclusion criteria. In study 2, we screened 58 patients, of which 51 were included, 5 declined participation, and 2 were excluded because they did not fulfill inclusion criteria. Therefore, we developed and tested a prototype app (study 1) following in part the suggested design guidelines by Jaatun et al, that is, using action buttons instead of icons, limiting written textual instruction, avoiding rapid changes on the screen, and using a paper metaphor [11]. Usability results, obtained through questionnaires and user observation, led to several improvements of the user interface and other parts of the app, which were then tested again in a similar sample (study 2). Using the improved version of the app, we further conducted a test-retest analysis of symptom drawing reproducibility in chronic pain patients (pain duration ≥3 months). The mean age was 56.2 (SD 16.1) years in study 1 and 60.4 (SD 15.7) years in study 2. There were no significant differences between both study populations regarding age, sex, body mass index, educational level, number of pain clusters, years of pain treatment, number of previous therapeutic consultations, and usage frequency of tablet computers or comparable devices (Table 1). (JMIR Mhealth Uhealth 2018;6(5):e127) doi: 10.2196/mhealth.8409 JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 1 (page number not for citation purposes) http://mhealth.jmir.org/2018/5/e127/ JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 1 (page number not for citation purposes) JMIR MHEALTH AND UHEALTH Neubert et al JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 2 (page number not for citation purposes) http://mhealth.jmir.org/2018/5/e127/ Introduction All participating doctors had been briefly trained by one of the authors (TN) on how to use the app. General Design The software app was organized into 3 different modules: drawing instructions, symptom specification, and drawing (Figure 1). App versions for patients and doctors used the same modules but with different content. In the following sections, we will describe all elements that were left unchanged between studies 1 and 2. Of the 4 anesthesiologists involved in study 2, 2 were pain specialists and 2 were in training for pain specialization. This time the specialists examined all 51 patients, of which 35 were additionally examined by a specialist in training. Doctors All doctors who evaluated the app were anesthesiologists with at least 5 years of clinical experience. Moreover, 6 of them participated in study 1, of which 2 were pain specialists and 4 were in training for pain specialization. The specialists examined 48 of the study patients, whereas those in training examined 30. Study Design The project comprised 2 consecutive studies: study 1 aimed at evaluating the usability of a prototype of our app. Study 2 was XSL•FO RenderX JMIR MHEALTH AND UHEALTH Table 1. Demographics of our study populations. P valuea Study 2 Study 1 Characteristic .19 60.4 (15.7) 56.2 (16.1) Age (years), mean (SD) Age range, n (%) 4 (8) 9 (17) 18-39 24 (47) 21 (40) 40-59 16 (31) 20 (38) 60-79 7 (14) 2 (4) 80+ .59 34 (67) 32 (62) Women, n (%) .67 27.0 (6.8) 27.6 (7.4) BMIb (kg/m²), mean (SD) .25 2.4 (0.8) 2.7 (1.1) Education level ISCEDc 1997, mean (SD) Number of pain clusters, mean (SD) .12 5.5 (7.3) 3.7 (4.5) Front .16 4.9 (5.7) 3.6 (3.4) Back .06 4.5 (5.2) 2.9 (3.4) Left .07 4.2 (5.4) 2.7 (3.0) Right .70 3.8 (2.2) 4.0 (1.8) Years of pain treatment, mean (SD) .18 14.1 (17.7) 10.2 (11.1) Number of previous therapeutic consultations, mean (SD) .18 Usage of comparable electronic devices, n (%) 32 (64) 30 (58) Daily 6 (12) 6 (12) 3-4 times/week 2 (4) 4 (8) 1-2 times/week 0 (0) 0 (0) 1-2 times/month 1 (2) 7 (13) Almost never 9 (18) 5 (10) Never aTwo-tailed t test or chi-square test. bBMI: body mass index. Neubert et al JMIR MHEALTH AND UHEALTH Table 1. Demographics of our study populations. aTwo-tailed t test or chi-square test. bBMI: body mass index. cISCED: International Standard Classification of Education. interactions with a separate inductive digitizer, which allows for a higher resolution while eliminating unwanted activation of the screen, for example, by the palm. JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 3 (page number not for citation purposes) http://mhealth.jmir.org/2018/5/e127/ Specification of Diagnostic Findings the body outline showing the respective body region. Users were further asked to color every point of the body outline where a certain symptom or finding was present. Other possible ways to mark a body region, such as hatching, ticking, or marking by symbols, like arrows, were explicitly prohibited. Finally, users were told to specify the symptom or finding by choosing descriptors from a list. Then, the use of the visual analog scale (VAS) and the rating of depth was explained. The doctors’ version of the app had an additional variation of the symptom specification module to enter common diagnostic findings in a bodily examination. This screen contained German translations of the following findings: allodynia, anesthesia, atrophy, dysesthesia, hyperalgesia, hyperhydrosis, and hypesthesia. The VAS and depth rating was similar to that of the symptom specification module. Drawing Instructions All symptom drawing data were collected on a Samsung Galaxy Note 2014 edition 10.1 (SM-P600) tablet computer running on Android 4.1.2 (study 1) or Android 5.1.1 (study 2). This tablet has a 10.1-inch touch screen with a resolution of 800×1280 pixels and is equipped with an electronic pen (stylus) that was used for all data entry. In contrast to entering data by finger, which uses the capacitive touchscreen, the tablet records stylus The first screen instructed the user how to make a correct symptom drawing. Following the suggested design guidelines by Jaatun et al [11], we used a paper metaphor and limited the written textual instructions as much as possible. Central elements were instructions to draw every symptom or finding that users found disturbing or abnormal and to draw it on each view of XSL•FO RenderX JMIR MHEALTH AND UHEALTH Neubert et al Improvements We made several improvements to the graphical user interface (GUI) of the prototype app, most of which were inspired by the results of the usability assessment of study 1 as well as requests from the doctors. Furthermore, we completely abandoned the use of pop-up windows as suggested by Jaatun et al [11]. Drawing Instructions Changes to the drawing instructions largely reflect the changes made to the other modules, for example, the newly added VAS of the drawing module (see below). In addition, we added a short explanation of the different drawing tools. As a general rule, we tried to move as much information as possible from instructions to symptom specification, because it seemed a more natural place to explain the choice of descriptors, symptom depth, and the use of the VAS and reduced the amount of information that needed to be memorized by the user. Finally, we reduced ambiguity of the app results for patients by specifying the time interval of the symptoms to be drawn (last 4 weeks). JMIR MHEALTH AND UHEALTH JMIR MHEALTH AND UHEALTH Neubert et al (“strongest imaginable intensity” and “strongest imaginable burden”) and anchored by numbers from 0 to 10. All changes were applied after consulting the participating doctors. Drawing Module Several profound design changes were made to the drawing module, all inspired by user feedback. To allow users to indicate local differences in symptom or finding intensity, we added a VAS to the drawing module. Different intensities in the drawing were indicated by different saturation values of the drawing color. We also changed the body outline and the way it was presented. First, we added the possibility to choose the gender of the outline (female, male, not specified). Sex-related changes to the outline were kept as small as possible to maintain comparability between the drawings from the different sexes. Specification of Diagnostic Findings In study 2, finding specification for doctors was also expanded. Besides the possibility to choose multiple symptom descriptors and depth categories, the second app contained an expanded list of findings (Table 2). In addition, doctors were able to add their own descriptors, and the depth descriptor were modified in the same way as for the patients’ version. Specification of Symptoms Many patients asked for the possibility to choose more than one descriptor for the single symptom, which is why we added this feature in the final version. Patients were also allowed to add their own descriptors if they were not happy with the available choices. Furthermore, the list of depth descriptors was changed following patients’requests. As many of them found it difficult to localize the depth of their symptoms in either “skin” or “muscle,” the term “skin” was split into “on the skin” and “beneath the skin,” whereas the other terms were left unchanged. Second, we changed the mode of presentation. While the four views (front, back, left, right) of the body outline had been presented on one screen in the prototype (Figure 1), the final app showed each view on a separate consecutive screen and users had to click through each of them. The motivation for this was that it allowed us to double the available screen area for drawing and to encourage patients to make use of all available body views. Symptom specification was expanded relative to study 1, which was largely motivated by considerations other than usability. Briefly, we asked for each symptom for the maximal and minimal intensity, if the symptom was currently present, the time of day when the symptom was worst (in intervals of 6 hours), and the perceived burden associated with the symptom. The perceived burden replaced the classification into major or minor symptom, because for the majority of the patients all symptoms were rated as major symptom. Maximal and minimal symptom intensity as well as perceived burden were rated on a VAS ranging from 0 (“no symptom” and “no burden”) to 10 We also changed the controls (ie, icons) at the bottom of the screen. Buttons for zoom (magnification glass) and scrolling (arrows) were removed because they had only been used by a minority of users. Furthermore, we reduced the line drawing tool to one thickness and added an eraser tool. Drawing Module After acknowledging the drawing instructions, a new screen was displayed asking users to specify any pain-related symptom in an iterative process. They were first asked to choose the type of sensation from the following list of descriptors (in German): burning, cold, cramping, dull, electric, heavy, hot, numb, pressing, pricking, radiating, shooting, stabbing, tearing, tender, throbbing, tingling, and tugging. In the next step, they rated the intensity of the sensation on a VAS, ranging from “no sensation” to “strongest imaginable intensity.” Next, they entered the perceived depth of the sensations by choosing one of the following descriptors: skin, muscle, organ, and bone. The initial version of the app contained an additional classification of the symptom into major or minor symptom. Following this textual specification, the drawing module was initialized. In the final module, users were shown a body outline to draw the location of the symptom or finding specified in the previous module. It had been specifically developed for this purpose based on photographs of a human body. The sex of the initial version was undetermined. Although previous screens allowed data entry by finger, drawings could only be made using the tablet’s stylus. Users were free to choose from a set of drawing tools (line, autofilled shape, or undo). Drawing was restricted to within the borders of the body outline. After finishing the drawing, users could either choose to end data entry or to add another symptom, which would bring them back to symptom specification. Figure 1. General structure of both app versions. Users were first instructed on how to make a correct symptom drawing (drawing instructions). Then, an iterative process was started, in which users characterized each of their symptoms (symptom specification). Finally, users were asked to mark the location and extent of the symptom on a body outline (drawing). JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 4 (page number not for citation purposes) http://mhealth.jmir.org/2018/5/e127/ JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 5 (page number not for citation purposes) Reproducibility Analysis We observed doctors’ evaluation during data entry at several timepoints of the project and asked them to report any problems or ideas for improvement. These were collected in a list and later analyzed. Furthermore, a meeting with the participating doctors was arranged after the first study to discuss the study results and plans for app improvements. We used a script written in Python 2.7 (Python Software Foundation [22]) to transform image data originally saved as Portable Network Graphics into Neuroimaging Informatics Technology Initiative file format [23]. Tools from FMRIB Software Library [24] were used to extract image information, such as symptom extent (number of pixels), number of clusters, intersection, and union of symptom clusters, all of which were restricted to within the body outline. We calculated Jaccard index of symptom patterns as well as a two-way, mixed model ICC (ICC(3,1); according to Shrout and Fleiss classification [25]) for symptom extent (overall number of pixels) and number of symptom clusters for each of the 25 test-retest pairs using Microsoft Excel (Microsoft Corporation) and Real Statistics Resource Pack software (Release 5.4.1) [26]. Results were calculated independently for each body view. We then assessed the maximum and average values of all body views for each patient. Body views not used by the patients in both test and retest were excluded from the analysis. In case the patient drew multiple symptoms, these were merged for reproducibility analysis and the maximum VAS value was used for each pixel. Symptom extent was further plotted as a Bland-Altman plot (ie, mean difference of the drawings of each patient against the mean of both drawings) [27] using Microsoft Excel (Microsoft Corporation). At the end of study 2, we evaluated usability from the doctors’ perspective through a Web-based survey comprising the following questionnaires: The System Usability Scale (SUS) [16] is a questionnaire for measuring usability for hard- and software products. It consists of 10 items and its results range from 0 to 100. Adjective rating scales were used for better interpretability [17]. The Attrakdiff 2 questionnaire [18] measures pragmatic and hedonic quality of a product. It consists of 4 subcategories: pragmatic quality, hedonic quality identity, hedonic quality stimulation, and attractiveness. The evaluation of these attributes is based on the ratings of 28 items, each of which is an adjective rating scale, ranging from −3 to 3. Usability Assessment Usability of the app and data acquisition method were assessed separately in patients (Aim 1) and doctors (Aim 2). Table 2. Specification of diagnostic findings for doctors (used in study 2). Diagnostic finding Category Burning, cold, cramping, dull, electric, heavy, hot, numb, pressing, pricking, radiating, shooting, stabbing, tender, throbbing, tingling, tugging, other Pain/paresthesia On the skin, beneath the skin, muscle, organ, bone Symptom depth Allodynia, analgesia, anesthesia, dysesthesia, hypoesthesia, hyperalgesia, hypoalgesia, pallanesthesia, pallhypesthesia, ther- manesthesia, thermhypesthesia, other Skin (sensitivity) Cyanosis, hyperthermia, hypothermia, pallor, redness, swelling, other Skin (perfusion) Anhidrosis, atrophy, hyperhidrosis, hypertrophy, piloerection, other Skin (autonomic) Allodynia, atrophy, disturbed proprioception, fasciculation, hyperalgesia, hypotonia, muscular defense, myogelosis, rebound tenderness, rigor, spasm, tenderness, other Muscle Tenderness, hypertrophy, induration, other Organ Table 2. Specification of diagnostic findings for doctors (used in study 2). JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 5 (page number not for citation purposes) http://mhealth.jmir.org/2018/5/e127/ http://mhealth.jmir.org/2018/5/e127/ XSL•FO RenderX XSL•FO RenderX JMIR MHEALTH AND UHEALTH Neubert et al Patients’ Evaluation questionnaires of the pain outpatient department. When preparing their first drawing, patients were not aware that they would have to draw a second one. To avoid interference with clinical routines and bias by the consultation, only those patients who had waiting periods of more than 20 min were included for a second symptom drawing. To evaluate the app and the tablet-based data acquisition, we designed a usability questionnaire. It contained a common part aimed at comparing usability between studies 1 and 2 and an individual part with items specific to each of the studies. The common part consisted of 8 Likert-type questions (possible answers from 0 to 10) and 2 open questions with free text answers. The individual part contained 3 dichotomous and 1 multiple-choice question for study 1 and one Likert-type question for study 2. A translated version of the 2 questionnaires can be found in Multimedia Appendix 2. Reproducibility Analysis The ISONORM 9241/10 questionnaire [19,20] assesses ergonomic principles for software dialogues according to ISO standard 9241 part 10. It consists of 7 categories: suitability for the task, self-descriptiveness, controllability, conformity with user expectations, error tolerance, suitability for individualization, and suitability for learning. Each category is evaluated through five 7-step items, ranging from −3 to 3. Usability Analysis Mean values, SDs, and two-tailed t tests for all types of questionnaires for patients and doctors were calculated using Microsoft Excel (Microsoft Corporation, Redmont, WA). JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 6 (page number not for citation purposes) http://mhealth.jmir.org/2018/5/e127/ Patients’ Evaluation (0=not comprehensible, 10=very com- prehensible) aStatistically significant difference (P<.05). Table 4. Usability assessment by doctors. Result (SD) Questionnaire 75.63 (19.51) System Usability Scale (range 0 to 100) Attrakdiff 2 (Range −3 to 3) 1.07 (1.41) Pragmatic quality 1.14 (1.08) Hedonic quality: identity 1.25 (1.00) Hedonic quality: stimulation 1.14 (0.93) Attractiveness ISONORM 9241/10 (range −3 to 3) 1.00 (1.62) Suitability for the task 0.95 (1.50) Self-descriptiveness −0.05 (1.43) Controllability 1.25 (1.59) Conformity with user expectations 0.65 (1.31) Error tolerance −0.15 (1.79) Suitability for individualization 1.80 (1.24) Suitability for learning JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 7 http://mhealth.jmir.org/2018/5/e127/ (page number not for citation purposes) FO Table 3. Usability assessment by patients comparing app versions from study 1 to study 2. P value Study 2 Study 1 Likert-type questions Mean (SD) N Mean (SD) N .84 7.20 (3.05) 51 7.31 (2.33) 52 How precisely does your drawing represent your actual sensations? (0=very imprecisely, 10=very precisely) .003a 1.86 (2.16) 51 3.38 (2.89) 52 How difficult was it to draw your sensations? (0=not difficult at all, 10=very difficult) .007a 8.73 (1.71) 51 7.54 (2.59) 52 How well could you identify yourself with the body outline? (0=not at all, 10=very well) .21 7.20 (2.47) 51 6.58 (2.54) 52 How precisely do the chosen terms describe the nature of your sensations? (0=very imprecisely, 10=very precisely) .02a 3.27 (2.77) 51 4.71 (3.18) 52 How difficult was it to evaluate the depth of your sensations (ie, skin, muscle, etc)? (0=not difficult at all, 10=very difficult) .45 7.10 (3.27) 51 7.52 (2.30) 52 How precisely do you rate your drawing with the electronic pen in com- parison with a pencil drawing? (0=very imprecise, 10=very precise) .62 1.65 (2.21) 51 1.43 (2.18) 51 How much physical or mental stress was the drawing of your sensations? (0=no stress, 10=very much stress) .18 8.32 (2.07) 50 7.71 (2.41) 52 How comprehensible were the drawing instructions (eg, drawing examples and written instructions) for you? (0=not comprehensible, 10=very com- prehensible) Table 3. Usability assessment by patients comparing app versions from study 1 to study 2. Table 4. Usability assessment by doctors. Patients’ Evaluation To evaluate test-retest reliability of our acquisition method (final app version on tablet PC) for symptom drawings, we planned to include 25 of our patients in the second study. The rational for this sample size was that previous studies had shown excellent test-retest reliability of pain extent with intraclass correlation coefficients (ICCs) between 0.92 and 0.97 [6]. Aiming at a 95% CI width of 0.1 with an alpha level of .05, we used formula 6 from Giraudeau and Mary [21] for our sample size estimation, which showed that 15 patients would be enough. As we planned to run additional reproducibility analyses for the number of clusters and the symptom pattern, we decided to include 25 patients. Thus, we asked 26 of our 51 patients to repeat their data entry after finishing the first one (1 patient had to be excluded because the image was not saved by the tablet PC). The second data entry was started 20 min after the first one, a period during which participants filled out other pain A total of 52 questionnaires entered the final analysis for study 1 and 51 for study 2. In study 1, 87% (45/52) of the study patients were content with the body outline. In total, 8% (4/52) of the patients proposed changes in its size and 4% (2/52) requested gender-specific changes. Moreover, 75% (39/52) of the patients agreed with the available choice of descriptors, whereas 23% (12/52) asked for additional or different terms to describe their sensations. In addition, 71% (37/52) of the patients were contempt with the terms to describe the depth of their sensations, whereas 27% (14/52) were not. Several patients used the free text option to suggest adding a multiselect option for sensations and depth descriptors. Furthermore, free text entries demanded the possibility to rate multiple symptom intensities over the day or criticized the restriction of the drawings to within the borders of the body outline. All translated free text answers XSL•FO RenderX XSL•FO RenderX JMIR MHEALTH AND UHEALTH Neubert et al 7.54 (SD 2.59) to 8.73 (SD 1.71; P=.007), and (3) the difficulty to select a depth descriptor decreased from 4.71 (SD 3.18) to 3.27 (SD 2.77; P=.02). 7.54 (SD 2.59) to 8.73 (SD 1.71; P=.007), and (3) the difficulty to select a depth descriptor decreased from 4.71 (SD 3.18) to 3.27 (SD 2.77; P=.02). of patients’usability questionnaire can be found in Multimedia Appendix 3. JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 7 (page number not for citation purposes) http://mhealth.jmir.org/2018/5/e127/ JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 7 Patients’ Evaluation Finally, 23% (12/52) of all patients of study 1 stated that they had used the zooming option. However, only 6% (3/52) of all participating patients used the magnification buttons for this. In study 2, 4% (2/51) of the patients found the size of the body outline too small. One patient of 51 (2%) reported problems using the VAS while drawing and another one proposed to use a table to support the tablet during data collection. Finally, the difficulty of drawing the symptom pattern from different angles of the human body was rated as 1.78 (SD 2.16) on a Likert-type scale from 0 (“not difficult at all”) to 10 (“very difficult”). The part of the questionnaire comparing the app versions of studies 1 and 2 showed significant differences for 3 of the 8 Likert-type items, indicating improved usability of the final app (Table 3): (1) The difficulty of the drawing process decreased from 3.38 (SD 2.89) to 1.86 (SD 2.16; P=.003), (2) the ability to identify oneself with the given body outline increased from to identify oneself with the given body outline increased from Table 3. Usability assessment by patients comparing app versions from study 1 to study 2. P value Study 2 Study 1 Likert-type questions Mean (SD) N Mean (SD) N .84 7.20 (3.05) 51 7.31 (2.33) 52 How precisely does your drawing represent your actual sensations? (0=very imprecisely, 10=very precisely) .003a 1.86 (2.16) 51 3.38 (2.89) 52 How difficult was it to draw your sensations? (0=not difficult at all, 10=very difficult) .007a 8.73 (1.71) 51 7.54 (2.59) 52 How well could you identify yourself with the body outline? (0=not at all, 10=very well) .21 7.20 (2.47) 51 6.58 (2.54) 52 How precisely do the chosen terms describe the nature of your sensations? (0=very imprecisely, 10=very precisely) .02a 3.27 (2.77) 51 4.71 (3.18) 52 How difficult was it to evaluate the depth of your sensations (ie, skin, muscle, etc)? (0=not difficult at all, 10=very difficult) .45 7.10 (3.27) 51 7.52 (2.30) 52 How precisely do you rate your drawing with the electronic pen in com- parison with a pencil drawing? (0=very imprecise, 10=very precise) .62 1.65 (2.21) 51 1.43 (2.18) 51 How much physical or mental stress was the drawing of your sensations? (0=no stress, 10=very much stress) .18 8.32 (2.07) 50 7.71 (2.41) 52 How comprehensible were the drawing instructions (eg, drawing examples and written instructions) for you? \| \| \| \| (page number not for citation purposes) JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 7 http://mhealth.jmir.org/2018/5/e127/ (page number not for citation purposes) FO JMIR MHEALTH AND UHEALTH JMIR MHEALTH AND UHEALTH Neubert et al categories “suitability for individualization” and “controllability” the results were slightly below the average. categories “suitability for individualization” and “controllability” the results were slightly below the average. Doctors’ Evaluation The results of the doctors’ usability assessment are displayed in (Table 4). We collected data through the Web-based questionnaire from the 4 doctors participating in study 2. The mean score on the SUS was 75.63 (SD 19.51), indicating good usability. The subscales of the Attrakdiff questionnaire all received ratings between 1.07 and 1.25. Both the hedonic (self-centered) and pragmatic (action-oriented) quality of the tablet app had similar values. There was no indication of hedonic or pragmatic dominance in the final app. Patients’ Evaluation Result (SD) Questionnaire 75.63 (19.51) System Usability Scale (range 0 to 100) Attrakdiff 2 (Range −3 to 3) 1.07 (1.41) Pragmatic quality 1.14 (1.08) Hedonic quality: identity 1.25 (1.00) Hedonic quality: stimulation 1.14 (0.93) Attractiveness ISONORM 9241/10 (range −3 to 3) 1.00 (1.62) Suitability for the task 0.95 (1.50) Self-descriptiveness −0.05 (1.43) Controllability 1.25 (1.59) Conformity with user expectations 0.65 (1.31) Error tolerance −0.15 (1.79) Suitability for individualization 1.80 (1.24) Suitability for learning JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 7 http://mhealth.jmir.org/2018/5/e127/ (page number not for citation purposes) FO XSL•FO RenderX aICC: intraclass correlation coefficient. JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 8 (page number not for citation purposes) Reproducibility Analysis Results of the test-retest analyses are shown in Table 5 and Figures 2-4. The Jaccard index was 0.47, indicating a mean overlap between test and retest symptom patterns of almost 50%. The ICC showed excellent reproducibility for symptom extent (ICC=0.92) and good reproducibility for Cluster count (ICC=0.70). Bland-Altman plots for symptom extent are shown in Figure 2 and Figure 3. There was no indication of a systematic difference between the measurements. Patients who drew larger areas also showed higher variability between test and retest drawings. Assessment using the ISONORM 9241/10 questionnaire showed good results in the categories “suitability for learning,” “suitability for the task,” “self-descriptiveness,” “conformity with user expectations,” and “error tolerance.” Only in the Table 5. Test-retest reliability. Result Analysis 0.47 (0.22) Jaccard index of symptom pattern (SD) ICCa of symptom extent (95% CI) 0.92 (0.88-0.95) Whole drawing (all body views) Single views 0.93 (0.84-0.97) Front 0.90 (0.78-0.96) Back 0.94 (0.86-0.97) Left 0.92 (0.82-0.97) Right ICC number of symptom clusters (95% CI) 0.70 (0.58-0.79) Whole drawing (all body views) Single views 0.66 (0.36-0.83) Front 0.56 (0.20-0.79) Back 0.75 (0.49-0.89) Left 0.87 (0.73-0.94) Right aICC: intraclass correlation coefficient. Table 5. Test-retest reliability. http://mhealth.jmir.org/2018/5/e127/ XSL•FO RenderX Discussion We have developed a new tablet-based app (SymptomMapper) to collect electronic drawings of pain and related bodily symptoms. Following the suggested design guidelines by Jaatun et al [11], we limited written textual instruction, used a paper metaphor, and avoided pop-ups and other fast changes on the screen. Two versions of the app were tested for their usability and reproducibility in a sample of chronic pain patients and their treating doctors. Neubert et al The latter option was used by the majority of patients in study 2. In total, 26 out of 51 patients (51%) selected more than one depth category. http://mhealth.jmir.org/2018/5/e127/ JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 10 (page number not for citation purposes) Neubert et al The change from a genderless to a gender- the very beginning. In the famous McGill pain questionnaire, the letters E and I were used in the pain drawing part to distinguish between “external” and “internal” pain [3], whereas Margoles used the letter D to identify deep pain [28]. Jamison et al tested depth assessment within a three-dimensional assessment of pain drawings [29], and recently, Tucker et al have developed a visual rating instrument to assess the depth of experimental back pain by calculating the “percentage of depth to center” [30]. They could show that depth and lateral position may be the most critical descriptors to determine the source of acute lumbar muscular pain. Indeed, the differentiation between different layers is not only significant in regard to diagnostic purposes but also to different therapeutic approaches, such as in acupuncture, where needles are placed in different structures according to the underlying condition. Concerning evaluation of symptom depth, we made 2 related improvements, namely, splitting the depth category “skin” into “on the skin” and “beneath the skin,” and adding the possibility to choose multiple depth descriptors at once. The latter option was used by the majority of patients in study 2. In total, 26 out of 51 patients (51%) selected more than one depth category. the very beginning. In the famous McGill pain questionnaire, the very beginning. In the famous McGill pain questionnaire, the letters E and I were used in the pain drawing part to distinguish between “external” and “internal” pain [3], whereas Margoles used the letter D to identify deep pain [28]. Jamison et al tested depth assessment within a three-dimensional assessment of pain drawings [29], and recently, Tucker et al have developed a visual rating instrument to assess the depth of experimental back pain by calculating the “percentage of depth to center” [30]. They could show that depth and lateral position may be the most critical descriptors to determine the source of acute lumbar muscular pain. Indeed, the differentiation between different layers is not only significant in regard to diagnostic purposes but also to different therapeutic approaches, such as in acupuncture, where needles are placed in different structures according to the underlying condition. Concerning evaluation of symptom depth, we made 2 related improvements, namely, splitting the depth category “skin” into “on the skin” and “beneath the skin,” and adding the possibility to choose multiple depth descriptors at once. Neubert et al Concerning identification with the body outline and overall difficultness of the drawing process, we believe that the improvements seen here were largely due to 2 major modifications we made to the drawing module, namely, the introduction of gender-specific body outlines and the consecutive rather than joint presentation of the single body views. The change from a genderless to a gender-specific body me clinical judgment. ablet-based app (SymptomMapper) ings of pain and related bodily ggested design guidelines by Jaatun en textual instruction, used a paper -ups and other fast changes on the app were tested for their usability mple of chronic pain patients and ring the pilot app from study 1 with 2 showed 3 areas of significant he patients: evaluation of symptom the body outline, and overall process. Although we did not assess of the app led to a particular we will speculate in the following causes and discuss them in the light t in depth evaluation, we must note rely superficial, a pain map has to imensional geometry of the painful ional surface [5]. It can be assumed n why depth assessment has never the very beginning. In the famous McGill pain q the letters E and I were used in the pain dra distinguish between “external” and “internal” pai Margoles used the letter D to identify deep pain et al tested depth assessment within a three assessment of pain drawings [29], and recently have developed a visual rating instrument to ass of experimental back pain by calculating the “ depth to center” [30]. They could show that dep position may be the most critical descriptors to source of acute lumbar muscular pain. Indeed, the between different layers is not only significant diagnostic purposes but also to different therapeut such as in acupuncture, where needles are place structures according to the underlying condition evaluation of symptom depth, we made 2 related im namely, splitting the depth category “skin” into and “beneath the skin,” and adding the possibil multiple depth descriptors at once. The latter op by the majority of patients in study 2. In total, patients (51%) selected more than one depth cate Concerning identification with the body outlin difficultness of the drawing process, we bel improvements seen here were largely due modifications we made to the drawing module introduction of gender-specific body outlin consecutive rather than joint presentation of th views. Neubert et al Figure 2. Bland-Altman plot of symptom extent. The central bold lines represent the mean difference. The dotted lines represent the 95% upper and lower limits of agreement. The mean symptom extent of the first and second symptom drawing (D1 and D2) is plotted against the difference in symptom extent between D1 and D2. Figure 3. Bland-Altman plot of symptom extent. The central bold lines represent the mean difference. The dotted lines represent the 95% upper and lower limits of agreement. The mean symptom extent of first and second symptom drawing (D1 and D2) is plotted against the percentual difference (of the mean) between D1 and D1. Figure 3. Bland-Altman plot of symptom extent. The central bold lines represent the mean difference. The dotted lines represent the 95% upper and lower limits of agreement. The mean symptom extent of first and second symptom drawing (D1 and D2) is plotted against the percentual difference (of the mean) between D1 and D1. JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 9 http://mhealth.jmir.org/2018/5/e127/ (page number not for citation purposes) FO erX JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 9 (page number not for citation purposes) JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 9 (page number not for citation purposes) http://mhealth.jmir.org/2018/5/e127/ JMIR MHEALTH AND UHEALTH Neubert et al Figure 4. Test-retest reliability results and problems with the Jaccard index exemplified by symptom drawings of 3 different patients: (a) low, (b) average, (c) high reliability. The first drawing (D1) of each patient is colored in red and the second drawing (D2) in blue. Purple color indicates the overlap of the 2 drawings. Jaccard indices calculated from the consecutive drawings are reported below each drawing. From a clinical standpoint, D1 and D2 would still lead to the same clinical judgment. p g g p g p , and D2 would still lead to the same clinical judgment. Discussion We have developed a new tablet-based app (SymptomMapper) to collect electronic drawings of pain and related bodily symptoms. Following the suggested design guidelines by Jaatun et al [11], we limited written textual instruction, used a paper metaphor, and avoided pop-ups and other fast changes on the screen. Neubert et al Two versions of the app were tested for their usability and reproducibility in a sample of chronic pain patients and their treating doctors. Usability Evaluation Patients’ Evaluation Usability assessment comparing the pilot app from study 1 with the final app from study 2 showed 3 areas of significant improvement as rated by the patients: evaluation of symptom depth, identification with the body outline, and overall difficultness of the drawing process. Although we did not assess explicitly which changes of the app led to a particular improvement in usability, we will speculate in the following sections on the most likely causes and discuss them in the light of the relevant literature. Regarding the improvement in depth evaluation, we must note that unless the pain is entirely superficial, a pain map has to display the complex three-dimensional geometry of the painful area onto a flat, two-dimensional surface [5]. It can be assumed that this is the main reason why depth assessment has never played a major role in pain drawings despite being used from the very beginning. In the famous McGill pain questionnaire, the letters E and I were used in the pain drawing part to distinguish between “external” and “internal” pain [3], whereas Margoles used the letter D to identify deep pain [28]. Jamison et al tested depth assessment within a three-dimensional assessment of pain drawings [29], and recently, Tucker et al have developed a visual rating instrument to assess the depth of experimental back pain by calculating the “percentage of depth to center” [30]. They could show that depth and lateral position may be the most critical descriptors to determine the source of acute lumbar muscular pain. Indeed, the differentiation between different layers is not only significant in regard to diagnostic purposes but also to different therapeutic approaches, such as in acupuncture, where needles are placed in different structures according to the underlying condition. Concerning evaluation of symptom depth, we made 2 related improvements, namely, splitting the depth category “skin” into “on the skin” and “beneath the skin,” and adding the possibility to choose multiple depth descriptors at once. The latter option was used by the majority of patients in study 2. In total, 26 out of 51 patients (51%) selected more than one depth category. Doctors’ Evaluation Our results closely replicate a study by Barbero et al [6], who in 2 samples of pain patients (chronic low back pain and neck pain) reported an ICC of 0.92 and 0.97, respectively, for pain extent and a Jaccard index of 0.46 and 0.49, respectively, for pain location. We can also confirm their observation that the difference between the first and the second drawing increases with the total number of pixels drawn, whereas the percentual difference is constant. As Barbero et al note further, it is questionable if the Jaccard index is the optimal measure to assess the reliability of pain location as it demands very high precision in pain reporting. We completely agree with the authors here. Figure 4 shows the test-retest results of 3 of our study patients to illustrate this. There is no doubt that the first and second drawings of patients (b) and (c) with Jaccard indices of 0.62 (average) and 0.83 (high) would be considered identical from a clinical point of view. However, although a Jaccard index of 0.08 as exhibited by patient (a) indicates a very low test-retest reliability, his drawings still seem to convey the same clinical information. Thus, further investigations on symptom drawings and their association with clinical judgment are warranted. Furthermore, more useful measures of similarity that do not rely on exact overlap are needed. At the end of study 2, the 4 participating doctors were asked to evaluate the tablet app through a Web-based usability survey consisting of the SUS, Attrakdiff 2, and ISONORM 9241/10 questionnaires. A score of 75.63 on the SUS indicates an overall good usability of the final app [17,32], which is in line with the results from the patients’ evaluation. With the Attrakdiff 2 questionnaire, we assessed pragmatic and hedonic qualities of the app, 2 dimensions that are independent from each other. Pragmatic quality perception evaluates the effectiveness of a product (task-related), whereas hedonic quality evaluates how the user-product interaction is stimulating the user and how the product is communicating the identity of the user (nontask-related) [33,34]. Attractiveness is the perceived property that is influenced by both pragmatic and hedonic qualities. However, although the pragmatic and hedonic qualities of a product are usually not altered by repeated use, its attractiveness can change, which Hassenzahl explains by a different weighting of these qualities based on the intention of the product usage [35]. Reproducibility Analysis After testing and improving the usability of our app, we used its final version to assess test-retest reliability of symptom drawings by a typical sample of chronic pain patients consulting a pain outpatient clinic. The analysis of pairs of consecutive drawings separated by 20 min and drawn by the same patients showed excellent reproducibility for symptom extent (ICC=0.92) and fair reproducibility for the exact symptom pattern (Jaccard index: 0.47). It is worth noting that we used 4 instead of the usual 2 views of the human. Although this complicates the drawing process, it allows for much more detailed assessment of lateral body regions. To our knowledge, this was the first study analyzing test-retest reliability for more than 2 body views. On the other hand, the consecutive presentation of the body views constitutes a more guided approach compared with the joint presentation allowing patients to focus on one view at a time. This may also have improved identification with the body outline as compared with the pilot app. Finally, the size of the body outline was almost twice as large in the consecutive presentation approach as compared with joint presentation. Interestingly, however, the enlargement of the body outline did not have an effect on patients’ perceived exactness of the drawings. Doctors’ Evaluation We expected higher pragmatic than hedonic ratings, because the layout of the app was clearly task-oriented. However, the ratings of hedonic and pragmatic quality both showed comparable ratings around 1 on the scale from −3 to 3. According to these results, the final app as rated by doctors was neither a solitary “self-product” nor a pure “act-product” [34]. In general, the app received positive evaluations in all categories of the Attrakdiff 2. Patients’ Evaluation Usability assessment comparing the pilot app from study 1 with the final app from study 2 showed 3 areas of significant improvement as rated by the patients: evaluation of symptom depth, identification with the body outline, and overall difficultness of the drawing process. Although we did not assess explicitly which changes of the app led to a particular improvement in usability, we will speculate in the following sections on the most likely causes and discuss them in the light of the relevant literature. Concerning identification with the body outline and overall difficultness of the drawing process, we believe that the improvements seen here were largely due to 2 major modifications we made to the drawing module, namely, the introduction of gender-specific body outlines and the consecutive rather than joint presentation of the single body views. The change from a genderless to a gender-specific body Regarding the improvement in depth evaluation, we must note that unless the pain is entirely superficial, a pain map has to display the complex three-dimensional geometry of the painful area onto a flat, two-dimensional surface [5]. It can be assumed that this is the main reason why depth assessment has never played a major role in pain drawings despite being used from XSL•FO RenderX JMIR MHEALTH AND UHEALTH Neubert et al order of by fixing the symptom descriptor(s) once the drawing starts. A possible way to improve this in future releases would be to add features that allow doctors to circumvent some of the restrictions and to enter data more freely. outline had been requested by patients and may have improved patients’ ability to identify themselves with the body outline. Egsgaard et al have shown that gender aspects of body outlines can influence the quality of the drawing as well as the patients’ drawing experience [31]. In their study, 85% of the female study population preferred a female body chart. outline had been requested by patients and may have improved patients’ ability to identify themselves with the body outline. Egsgaard et al have shown that gender aspects of body outlines can influence the quality of the drawing as well as the patients’ drawing experience [31]. In their study, 85% of the female study population preferred a female body chart. http://mhealth.jmir.org/2018/5/e127/ JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 11 (page number not for citation purposes) Multimedia Appendix 1 GRRAS checklist for reporting of studies of reliability and agreement. [PDF File (Adobe PDF File), 115KB-Multimedia Appendix 1] GRRAS checklist for reporting of studies of reliability and agreement. JMIR MHEALTH AND UHEALTH JMIR MHEALTH AND UHEALTH Neubert et al the doctors. The occupation with different aspects of their pain induced by the questionnaires may have influenced the second drawing, for example, by reminding patients of previously forgotten pain foci. Second, we cannot estimate the effect of learning. Roach et al showed that patients may complete pain drawings more reliably after they have been exposed to them several times [36]. In our study, patients only drew twice, and none of them underwent a training for using the app correctly, except for the drawing instructions given on the first screen. Finally, some patients reported during their second drawing that they had forgotten a symptom or complete body view in the first drawing. Despite the negative impact on test-retest reliability, we decided against excluding these patients, because their behavior probably reflects that of general pain patients. Conflicts of Interest None declared. Acknowledgments The authors would like to thank the Horst Görtz Foundation for providing financial support. Conclusions We developed an app for symptom drawing acquisition and assessed the usability of it in a sample of chronic pain patients and their treating doctors. We measured increases in usability of the improved app in terms of identification with the body outline, symptom depth evaluation, and difficultness of the drawing process according to patients’ evaluation (Aim 1). Furthermore, usability evaluation through treating doctors showed good overall usability and balanced hedonic and pragmatic values (Aim 2). Test-retest reliability of symptom drawings by chronic pain patients showed fair to excellent reproducibility for symptom pattern, symptom extent, and number of symptom clusters (Aim 3). Patients’ usability evaluation is an important factor when designing apps for mobile or eHealth apps and should not be neglected. [PDF File (Adobe PDF File), 99KB-Multimedia Appendix 2] [PDF File (Adobe PDF File), 99KB-Multimedia Appendix 2] [PDF File (Adobe PDF File), 99KB-Multimedia Appendix 2] [PDF File (Adobe PDF File), 25KB-Multimedia Appendix 3] [PDF File (Adobe PDF File), 25KB-Multimedia Appendix 3] References 1. 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Limitations As in every study, we must note some limitations. Our attempt to quantify symptom depth relied on verbal descriptors, which may have led to mistakes arising from different interpretations of expressions, such as “beneath the skin,” whose German translation may have been interpreted by some as meaning “in every tissue layer beneath the skin.” Descriptors should be carefully checked for all possible meanings they convey. Furthermore, there are alternative approaches to assess symptom depth, for example, the aforementioned visual rating of the “percentage of depth to center” by Tucker et al [30] or depth assessment based on three-dimensional pain drawings as used by Jamison et al [29]. The ISONORM 9241/10-questionnaire evaluates several aspects of software usability including suitability for the task, controllability, conformity with user expectations, and error tolerance. All categories were evaluated above average by our doctors, except for “suitability for individualization” and “controllability.” Both categories received slightly negative ratings, which may reflect the fact that doctors and patients used the same app in our study that only differed in the lists of available symptoms. As one of the design goals of this app was to increase homogeneity and validity of symptom drawings, we tried to reduce sources of between-subject variability that are due to unnecessary freedom in the drawing process. Therefore, the final app version used a rather rigid succession of data entry steps, for example, by presenting all body views in a defined Our test-retest results may have been biased by several uncontrolled factors. 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[doi: 10.1145/332040.332432] JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 13 (page number not for citation purposes) http://mhealth.jmir.org/2018/5/e127/ XSL•FO RenderX XSL•FO RenderX JMIR MHEALTH AND UHEALTH JMIR Mhealth Uhealth 2018 \| vol. 6 \| iss. 5 \| e127 \| p. 14 (page number not for citation purposes) Abbreviations Edited by G Eysenbach; submitted 14.07.17; peer-reviewed by M Barbero, H Devan, B Thompson; comments to author 05.10.17; revised version received 19.01.18; accepted 28.03.18; published 30.05.18 Please cite as: Neubert TA, Dusch M, Karst M, Beissner F Designing a Tablet-Based Software App for Mapping Bodily Symptoms: Usability Evaluation and Reproducibility Analysis JMIR Mhealth Uhealth 2018;6(5):e127 URL: http://mhealth.jmir.org/2018/5/e127/ doi: 10.2196/mhealth.8409 PMID: ©Till-Ansgar Neubert, Martin Dusch, Matthias Karst, Florian Beissner. Originally published in JMIR Mhealth and Uhealth (http://mhealth.jmir.org), 30.05.2018. 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https://openalex.org/W2148178758	https://epub.ub.uni-muenchen.de/24287/1/oa_24287.pdf	English	null	Fiducial and differential cross sections of Higgs boson production measured in the four-lepton decay channel in pp collisions at<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.gif" overflow="scroll"><mml:msqrt><mml:mi>s</mml:mi></mml:msqrt><mml:mo>=</mml:mo><mml:mn>8</mml:mn><mml:mtext> </mml:mtext><mml:mtext>TeV</mml:mtext></mml:math>with the ATLAS detector	Physics letters. B	2,014	cc-by	25,537	pipe. The x-axis points from the IP to the centre of the LHC ring, and the y-axis points upward. Cylindrical coordinates (r, φ) are used in the transverse plane, φ being the azimuthal angle around the beam pipe. The pseudorapidity is deﬁned in terms of the polar angle θ as η = − ln[tan(θ/2)]. a r t i c l e i n f o Article history: Received 14 August 2014 Received in revised form 10 September 2014 Accepted 23 September 2014 Available online 28 September 2014 Editor: W.-D. Schlatter Measurements of ﬁducial and differential cross sections of Higgs boson production in the H →Z Z∗→4ℓ decay channel are presented. The cross sections are determined within a ﬁducial phase space and corrected for detection eﬃciency and resolution effects. They are based on 20.3 fb−1 of pp collision data, produced at √ s = 8 TeV centre-of-mass energy at the LHC and recorded by the ATLAS detector. The differential measurements are performed in bins of transverse momentum and rapidity of the four- lepton system, the invariant mass of the subleading lepton pair and the decay angle of the leading lepton pair with respect to the beam line in the four-lepton rest frame, as well as the number of jets and the transverse momentum of the leading jet. The measured cross sections are compared to selected theoretical calculations of the Standard Model expectations. No signiﬁcant deviation from any of the tested predictions is found. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by SCOAP3. 1. Introduction tector covering the pseudorapidity range \|η\| < 2.5 surrounded by a superconducting solenoid, electromagnetic and hadronic calorime- ters, and an external muon spectrometer with large superconduct- ing toroidal magnets. In 2012 the ATLAS and CMS Collaborations announced the dis- covery of a new particle [1,2] in the search for the Standard Model (SM) Higgs boson [3–8] at the CERN Large Hadron Collider (LHC) [9]. Since this discovery, the particle’s mass mH was mea- sured by the ATLAS and CMS Collaborations [10–12]. The result of the ATLAS measurement based on 25 fb−1 of data collected at centre-of-mass energies of 7 TeV and 8 TeV is 125.36 ± 0.41 GeV. Tests of the couplings and spin/CP quantum numbers have been re- ported by both collaborations [11,13,14] and show agreement with the predicted scalar nature of the SM Higgs boson. Fiducial cross sections are quoted to minimize the model de- pendence of the acceptance corrections related to the extrapolation to phase-space regions not covered by the detector. The measured ﬁducial cross sections are corrected for detector effects to be di- rectly compared to theoretical calculations. The differential measurements are performed in several ob- servables related to the Higgs boson production and decay. These include the transverse momentum pT,H and rapidity \|yH\| of the Higgs boson, the invariant mass of the subleading lepton pair m34 (the leading and subleading lepton pairs are deﬁned in Section 3) and the magnitude of the cosine of the decay angle of the lead- ing lepton pair in the four-lepton rest frame with respect to the beam axis \| cosθ∗\|. The number of jets njets and the transverse momentum of the leading jet pT,jet are also included. The dis- tribution of the pT,H observable is sensitive to the Higgs boson production mechanisms as well as spin/CP quantum numbers, and can be used to test perturbative QCD predictions. This distribution In this Letter, measurements of ﬁducial and differential pro- duction cross sections for the H →Z Z∗→4ℓdecay channel are reported and compared to selected theoretical calculations. The event selection and the background determination are the same as in Ref. [15], where a detailed description is given. For this mea- surement, an integrated luminosity of 20.3 fb−1 of pp collisions is analyzed. The data were collected at the LHC at a centre-of-mass energy of √ s = 8 TeV and recorded with the ATLAS detector [16]. Fiducial and differential cross sections of Higgs boson production measured in the four-lepton decay channel in pp collisions at √ s = 8 TeV with the ATLAS detector Fiducial and differential cross sections of Higgs boson production measured in the four-lepton decay channel in pp collisions at √ s = 8 TeV with the ATLAS detector .ATLAS Collaboration ⋆ ⋆E-mail address: atlas.publications@cern.ch. 1 ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) at the centre of the detector and the z-axis along the beam http://dx.doi.org/10.1016/j.physletb.2014.09.054 0370-2693/Published by Elsevier B.V. This is an open access article under the CC BY lice ⋆E-mail address: atlas.publications@cern.ch. 1 ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) at the centre of the detector and the z-axis along the beam pipe. The x axis points from the IP to the centre of the LHC points upward. Cylindrical coordinates (r, φ) are used in the being the azimuthal angle around the beam pipe. The pseudo terms of the polar angle θ as η = − ln[tan(θ/2)]. http://dx.doi.org/10.1016/j.physletb.2014.09.054 0370-2693/Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by SC Physics Letters B 738 (2014) 234–253 Physics Letters B 738 (2014) 234–253 /j p y Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by SCOAP3 ⋆E-mail address: atlas.publications@cern.ch. 1 ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) at the centre of the detector and the z-axis along the beam 2. Theoretical predictions and simulated samples The Higgs boson production cross sections and decay branching fractions as well as their uncertainties are taken from Refs. [21,22]. The cross sections for the gluon-fusion (ggF) process have been calculated to next-to-leading order (NLO) [23–25], and next-to- next-to-leading order (NNLO) [26–28] in QCD with additional next- to-next-to-leading logarithm (NNLL) soft-gluon resummation [29]. The cross section values have been modiﬁed to include NLO elec- troweak (EW) radiative corrections, assuming factorization be- tween QCD and EW effects [30–34]. The cross sections for the vector-boson fusion (VBF) processes are calculated with full NLO QCD and EW corrections [35–37], and approximate NNLO QCD cor- rections are included [38]. The cross sections for the associated W H/Z H production processes (V H) are calculated at NLO [39] and at NNLO [40] in QCD, and NLO EW radiative corrections [41] are applied. The cross sections for associated Higgs boson produc- tion with a t¯t pair (t¯tH) are calculated at NLO in QCD [42–45]. Events with at least four leptons are selected with single-lepton and dilepton triggers. The transverse momentum and transverse energy thresholds for the single-muon and single-electron triggers are 24 GeV. Two dimuon triggers are used, one with symmetric thresholds at 13 GeV and the other with asymmetric thresholds at 18 GeV and 8 GeV. For the dielectron trigger the symmetric thresh- olds are 12 GeV. Furthermore there is an electron–muon trigger with thresholds at 12 GeV (electron) and 8 GeV (muon). Higgs boson candidates are formed by selecting two same- ﬂavour opposite-sign (SFOS) lepton pairs (a lepton quadruplet). The leptons must satisfy identiﬁcation, impact parameter, and track- based and calorimeter-based isolation criteria. Each muon (elec- tron) must satisfy transverse momentum pT > 6 GeV (transverse energy ET > 7 GeV) and be in the pseudorapidity range \|η\| < 2.7 (2.47). The highest-pT lepton in the quadruplet must satisfy pT > 20 GeV, and the second (third) lepton in pT order must satisfy pT > 15 (10) GeV. The leptons are required to be separated from each other by R ≡ (η)2 + (φ)2 > 0.1 (0.2) when having the same (different) lepton ﬂavours. The Higgs boson branching fractions for decays to four-lepton ﬁnal states are provided by Prophecy4f [46,47], which implements the complete NLO QCD + EW corrections and interference effects between identical ﬁnal-state fermions. 2. Theoretical predictions and simulated samples The H →Z Z∗→4ℓsignal is modelled using the Powheg Monte Carlo (MC) event generator [48–52], which calculates sep- arately the ggF and VBF production mechanisms with matrix ele- ments up to NLO. The description of the Higgs boson transverse momentum spectrum in the ggF process is adjusted to follow the calculation in Refs. [19,20], which includes QCD corrections up to NLO and QCD soft-gluon resummations up to NNLL, as well as ﬁ- nite quark masses [53]. Powheg is interfaced to Pythia8 [54] for showering and hadronization, which in turn is interfaced to Pho- tos [55,56] to model photon radiation in the ﬁnal state. Pythia8 is used to simulate V H and t¯tH production. The response of the ATLAS detector is modelled in a simulation [57] based on GEANT4 [58]. Multiple quadruplets within a single event are possible: for four muons or four electrons there are two ways to pair the masses, and for ﬁve or more leptons there are multiple combinations. The quadruplet selection is done separately in each channel: 4μ, 2e2μ, 2μ2e, 4e, keeping only a single quadruplet per channel. Here the ﬁrst ﬂavour index refers to the leading lepton pair, which is the pair with the invariant mass m12 closest to the Z boson mass [61]. The invariant mass m12 is required to be between 50 GeV and 106 GeV. The subleading pair of each channel is chosen as the remaining pair with mass m34 closest to the Z boson mass and satisfying the requirement 12 < m34 < 115 GeV. Finally, if more than one channel has a quadruplet passing the selection, the chan- nel with the highest expected signal rate is kept, in the order: 4μ, 2e2μ, 2μ2e, 4e. A J/ψ veto is applied: m(ℓi, ℓj) > 5 GeV for SFOS lepton pairs. Only events with a four-lepton invariant mass in the range 118–129 GeV are kept. This requirement deﬁnes the signal mass window and was chosen by minimizing the expected uncer- tainty on the total signal yield determination, taking into account the experimental uncertainty on the Higgs boson mass. The measured ﬁducial cross-section distributions are compared to three ggF theoretical calculations: Powheg without the adjust- ments to the pT,H spectrum described above, Powheg interfaced to Minlo (Multi-scale improved NLO) [59] and HRes2 (v.2.2) [19,20]. Powheg with Minlo provides predictions for jet-related variables at NLO for Higgs boson production in association with one jet. 1. Introduction The ATLAS detector covers the pseudorapidity range \|η\| < 4.9 and the full azimuthal angle φ.1 It consists of an inner tracking de- ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 235 the total cross section are 87.3% (ggF), 7.1% (VBF), 3.1% (W H), 1.9% (Z H) and 0.6% (t¯tH), respectively. the total cross section are 87.3% (ggF), 7.1% (VBF), 3.1% (W H), 1.9% (Z H) and 0.6% (t¯tH), respectively. has been studied extensively and precise predictions exist (see e.g. Refs. [17–21]), including the effect of ﬁnite quark masses. The dis- tribution of the \|yH\| observable can be used to probe the parton distribution functions (PDFs) of the proton. The distributions of the decay variables m34 and \| cosθ∗\| are sensitive to the Lagrangian structure of Higgs boson interactions, e.g. spin/CP quantum num- bers and higher-dimensional operators. The jet multiplicity and transverse momentum distributions are sensitive to QCD radiation effects and to the relative rates of Higgs boson production modes. The distribution of the transverse momentum of the leading jet probes quark and gluon radiation. All theoretical predictions are computed for a SM Higgs boson with mass 125.4 GeV. They are normalized to the most precise SM inclusive cross-section predictions currently available [60], cor- rected for the ﬁducial acceptance derived from the simulation. The Z Z, W Z, t¯t and Z + jets background events are mod- elled using the simulated samples and cross sections described in Ref. [15]. 3. Event selection The detector level physics object deﬁnitions of muons, elec- trons, and jets, and the event selection applied in this analysis are the same as in Ref. [15], with the exception of the jet selection and the additional requirement on the four-lepton invariant mass described below. A brief overview is given in this section. 6. Observed differential yields and unfolding The extraction of the signal yield for the measurement of the ﬁducial cross section is performed through a ﬁt to the m4ℓdistri- bution using shape templates for the signal and background contri- butions [15]. In this ﬁt, the Higgs boson mass is ﬁxed to 125.4 GeV and the parameter of interest is the total number of signal events. The extracted number of observed signal events in the mass win- dow is 23.7+5.9 −5.3(stat.) ± 0.6(syst.). Jets are selected by requiring pT > 30 GeV, \|y\| < 4.4 and R(jet, electron) > 0.2. Muons (electrons) must satisfy pT > 6 (7) GeV and \|η\| < 2.7 (2.47). Events in which at least one of the Z bosons decays into τ leptons are removed. Quadruplets are formed from two pairs of SFOS leptons. The leptons are paired as in Sec- tion 3, including the possibility of incorrectly pairing the leptons, which happens in about 5% of the selected events for a SM Higgs boson with mass 125.4 GeV. The leading pair is deﬁned as the SFOS lepton pair with invariant mass m12 closest to the Z boson mass and the subleading pair is deﬁned as the remaining SFOS lepton pair with invariant mass m34 closest to the Z boson mass. 5.3 In the differential cross-section measurements, given the low number of signal events expected in each measured bin i, the sig- nal yields nsig i are determined by subtracting the expected number of background events from the observed number of events. This is done within the mass window for each bin of the observable of interest. The total number of observed events in the mass win- dow is 34 and the extracted signal yield is 25.1+6.3 −5.4(stat.)+0.6 −0.4(syst.) events. The difference between the number of signal events extracted with the two methods is mainly due to ﬁxing the Higgs boson mass to 125.4 GeV in the ﬁt method. As reported in Ref. [10], the best ﬁt mass in the H →Z Z∗→4ℓchannel alone is 124.5 GeV, causing smaller weights for some events in the ﬁt. The three highest-pT leptons in the quadruplet are required to have pT > 20, 15, 10 GeV, respectively, and the lepton pairs must have 50 < m12 < 106 GeV and 12 < m34 < 115 GeV. The separation between the leptons is required to be R(ℓi, ℓj) > 0.1 (0.2) for same- (different-) ﬂavour leptons. Table 1 List of selection cuts which deﬁne the ﬁducial region of the cross section measure- ment. The same ﬂavour opposite sign lepton pairs are denoted as SFOS, the leading lepton pair mass as m12, and the subleading lepton pair mass as m34. The background estimates used in this analysis are described in detail in Ref. [15]. The irreducible Z Z and the reducible W Z background contributions are estimated using simulated samples normalized to NLO predictions. For the jet-related variables, the simulation predictions are compared to data for m4ℓ> 190 GeV where the Z Z background process is dominant; shape differences between the distributions in data and simulation are used to esti- mate systematic uncertainties. Lepton selection Muons: pT > 6 GeV, \|η\| < 2.7 Electrons: pT > 7 GeV, \|η\| < 2.47 Lepton pairing Leading pair: SFOS lepton pair with smallest \|mZ −mℓℓ\| Subleading pair: Remaining SFOS lepton pair with smallest \|mZ −mℓℓ\| Event selection Lepton kinematics: pT > 20, 15, 10 GeV Mass requirements: 50 < m12 < 106 GeV, 12 < m34 < 115 GeV Lepton separation: R(ℓi, ℓj) > 0.1 (0.2) for same- (different-) ﬂavour leptons J/ψ veto: m(ℓi,ℓj) > 5 GeV for all SFOS lepton pairs Mass window: 118 < m4ℓ< 129 GeV The reducible Z + jets and t¯t background contributions are es- timated with data-driven methods. Their normalizations are ob- tained from data control regions and extrapolated to the signal region using transfer factors. The ℓℓ + μμ ﬁnal state is dominated by Z + heavy-ﬂavour jets and the ℓℓ + ee ﬁnal state by Z + light- ﬂavour jets. The misidentiﬁcation of light-ﬂavour jets as electrons is diﬃcult to model in the simulation. Therefore the distributions for ℓℓ + ee are taken from data control regions and extrapolated to the signal region, while the background distributions for ℓℓ + μμ are taken from simulated samples. mreco 34 , the magnitude of the cosine of the decay angle of the lead- ing lepton pair in the four-lepton rest frame with respect to the beam axis \| cosθ∗reco\|, the number of jets nreco jets , and the transverse momentum of the leading jet preco T,jet. In order to distinguish them from the unfolded variables used in the cross section bin deﬁni- tion, they are labelled with “reco”. 2. Theoretical predictions and simulated samples The HRes2 program computes ﬁxed-order cross sections for ggF SM Higgs boson production up to NNLO. All-order resummation of soft-gluon effects at small transverse momenta is consistently included up to NNLL, using dynamic factorization and resumma- tion scales. The program implements top- and bottom-quark mass dependence up to NLL + NLO. At NNLL + NNLO level only the top- quark contribution is considered. HRes2 does not perform shower- ing and QED ﬁnal-state radiation effects are not included. Jets are reconstructed from topological clusters of calorimeter cells using the anti-kt algorithm [62] with the distance parameter R = 0.4. In this analysis, jets [63] are selected by requiring pT > 30 GeV, \|y\| < 4.4 and, in order to avoid double counting of elec- trons that are also reconstructed as jets, R(jet, electron) > 0.2. Jets are reconstructed from topological clusters of calorimeter cells using the anti-kt algorithm [62] with the distance parameter R = 0.4. In this analysis, jets [63] are selected by requiring pT > 30 GeV, \|y\| < 4.4 and, in order to avoid double counting of elec- trons that are also reconstructed as jets, R(jet, electron) > 0.2. The events are divided into bins of the variables of interest, which are computed with the reconstructed four-momenta of the selected lepton quadruplets or from the reconstructed jets: the transverse momentum preco T,H and the rapidity \|yreco H \| of the four- lepton system, the invariant mass of the subleading lepton pair The events are divided into bins of the variables of interest, which are computed with the reconstructed four-momenta of the selected lepton quadruplets or from the reconstructed jets: the transverse momentum preco T,H and the rapidity \|yreco H \| of the four- lepton system, the invariant mass of the subleading lepton pair The contributions from the other production modes are added to the ggF predictions. At a centre-of-mass energy of 8 TeV and for a Higgs boson mass of 125.4 GeV, their relative contributions to ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 236 Table 1 After the analysis selection about 9 background events are ex- pected: 6.7 events from irreducible Z Z and 2.2 events from the reducible background. The observed distributions compared to the signal and back- ground expectations for the six reconstructed observables preco T,H \|yreco H \|, mreco 34 , \| cosθ∗reco\|, nreco jets , and preco T,jet are shown in Fig. 1. The signal prediction includes VBF, Z H, W H, t¯tH, and the Powheg ggF calculation for a Higgs boson with mH = 125 GeV and is nor- malized to the most precise SM inclusive cross-section calculation currently available [60]. 4. Deﬁnition of the ﬁducial region The ﬁducial selection, outlined in Table 1, is designed to repli- cate at simulation level, before applying detector effects, the anal- ysis selection as closely as possible in order to minimize model- dependent acceptance effects on the measured cross sections. The ﬁducial selection is applied to electrons and muons orig- inating from vector-boson decays before they emit photon radi- ation, referred to as Born-level leptons. An alternative approach would be to correct the lepton momenta by adding ﬁnal-state radiation photons within a cone of size R < 0.1 around each lep- ton (dressing). For this analysis the acceptance difference between Born and dressed-lepton deﬁnitions is less than 0.5%. Particle-level jets are reconstructed from all stable particles except muons and neutrinos using the anti-kt algorithm with the distance parameter R = 0.4. 6. Observed differential yields and unfolding A J/ψ veto is applied: m(ℓi, ℓj) > 5 GeV for all SFOS lepton pairs. Fur- thermore, the mass of the four-lepton system m4ℓmust be close to mH, i.e. 118 < m4ℓ< 129 GeV. After subtracting the background, the measured signal yields are corrected for detector eﬃciency and resolution effects. This unfolding is performed using correction factors derived from sim- ulated SM signal samples. The correction factor in the i-th bin is calculated as For a SM Higgs boson mass of 125.4 GeV, the acceptance of the ﬁducial selection (with respect to the full phase space of H →Z Z∗→2ℓ2ℓ′, where ℓ, ℓ′ = e, μ) is 45.7%. The number of events passing the event selection divided by the number of events passing the ﬁducial selection is 55.3%; about 1% of the events pass- ing the event selection do not pass the ﬁducial selection. ci = Nreco i Nﬁd i , where Nreco i is the number of reconstructed events in the i-th bin of the observed distribution and Nﬁd i is the number of events in ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 Data yield distributions for the transverse momentum preco T,H and the rapidity \|yreco H \| of the four-lepton system, the invariant mass of the subleading lepton p gnitude of the cosine of the decay angle of the leading lepton pair in the four-lepton rest frame with respect to the beam axis \| cosθ∗reco\|, the number of transverse momentum of the leading jet preco T,jet compared to signal and background expectations. The signal prediction includes VBF, Z H, W H, t¯tH, and the culation for a Higgs boson with mH = 125 GeV and is normalized to the most precise SM inclusive cross-section calculation currently available [60]. The hatc the systematic uncertainties on the backgrounds. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web versi 237 Fig. 1. Table 2 Summary of the relative systematic uncertainties on the total back- ground contribution (top rows) and on the parameters that enter the signal extraction (bottom rows). The ranges indicate the variation across observables and bins. The systematic uncertainties on the lepton trigger, reconstruc- tion and identiﬁcation eﬃciencies [66,67] are propagated to the signal correction factors and the Z Z∗background, taking into ac- count correlations. For the correction factors, systematic uncer- tainties are assigned on the jet resolution and energy scales. The largest systematic uncertainty is due to the uncertainty in the jet ﬂavour composition [63,68,69]. Systematic uncertainties (%) Background Luminosity 1.4–2.3 Reducible background 1.6–34 Experimental, leptons 1.3–2.3 PDF/scale 3.0–24 Correction factors/conversion to σ Luminosity 2.8 Experimental, leptons 2.1–2.6 Experimental, jets 2.7–13 Production process 0.1–15 Higgs boson mass 0.4–2.7 Systematic uncertainties (%) The uncertainties on the correction factors due to PDF choice as well as QCD renormalization and factorization scale variations are evaluated in signal samples using the procedure described in Ref. [15] and found to be negligible. A similar procedure is followed for most variables for the irreducible Z Z background. For the jet-related observables an uncertainty is derived instead by comparing the data with the predicted Z Z distributions for m4ℓ> 190 GeV, after normalizing the MC estimate to the observed data yield. The systematic uncertainty is estimated as the larger of the data-MC difference and the statistical uncertainty on the data. This systematic uncertainty accounts for both the theoretical and experimental uncertainties in the modelling of the Z Z jet dis- tributions. Systematic uncertainties due to the modelling of QED ﬁnal-state radiation are found to be negligible with respect to the total uncertainty. ﬁducial objects, the correction factors for t¯tH differ from those for the other production modes. For each bin, the number of expected background events, the number of observed events, the luminosity, and the correction factors are used to calculate a proﬁle likelihood ratio [64]. The likelihood includes shape and normalization uncertainties of back- grounds and correction factors as nuisance parameters. For each variable all bins are included in the likelihood and correlations of uncertainties between the different bins and between backgrounds and correction factors are taken into account. The cross sections are extracted for each bin by minimizing twice the negative loga- rithm of the proﬁle likelihood ratio −2 lnΛ. The cross section in the ﬁducial region described in Table 1 is σ ﬁd tot = 2.11+0.53 −0.47(stat.) ± 0.08(syst.) fb. The cross section in the ﬁducial region described in Table 1 is σ ﬁd tot = 2.11+0.53 −0.47(stat.) ± 0.08(syst.) fb. Table 2 The uncertainties on the cross sections are also estimated using −2 ln Λ by evaluating its variation as a function of the parameter of interest (the cross section value in each bin). Under the asymptotic assumption [64], −2 lnΛ behaves as a χ 2 distribution with one degree of freedom. For some of the ﬁtted intervals, due to the low number of events, the distribution of the proﬁle likelihood ratio does not follow a χ 2 distribution and the uncertainties are derived using pseudo- experiments. The correction factors are calculated assuming the predicted relative cross sections of the different Higgs production modes. The corresponding systematic uncertainty is evaluated by varying these predictions within the current experimental bounds [14]. The VBF and V H fractions are varied by factors of 0.5 and 2 with respect to the SM prediction and the t¯tH fraction is varied by factors of 0 and 5. The experimental uncertainty on mH [10] is propagated to the correction factors by studying their dependence on the Higgs bo- son mass. The systematic uncertainties on the theoretical predictions in- clude the PDF and QCD scale choices as well as the uncertainty on the H →Z Z∗branching fraction [60]. The procedure described in Ref. [70] is used to evaluate the scale uncertainties of the predicted njets distribution. The compatibility between the measured cross sections and the theoretical predictions is evaluated by computing the differ- ence between the value of −2 lnΛ at the best-ﬁt value and the value obtained by ﬁxing the cross sections in all bins to the ones predicted by theory. Under the asymptotic assumption [64], this statistical observable behaves as a χ 2 with the number of degrees of freedom equal to the number of bins; it is used as a test statis- tic to compute the p-values quantifying the compatibility between the observed distributions and the predictions. For all measured observables the asymptotic assumption is veriﬁed with pseudo- experiments. j The upper edges of the uncertainty ranges in Table 2 are in most cases due to the highest bins in the njets and pT,jet distribu- tions. The background systematic uncertainties are large in some bins due to the limited statistics in the data control regions. 6. Observed differential yields and unfolding Data yield distributions for the transverse momentum preco T,H and the rapidity \|yreco H \| of the four-lepton system, the invariant mass of the subleading lepton pair mreco 34 , the magnitude of the cosine of the decay angle of the leading lepton pair in the four-lepton rest frame with respect to the beam axis \| cosθ∗reco\|, the number of jets nreco jets , and the transverse momentum of the leading jet preco T,jet compared to signal and background expectations. The signal prediction includes VBF, Z H, W H, t¯tH, and the Powheg ggF calculation for a Higgs boson with mH = 125 GeV and is normalized to the most precise SM inclusive cross-section calculation currently available [60]. The hatched areas denote the systematic uncertainties on the backgrounds. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) the i-th bin of the particle-level distribution, within the ﬁducial region. The correction factors used in this analysis are obtained from simulated samples for all SM Higgs production modes, using the relative rates as predicted by the SM. The inclusive correction fac- tor is c = 0.553 ± 0.002(stat.) ± 0.015(syst.). The correction factors for the different production modes are 0.553 (ggF), 0.572 (VBF), 0.535 (W H), 0.551 (Z H) and 0.417 (t¯tH). In t¯tH production the Higgs boson is accompanied by light- and heavy-ﬂavour jets as well as possible additional leptons from the top-quark decays. Since lepton isolation is applied to the reconstructed but not the The unfolded signal yield in each bin is then converted into a differential ﬁducial cross section via dσﬁd,i dxi = nsig i ci · Lint · xi , dσﬁd,i dxi = nsig i ci · Lint · xi , where xi is the bin width and Lint the integrated luminosity. where xi is the bin width and Lint the integrated luminosity. ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 238 Systematic uncertainties on the data-driven estimate of the re- ducible backgrounds are assigned both to the normalization and the shapes of the distributions by varying the estimation methods [15]. 7. Systematic uncertainties σ ﬁd tot = 2.11+0.53 −0.47(stat.) ± 0.08(syst.) fb. Systematic uncertainties are calculated for the estimated back- grounds, the correction factors, and the SM theoretical predictions; the latter only have an impact on the quantitative comparison of the measurements with different predictions. An overview of the systematic uncertainties on the total background prediction and the correction factors is shown in Table 2. The theoretical prediction from Ref. [60] for a Higgs boson mass of 125.4 GeV is 1.30 ± 0.13 fb. The differential cross sections as a function of pT,H, yH, m34, \| cosθ∗\|, njets, and pT,jet are shown in Fig. 2. For all variables and bins the total uncertainties on the cross-section measurements are dominated by statistical uncertainties. Powheg, Minlo and HRes2 calculations of ggF, added to VBF, Z H/W H and t¯tH (see Section 2), are overlaid. The HRes2 calculation was developed for modelling the Higgs kinematic variables and is only used for pT,H and yH. The theoretical calculations are normalized to the most precise SM inclusive cross-section predictions currently available [60]. The uncertainty on the integrated luminosity is propagated in a correlated way to the backgrounds evaluated from the MC predic- tions and to the unfolding, where it is used when converting the estimated unfolded signal yield into a ﬁducial cross section. This uncertainty is derived following the same methodology as that de- tailed in Ref. [65] from a preliminary calibration of the luminosity scale derived from beam-separation scans performed in November 2012. The p-values quantifying the compatibility between data and predictions, computed with the method described in Section 6, are shown in Table 3. No signiﬁcant discrepancy is observed. ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 239 ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 ntial unfolded cross sections for the transverse momentum pT,H and rapidity yH of the Higgs boson, the invariant mass of the subleading lepton pair the cosine of the decay angle of the leading lepton pair in the four-lepton rest frame with respect to the beam axis \| cosθ∗\|, the number of jets njets, mentum of the leading jet pT,jet in the H →Z Z∗→4ℓdecay channel compared to different theoretical calculations of the ggF process: Powheg, Mi ntributions from VBF, Z H/W H and t¯tH are determined as described in Section 2 and added to the ggF distributions. 7. Systematic uncertainties All theoretical calculations are no recise SM inclusive cross-section predictions currently available [60]. The error bars on the data points show the total (stat. ⊕syst.) uncertainty, while the systematic uncertainties. The bands of the theoretical prediction indicate the total uncertainty. (For interpretation of the references to colour in th ader is referred to the web version of this article.) i k l d Fig. 2. Differential unfolded cross sections for the transverse momentum pT,H and rapidity yH of the Higgs boson, the invariant mass of the subleading lepton pair m34, the magnitude of the cosine of the decay angle of the leading lepton pair in the four-lepton rest frame with respect to the beam axis \| cosθ∗\|, the number of jets njets, and the transverse momentum of the leading jet pT,jet in the H →Z Z∗→4ℓdecay channel compared to different theoretical calculations of the ggF process: Powheg, Minlo and HRes2. The contributions from VBF, Z H/W H and t¯tH are determined as described in Section 2 and added to the ggF distributions. All theoretical calculations are normalized to the most precise SM inclusive cross-section predictions currently available [60]. 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Berge 107, J .P. Benjamin 45, J.R. Bensinger 23, K. Benslama 132, S. Bentvelsen 107, D. Berge 107, j , J g , , , g , E. Bergeaas Kuutmann 168, N. Berger 5, F. Berghaus 171, J. Beringer 15, C. Bernard 22, P. Bernat 78, C B i 79 F U B l h 171 T B 77 P B t 129 C B t ll 85 G B t li 148a 148b j J g g . Bergeaas Kuutmann 168, N. Berger 5, F. Berghaus 171, J. Beringer 15, C. Bernard 22, . Bernius 79, F.U. Bernlochner 171, T. Berry 77, P. Berta 129, C. ATLAS Collaboration Fulsom 145, J. Fuster 169, C. Gabaldon 55, O. Gabizon 177, A. Gabrielli 20a,20b, A. Gabrielli 134a,134b, S. Gadatsch 107, S. Gadomski 49, G. Gagliardi 50a,50b, P. Gagnon 61, C. Galea 106, B. Galhardo 126a,126c, E.J. Gallas 120, V. Gallo 17, ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 243 P. Conde Muiño 126a,126b, E. Coniavitis 48, M.C. Conidi 12, S.H. Connell 147b, I.A. Connelly 77, S.M. Consonni 91a,91b, V. Consorti 48, S. Constantinescu 26a, C. Conta 121a,121b, G. Conti 57, F. Conventi 104a,i M. 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Davignon 80, A.R. Davison 78, P. Davison 78, Y. Davygora 58a, E. Dawe 144, I. Dawson 141, R.K. Daya-Ishmukhametova 86, K. De 8, R. de Asmundis 104a, S. De Castro 20a,20b, S. De Cecco 80, N. De Groot 106, P. de Jong 107, H. De la Torre 82, F. De Lorenzi 64, L. De Nooij 107, D. De Pedis 134a, A. De Salvo 134a, U. De Sanctis 151, A. De Santo 151, J.B. De Vivie De Regie 117, W.J. Dearnaley 72, R. Debbe 25, C. Debenedetti 139, B. Dechenaux 55, D.V. Dedovich 65, I. Deigaard 107, J. Del Peso 82, T. Del Prete 124a,124b, F. Deliot 138, C.M. Delitzsch 49, M. Deliyergiyev 75, A. Dell’Acqua 30, L. Dell’Asta 22, M. Dell’Orso 124a,124b, M. Della Pietra 104a,i, D. della Volpe 49, M. ATLAS Collaboration Delmastro 5, P.A. Delsart 55, C. Deluca 107, S. Demers 178, M. Demichev 65, A. Demilly 80, S.P. Denisov 130, D. Derendarz 39, J.E. Derkaoui 137d, F. Derue 80, P. Dervan 74, K. Desch 21, C. Deterre 42, P.O. Deviveiros 107, A. Dewhurst 131, S. Dhaliwal 107, A. Di Ciaccio 135a,135b, L. Di Ciaccio 5, A. 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Fedin 123,k, W. Fedorko 170, M. Fehling-Kaschek 48, S. Feigl 30, L. Feligioni 85, C. Feng 33d, E.J. Feng 6, H. Feng 89, A.B. Fenyuk 130, S. Fernandez Perez 30, S. Ferrag 53, J. Ferrando 53, A. Ferrari 168, P. Ferrari 107, R. Ferrari 121a, D.E. Ferreira de Lima 53, A. Ferrer 169, D. Ferrere 49, C. Ferretti 89, A. Ferretto Parodi 50a,50b, M. Fiascaris 31, F. Fiedler 83, A. Filipˇciˇc 75, M. Filipuzzi 42, F. Filthaut 106, M. Fincke-Keeler 171, K.D. Finelli 152, M.C.N. Fiolhais 126a,126c, L. Fiorini 169, A. Firan 40, A. Fischer 2, J. Fischer 177, W.C. Fisher 90, E.A. Fitzgerald 23, M. Flechl 48, I. Fleck 143, P. Fleischmann 89, S. Fleischmann 177, G.T. Fletcher 141, G. Fletcher 76, T. Flick 177, A. Floderus 81, L.R. Flores Castillo 60a, A.C. Florez Bustos 161b, M.J. Flowerdew 101, A. Formica 138, A. Forti 84, D. Fortin 161a, D. Fournier 117, H. Fox 72, S. Fracchia 12, P. Francavilla 80, M. Franchini 20a,20b, S. Franchino 30, D. Francis 30, L. Franconi 119, M. Franklin 57, b P. Conde Muiño , E. Coniavitis , M.C. Conidi , S.H. Connell , I.A. Connelly , S.M. Consonni 91a,91b, V. Consorti 48, S. Constantinescu 26a, C. Conta 121a,121b, G. Conti 57, F. Conventi 104a,i, M. Cooke 15, B.D. Cooper 78, A.M. Cooper-Sarkar 120, N.J. Cooper-Smith 77, K. Copic 15, T. Cornelissen 177, M. Corradi 20a, F. Corriveau 87,j, A. Corso-Radu 165, A. Cortes-Gonzalez 12, G. Cortiana 101, G. Costa 91a, M.J. Costa 169, D. Costanzo 141, D. Côté 8, G. Cottin 28, G. Cowan 77, B.E. Cox 84, K. Cranmer 110, G. Cree 29, S. Crépé-Renaudin 55, F. Crescioli 80, W.A. Cribbs 148a,148b, M. Crispin Ortuzar 120, M. Cristinziani 21, V. Croft 106, G. Crosetti 37a,37b, C.-M. Cuciuc 26a, T. Cuhadar Donszelmann 141, J. Cummings 178, M. Curatolo 47, C. Cuthbert 152, H. Czirr 143, P. Czodrowski 3, Z. Czyczula 178, S. D’Auria 53, M. D’Onofrio 74, M.J. Da Cunha Sargedas De Sousa 126a,126b, C. Da Via 84, W. Dabrowski 38a, A. Daﬁnca 120, T. Dai 89, O. Dale 14, F. Dallaire 95, C. Dallapiccola 86, M. Dam 36, A.C. Daniells 18, M. Dano Hoffmann 138, V. Dao 48, G. Darbo 50a, S. ATLAS Collaboration Bertella 85, G. Bertoli 1 124 124b 113 113 91 106 y Bertolucci 124a,124b, C. Bertsche 113, D. Bertsche 113, M.I. Besana 91a, G.J. Besjes 106 , , , , J j . Bessidskaia 148a,148b, M. Bessner 42, N. Besson 138, C. Betancourt 48, S. Bethke 101 M Bianchi 125 L Bianchini 23 M Bianco 30 O Biebel 100 S P Bieniek 78 K Bierw . Bessidskaia , M. Bessner , N. Besson , C. Betancourt , S. Bethke .M. Bianchi 125, L. Bianchini 23, M. Bianco 30, O. Biebel 100, S.P. Bieniek 78, K. Bierw , , , , , j , R.M. Bianchi 125, L. Bianchini 23, M. Bianco 30, O. Biebel 100, S.P. Bieniek 78, K. Bierwagen 54, J. Biesiada j R.M. Bianchi 125, L. Bianchini 23, M. Bianco 30, O. Biebel 100, S.P. Bieniek 78, K. Bierwagen 54, J. Biesiada 15, M Bi li i 136a J Bilb D M di b l 49 H Bil k 47 M Bi di 54 S Bi 117 A Bi l 19c g , J , , , , g C. Bini 134a,134b, C.W. Black 152, J.E. Black 145, K.M. Black 22, D. Blackburn 140, R.E. 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Da Via 84, W. Dabrowski 38a, A. Daﬁnca 120, T. Dai 89, O. Dale 14, F. Dallaire 95, C. Dallapiccola 86, M. Dam 36, A.C. Daniells 18, M. ATLAS Collaboration Dano Hoffmann 138, V. Dao 48, G. Darbo 50a, S. Darmora 8, J.A. Dassoulas 42, A. Dattagupta 61, W. Davey 21, C. David 171, T. Davidek 129, E. Davies 120,d, M. Davies 155, O. Davignon 80, A.R. Davison 78, P. Davison 78, Y. Davygora 58a, E. Dawe 144, I. Dawson 141, R.K. Daya-Ishmukhametova 86, K. De 8, R. de Asmundis 104a, S. De Castro 20a,20b, S. De Cecco 80, N. De Groot 106, P. de Jong 107, H. De la Torre 82, F. De Lorenzi 64, L. De Nooij 107, D. De Pedis 134a, A. De Salvo 134a, U. De Sanctis 151, A. De Santo 151, J.B. De Vivie De Regie 117, W.J. Dearnaley 72, R. Debbe 25, C. Debenedetti 139, B. Dechenaux 55, D.V. Dedovich 65, I. Deigaard 107, J. Del Peso 82, T. Del Prete 124a,124b, F. Deliot 138, C.M. Delitzsch 49, M. Deliyergiyev 75, A. Dell’Acqua 30, L. Dell’Asta 22, M. Dell’Orso 124a,124b, M. Della Pietra 104a,i, D. della Volpe 49, M. Delmastro 5, P.A. Delsart 55, C. Deluca 107, S. Demers 178, M. Demichev 65, A. Demilly 80, S.P. Denisov 130, D. Derendarz 39, J.E. Derkaoui 137d, F. Derue 80, P. Dervan 74, K. Desch 21, C. Deterre 42, P.O. Deviveiros 107, A. Dewhurst 131, S. Dhaliwal 107, A. Di Ciaccio 135a,135b, L. Di Ciaccio 5, A. Di Domenico 134a,134b, C. Di Donato 104a,104b, A. Di Girolamo 30, B. Di Girolamo 30, A. Di Mattia 154, B. Di Micco 136a,136b, R. Di Nardo 47, A. Di Simone 48, R. Di Sipio 20a,20b, D. Di Valentino 29, F.A. Dias 46, M.A. Diaz 32a, E.B. Diehl 89, J. Dietrich 42, T.A. Dietzsch 58a, S. Diglio 85, A. Dimitrievska 13a, J. Dingfelder 21, C. Dionisi 134a,134b, P. Dita 26a, S. Dita 26a, F. Dittus 30, F. Djama 85, T. Djobava 51b, J.I. Djuvsland 58a, M.A.B. do Vale 24c, A. Do Valle Wemans 126a,126g, D. Dobos 30, C. Doglioni 49, T. Doherty 53, T. Dohmae 157, J. Dolejsi 129, Z. Dolezal 129, B.A. Dolgoshein 98,∗, M. Donadelli 24d, S. Donati 124a,124b, P. Dondero 121a,121b, J. Donini 34, J. Dopke 131, A. Doria 104a, M.T. Dova 71, A.T. Doyle 53, M. Dris 10, J. Dubbert 89, S. Dube 15, E. Dubreuil 34, E. Duchovni 174, G. Duckeck 100, O.A. Ducu 26a, D. Duda 177, A. Dudarev 30, F. Dudziak 64, L. Duﬂot 117, L. Duguid 77, M. Dührssen 30, M. ATLAS Collaboration Dunford 58a, H. Duran Yildiz 4a, M. Düren 52, A. Durglishvili 51b, M. Dwuznik 38a, M. Dyndal 38a, J. Ebke 100, W. Edson 2, N.C. Edwards 46, W. Ehrenfeld 21, T. Eifert 145, G. Eigen 14, K. Einsweiler 15, T. Ekelof 168, M. El Kacimi 137c, M. Ellert 168, S. Elles 5, F. Ellinghaus 83, N. Ellis 30, J. Elmsheuser 100, M. Elsing 30, D. Emeliyanov 131, Y. Enari 157, O.C. Endner 83, M. Endo 118, R. Engelmann 150, J. Erdmann 178, A. Ereditato 17, D. Eriksson 148a, G. Ernis 177, J. Ernst 2, M. Ernst 25, J. Ernwein 138, D. Errede 167, S. Errede 167, E. Ertel 83, M. Escalier 117, H. Esch 43, C. Escobar 125, B. Esposito 47, A.I. Etienvre 138, E. Etzion 155, H. Evans 61, A. Ezhilov 123, L. 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Kamenshchikov 130, N. Kanaya 157, M. Kaneda 30, S. Kaneti 28, V.A. Kantserov 98, J. Kanzaki 66, B. Kaplan 110, A. Kapliy 31, D. Kar 53, K. Karakostas 10, N. Karastathis 10, M.J. Kareem 54, M. Karnevskiy 83, S.N. Karpov 65, Z.M. Karpova 65, K. Karthik 110, V. Kartvelishvili 72, A.N. Karyukhin 130, L. Kashif 175, G. Kasieczka 58b, R.D. Kass 111, A. Kastanas 14, Y. Kataoka 157, A. Katre 49, J. Katzy 42, V. Kaushik 7, K. Kawagoe 70, T. Kawamoto 157, G. Kawamura 54, S. Kazama 157, V.F. Kazanin 109, M.Y. Kazarinov 65, R. Keeler 171, R. Kehoe 40, M. Keil 54, J.S. Keller 42, J.J. Kempster 77, H. Keoshkerian 5, O. Kepka 127, B.P. Kerševan 75, S. Kersten 177, K. Kessoku 157, J. Keung 160, F. Khalil-zada 11, H. Khandanyan 148a,148b, A. Khanov 114, A. Khodinov 98, A. Khomich 58a, T.J. Khoo 28, G. Khoriauli 21, A. Khoroshilov 177, V. Khovanskiy 97, E. Khramov 65, J. Khubua 51b, H.Y. Kim 8, H. Kim 148a,148b, S.H. Kim 162, N. Kimura 173, O. Kind 16, B.T. King 74, M. King 169 R.S.B. King 120, S.B. 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Orlando 73a,73b, C. Oropeza Barrera 53, R.S. Orr 160, B. Osculati 50a,50b, R. Ospanov 122, G. Otero y Garzon 27, H. Otono 70, M. Ouchrif 137d, E.A. Ouellette 171, F. Ould-Saada 119, A. Ouraou 138, K.P. Oussoren 107, Q. Ouyang 33a, A. Ovcharova 15, M. Owen 84, V.E. Ozcan 19a, N. Ozturk 8, K. Pachal 120, A. Pacheco Pages 12, C. Padilla Aranda 12, M. Pagáˇcová 48, S. Pagan Griso 15, E. Paganis 141, C. Pahl 101, F. Paige 25, P. Pais 86, K. Pajchel 119, G. Palacino 161b, S. Palestini 30, M. Palka 38b, D. Pallin 34, A. Palma 126a,126b, J.D. Palmer 18, Y.B. Pan 175, E. Panagiotopoulou 10, J.G. Panduro Vazquez 77, P. Pani 107, N. Panikashvili 89, S. Panitkin 25, D. Pantea 26a, L. Paolozzi 135a,135b, Th.D. Papadopoulou 10, K. Papageorgiou 156,l, A. Paramonov 6, D. Paredes Hernandez 156, M.A. Parker 28, F. Parodi 50a,50b, J.A. Parsons 35, U. Parzefall 48, E. Pasqualucci 134a, S. Passaggio 50a, A. Passeri 136a, F. Pastore 136a,136b,∗, Fr. Pastore 77, G. Pásztor 29, S. Pataraia 177, N.D. Patel 152, J.R. Pater 84, S. Patricelli 104a,104b, T. Pauly 30, J. Pearce 171, L.E. Pedersen 36, M. Pedersen 119, S. Pedraza Lopez 169, R. Pedro 126a,126b, S.V. Peleganchuk 109, D. Pelikan 168, H. Peng 33b, B. Penning 31, J. Penwell 61, D.V. Perepelitsa 25, E. Perez Codina 161a, M.T. Pérez García-Estañ 169, V. Perez Reale 35, L. Perini 91a,91b, H. Pernegger 30, S. Perrella 104a,104b, R. Perrino 73a, R. Peschke 42, V.D. Peshekhonov 65, K. Peters 30, R.F.Y. Peters 84, B.A. Petersen 30, T.C. Petersen 36, E. Petit 42, A. Petridis 148a,148b, C. Petridou 156, E. Petrolo 134a, F. Petrucci 136a,136b, N.E. Pettersson 159, R. Pezoa 32b, P.W. Phillips 131, G. Piacquadio 145, E. Pianori 172, A. Picazio 49, E. Piccaro 76, M. Piccinini 20a,20b, R. Piegaia 27, D.T. Pignotti 111, J.E. Pilcher 31, A.D. Pilkington 78, J. Pina 126a,126b,126d, M. Pinamonti 166a,166c,ab, A. Pinder 120, J.L. Pinfold 3, A. Pingel 36, B. Pinto 126a, S. Pires 80, M. Pitt 174, C. Pizio 91a,91b, L. Plazak 146a, M.-A. Pleier 25, V. Pleskot 129, E. Plotnikova 65, P. ATLAS Collaboration Plucinski 148a,148b, D. Pluth 64, S. Poddar 58a, F. Podlyski 34, R. Poettgen 83, L. Poggioli 117, D. Pohl 21, M. Pohl 49, G. Polesello 121a, A. Policicchio 37a,37b, R. Polifka 160, A. Polini 20a, C.S. Pollard 45, V. Polychronakos 25, K. Pommès 30, L. Pontecorvo 134a, B.G. Pope 90, G.A. Popeneciu 26b, D.S. Popovic 13a, A. Poppleton 30, X. Portell Bueso 12, S. Pospisil 128, K. Potamianos 15, I.N. Potrap 65, C.J. Potter 151, C.T. Potter 116, G. Poulard 30, J. Poveda 61, V. Pozdnyakov 65, P. Pralavorio 85, A. Pranko 15, S. Prasad 30, R. Pravahan 8, S. Prell 64, D. Price 84, J. Price 74, L.E. Price 6, D. Prieur 125, M. Primavera 73a, M. Proissl 46, K. Prokoﬁev 47, F. Prokoshin 32b, E. Protopapadaki 138, S. Protopopescu 25, J. Proudfoot 6, M. Przybycien 38a, H. Przysiezniak 5, E. Ptacek 116, D. Puddu 136a,136b, E. Pueschel 86, D. Puldon 150, M. Purohit 25,ac, P. Puzo 117, J. Qian 89, G. Qin 53, Y. Qin 84, A. Quadt 54, D.R. 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Teixeira Dias Castanheira 76, ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 248 A. Salzburger 30, D. Sampsonidis 156, A. Sanchez 104a,104b, J. Sánchez 169, V. Sanchez Martinez 169, H. Sandaker 14, R.L. Sandbach 76, H.G. Sander 83, M.P. Sanders 100, M. Sandhoff 177, T. Sandoval 28, C. Sandoval 164, R. Sandstroem 101, D.P.C. Sankey 131, A. Sansoni 47, C. Santoni 34, R. Santonico 135a,135b, H. Santos 126a, I. Santoyo Castillo 151, K. Sapp 125, A. Sapronov 65, J.G. Saraiva 126a,126d, B. Sarrazin 21, G. Sartisohn 177, O. Sasaki 66, Y. Sasaki 157, G. Sauvage 5,∗, E. Sauvan 5, P. Savard 160,e, D.O. Savu 30, C. Sawyer 120, L. Sawyer 79,m, D.H. Saxon 53, J. Saxon 122, C. Sbarra 20a, A. Sbrizzi 20a,20b, T. Scanlon 78, D.A. Scannicchio 165, M. Scarcella 152, V. Scarfone 37a,37b, J. Schaarschmidt 174, P. Schacht 101, D. Schaefer 30, R. Schaefer 42, S. Schaepe 21, S. Schaetzel 58b, U. Schäfer 83, A.C. Schaffer 117, D. Schaile 100, R.D. Schamberger 150, V. Scharf 58a, V.A. Schegelsky 123, D. Scheirich 129, M. Schernau 165, M.I. Scherzer 35, C. Schiavi 50a,50b, J. Schieck 100, C. Schillo 48, M. Schioppa 37a,37b, S. Schlenker 30, E. Schmidt 48, K. Schmieden 30, C. Schmitt 83, S. Schmitt 58b, B. Schneider 17, Y.J. Schnellbach 74, U. Schnoor 44, L. Schoeffel 138, A. Schoening 58b, B.D. Schoenrock 90, A.L.S. Schorlemmer 54, M. Schott 83, D. Schouten 161a, J. Schovancova 25, S. Schramm 160, M. Schreyer 176, C. Schroeder 83, N. Schuh 83, M.J. Schultens 21, H.-C. Schultz-Coulon 58a, H. Schulz 16, M. Schumacher 48, B.A. Schumm 139, Ph. Schune 138, C. ATLAS Collaboration Schwanenberger 84, A. Schwartzman 145, T.A. Schwarz 89, Ph. Schwegler 101, Ph. Schwemling 138, R. Schwienhorst 90, J. Schwindling 138, T. Schwindt 21, M. Schwoerer 5, F.G. Sciacca 17, E. Scifo 117, G. Sciolla 23, W.G. Scott 131, F. Scuri 124a,124b, F. Scutti 21, J. Searcy 89, G. Sedov 42, E. Sedykh 123, S.C. Seidel 105, A. Seiden 139, F. Seifert 128, J.M. Seixas 24a, G. Sekhniaidze 104a, S.J. 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Sivoklokov 99, J. Sjölin 148a,148b, T.B. Sjursen 14, H.P. Skottowe 57, K.Yu. Skovpen 109, P. Skubic 113, M. Slater 18, T. Slavicek 128, M. Slawinska 107, K. Sliwa 163, V. Smakhtin 174, B.H. Smart 46, L. Smestad 14, S.Yu. Smirnov 98, Y. Smirnov 98, L.N. Smirnova 99,ae, O. Smirnova 81, K.M. Smith 53, M. Smizanska 72, K. Smolek 128, A.A. Snesarev 96, G. Snidero 76, S. Snyder 25, R. Sobie 171,j, F. Socher 44, A. Soffer 155, D.A. Soh 153,ad, C.A. Solans 30, M. Solar 128, J. Solc 128, E.Yu. Soldatov 98, U. Soldevila 169, A.A. Solodkov 130, A. Soloshenko 65, O.V. Solovyanov 130, V. Solovyev 123, P. Sommer 48, H.Y. Song 33b, N. Soni 1, A. Sood 15, A. Sopczak 128, B. Sopko 128, V. Sopko 128, V. Sorin 12, M. Sosebee 8, R. Soualah 166a,166c, P. Soueid 95, A.M. Soukharev 109,c, D. South 42, S. Spagnolo 73a,73b, F. ATLAS Collaboration Rauscher 100, T.C. Rave 48, T. Ravenscroft 53, M. Raymond 30, A.L. Read 119, N.P. Readioff 74, D.M. Rebuzzi 121a,121b, A. Redelbach 176, G. Redlinger 25, R. 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Rud 99, C. Rudolph 44, M.S. Rudolph 160, F. Rühr 48, A. Ruiz-Martinez 30, Z. Rurikova 48, N.A. Rusakovich 65, A. Ruschke 100, J.P. Rutherfoord 7, N. Ruthmann 48, Y.F. Ryabov 123, M. Rybar 129, G. Rybkin 117, N.C. Ryder 120, A.F. Saavedra 152, G. Sabato 107, S. Sacerdoti 27, A. Saddique 3, I. Sadeh 155, H.F-W. Sadrozinski 139, R. Sadykov 65, F. Safai Tehrani 134a, H. Sakamoto 157, Y. Sakurai 173, G. Salamanna 136a,136b, A. Salamon 135a, M. Saleem 113, D. Salek 107, P.H. Sales De Bruin 140, D. Salihagic 101, A. Salnikov 145, J. Salt 169, D. Salvatore 37a,37b, F. Salvatore 151, A. Salvucci 106, 248 ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 A. Salzburger 30, D. Sampsonidis 156, A. Sanchez 104a,104b, J. Sánchez 169, V. Sanchez Martinez 169, H. Sandaker 14, R.L. Sandbach 76, H.G. Sander 83, M.P. Sanders 100, M. Sandhoff 177, T. Sandoval 28, C. Sandoval 164, R. Sandstroem 101, D.P.C. Sankey 131, A. Sansoni 47, C. Santoni 34, R. Santonico 135a,135b, H. Santos 126a, I. Santoyo Castillo 151, K. Sapp 125, A. Sapronov 65, J.G. Saraiva 126a,126d, B. ATLAS Collaboration Sarrazin 21, G. Sartisohn 177, O. Sasaki 66, Y. Sasaki 157, G. Sauvage 5,∗, E. Sauvan 5, P. Savard 160,e, D.O. Savu 30, C. Sawyer 120, L. Sawyer 79,m, D.H. Saxon 53, J. Saxon 122, C. Sbarra 20a, A. Sbrizzi 20a,20b, T. Scanlon 78, D.A. Scannicchio 165, M. Scarcella 152, V. Scarfone 37a,37b, J. Schaarschmidt 174, P. Schacht 101, D. Schaefer 30, R. Schaefer 42, S. Schaepe 21, S. Schaetzel 58b, U. Schäfer 83, A.C. Schaffer 117, D. Schaile 100, R.D. Schamberger 150, V. Scharf 58a, V.A. Schegelsky 123, D. Scheirich 129, M. Schernau 165, M.I. Scherzer 35, C. Schiavi 50a,50b, J. Schieck 100, C. Schillo 48, M. Schioppa 37a,37b, S. Schlenker 30, E. Schmidt 48, K. Schmieden 30, C. Schmitt 83, S. Schmitt 58b, B. Schneider 17, Y.J. Schnellbach 74, U. Schnoor 44, L. Schoeffel 138, A. Schoening 58b, B.D. Schoenrock 90, A.L.S. Schorlemmer 54, M. Schott 83, D. Schouten 161a, J. Schovancova 25, S. Schramm 160, M. Schreyer 176, C. Schroeder 83, N. Schuh 83, M.J. Schultens 21, H.-C. Schultz-Coulon 58a, H. Schulz 16, M. Schumacher 48, B.A. Schumm 139, Ph. Schune 138, C. Schwanenberger 84, A. Schwartzman 145, T.A. Schwarz 89, Ph. Schwegler 101, Ph. Schwemling 138, R. Schwienhorst 90, J. Schwindling 138, T. Schwindt 21, M. Schwoerer 5, F.G. Sciacca 17, E. Scifo 117, G. Sciolla 23, W.G. Scott 131, F. Scuri 124a,124b, F. Scutti 21, J. Searcy 89, G. Sedov 42, E. Sedykh 123, S.C. Seidel 105, A. Seiden 139, F. Seifert 128, J.M. Seixas 24a, G. Sekhniaidze 104a, S.J. Sekula 40, K.E. Selbach 46, D.M. Seliverstov 123,∗, G. Sellers 74, N. Semprini-Cesari 20a,20b, C. Serfon 30, L. Serin 117, L. Serkin 54, T. Serre 85, R. Seuster 161a, H. Severini 113, T. Sﬁligoj 75, F. Sforza 101, A. Sfyrla 30, E. Shabalina 54, M. Shamim 116, L.Y. Shan 33a, R. Shang 167, J.T. Shank 22, M. Shapiro 15, P.B. Shatalov 97, K. Shaw 166a,166b, C.Y. Shehu 151, P. Sherwood 78, L. Shi 153,ad, S. Shimizu 67, C.O. Shimmin 165, M. Shimojima 102, M. Shiyakova 65, A. Shmeleva 96, M.J. Shochet 31, D. Short 120, S. Shrestha 64, E. Shulga 98, M.A. Shupe 7, S. Shushkevich 42, P. Sicho 127, O. Sidiropoulou 156, D. Sidorov 114, A. Sidoti 134a, F. Siegert 44, Dj. Sijacki 13a, J. Silva 126a,126d, Y. Silver 155, D. Silverstein 145, S.B. Silverstein 148a, V. Simak 128, O. Simard 5, Lj. ATLAS Collaboration Simic 13a, S. Simion 117, E. Simioni 83, B. Simmons 78, R. Simoniello 91a,91b, M. Simonyan 36, P. Sinervo 160, N.B. Sinev 116, V. Sipica 143, G. Siragusa 176, A. Sircar 79, A.N. Sisakyan 65,∗, S.Yu. Sivoklokov 99, J. Sjölin 148a,148b, T.B. Sjursen 14, H.P. Skottowe 57, K.Yu. Skovpen 109, P. Skubic 113, M. Slater 18, T. Slavicek 128, M. Slawinska 107, K. Sliwa 163, V. Smakhtin 174, B.H. Smart 46, L. Smestad 14, S.Yu. Smirnov 98, Y. Smirnov 98, L.N. Smirnova 99,ae, O. Smirnova 81, K.M. Smith 53, M. Smizanska 72, K. Smolek 128, A.A. Snesarev 96, G. Snidero 76, S. Snyder 25, R. Sobie 171,j, F. Socher 44, A. Soffer 155, D.A. Soh 153,ad, C.A. Solans 30, M. Solar 128, J. Solc 128, E.Yu. Soldatov 98, U. Soldevila 169, A.A. Solodkov 130, A. Soloshenko 65, O.V. Solovyanov 130, V. Solovyev 123, P. Sommer 48, H.Y. Song 33b, N. Soni 1, A. Sood 15, A. Sopczak 128, B. Sopko 128, V. Sopko 128, V. Sorin 12, M. Sosebee 8, R. Soualah 166a,166c, P. Soueid 95, A.M. Soukharev 109,c, D. South 42, S. Spagnolo 73a,73b, F. Spanò 77, W.R. Spearman 57, F. Spettel 101, R. Spighi 20a, G. Spigo 30, L.A. Spiller 88, M. Spousta 129, T. Spreitzer 160, B. Spurlock 8, R.D. St. Denis 53,∗, S. Staerz 44, J. Stahlman 122, R. Stamen 58a, S. Stamm 16, E. Stanecka 39, R.W. Stanek 6, C. Stanescu 136a, M. Stanescu-Bellu 42, M.M. Stanitzki 42, S. Stapnes 119, E.A. Starchenko 130, J. Stark 55, P. Staroba 127, P. Starovoitov 42, R. Staszewski 39, P. Stavina 146a,∗, P. Steinberg 25, B. Stelzer 144, H.J. Stelzer 30, O. Stelzer-Chilton 161a, H. Stenzel 52, S. Stern 101, G.A. Stewart 53, J.A. Stillings 21, M.C. Stockton 87, M. Stoebe 87, G. Stoicea 26a, P. Stolte 54, S. Stonjek 101, A.R. Stradling 8, A. Straessner 44, M.E. Stramaglia 17, J. Strandberg 149, S. Strandberg 148a,148b, A. Strandlie 119, E. Strauss 145, M. Strauss 113, P. Strizenec 146b, R. Ströhmer 176, D.M. Strom 116, R. Stroynowski 40, A. Strubig 106, S.A. Stucci 17, B. Stugu 14, N.A. Styles 42, D. Su 145, J. Su 125, R. Subramaniam 79, A. Succurro 12, Y. Sugaya 118, C. Suhr 108, M. Suk 128, V.V. Sulin 96, S. Sultansoy 4d, T. Sumida 68, S. Sun 57, X. Sun 33a, J.E. Sundermann 48, K. Suruliz 141, G. Susinno 37a,37b, M.R. Sutton 151, Y. ATLAS Collaboration Spanò 77, W.R. Spearman 57, F. Spettel 101, R. Spighi 20a, G. Spigo 30, L.A. Spiller 88, M. Spousta 129, T. Spreitzer 160, B. Spurlock 8, R.D. St. Denis 53,∗, S. Staerz 44, J. Stahlman 122, R. Stamen 58a, S. Stamm 16, E. Stanecka 39, R.W. Stanek 6, C. Stanescu 136a, M. Stanescu-Bellu 42, M.M. Stanitzki 42, S. Stapnes 119, E.A. Starchenko 130, J. Stark 55, P. Staroba 127, P. Starovoitov 42, R. Staszewski 39, P. Stavina 146a,∗, P. Steinberg 25, B. Stelzer 144, H.J. Stelzer 30, O. Stelzer-Chilton 161a, H. Stenzel 52, S. Stern 101, G.A. Stewart 53, J.A. Stillings 21, M.C. Stockton 87, M. Stoebe 87, G. Stoicea 26a, P. Stolte 54, S. Stonjek 101, A.R. Stradling 8, A. Straessner 44, M.E. Stramaglia 17, J. Strandberg 149, S. Strandberg 148a,148b, A. Strandlie 119, E. Strauss 145, M. Strauss 113, P. Strizenec 146b, R. Ströhmer 176, D.M. Strom 116, R. Stroynowski 40, A. Strubig 106, S.A. Stucci 17, B. Stugu 14, N.A. Styles 42, D. Su 145, J. Su 125, R. Subramaniam 79, A. Succurro 12, Y. Sugaya 118, C. Suhr 108, M. Suk 128, V.V. Sulin 96, S. Sultansoy 4d, T. Sumida 68, S. Sun 57, X. Sun 33a, J.E. Sundermann 48, K. Suruliz 141, G. Susinno 37a,37b, M.R. Sutton 151, Y. Suzuki 66, M. Svatos 127, S. Swedish 170, M. Swiatlowski 145, I. Sykora 146a, T. Sykora 129, D. Ta 90, C. Taccini 136a,136b, K. Tackmann 42, J. Taenzer 160, A. Taffard 165, R. Taﬁrout 161a, N. Taiblum 155, H. Takai 25, R. 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Unel 165, F.C. Ungaro 48, Y. Unno 66, C. Unverdorben 100, D. Urbaniec 35, P. Urquijo 88, G. Usai 8, A. Usanova 62, L. Vacavant 85, V. Vacek 128, B. Vachon 87, N. Valencic 107, S. Valentinetti 20a,20b, A. Valero 169, L. Valery 34, S. Valkar 129, E. Valladolid Gallego 169, S. Vallecorsa 49, J.A. Valls Ferrer 169, W. Van Den Wollenberg 107, P.C. Van Der Deijl 107, R. van der Geer 107, H. van der Graaf 107, R. Van Der Leeuw 107, D. van der Ster 30, N. van Eldik 30, P. van Gemmeren 6, J. Van Nieuwkoop 144, I. van Vulpen 107, M.C. van Woerden 30, M. Vanadia 134a,134b, W. Vandelli 30, R. Vanguri 122, A. Vaniachine 6, P. Vankov 42, F. Vannucci 80, G. Vardanyan 179, R. Vari 134a, E.W. Varnes 7, T. Varol 86, D. Varouchas 80, A. Vartapetian 8, K.E. Varvell 152, F. Vazeille 34, T. Vazquez Schroeder 54, J. Veatch 7, F. Veloso 126a,126c, S. Veneziano 134a, A. Ventura 73a,73b, D. Ventura 86, M. Venturi 171, N. Venturi 160, A. Venturini 23, V. Vercesi 121a, M. Verducci 134a,134b, W. Verkerke 107, J.C. ATLAS Collaboration Vermeulen 107, A. Vest 44, M.C. Vetterli 144,e, O. Viazlo 81, I. Vichou 167, T. Vickey 147c,ah, O.E. Vickey Boeriu 147c, G.H.A. Viehhauser 120, S. Viel 170, R. Vigne 30, M. Villa 20a,20b, M. Villaplana Perez 91a,91b, E. Vilucchi 47, M.G. Vincter 29, V.B. Vinogradov 65, J. Virzi 15, I. Vivarelli 151, F. Vives Vaque 3, S. Vlachos 10, D. Vladoiu 100, M. Vlasak 128, A. Vogel 21, M. Vogel 32a, P. Vokac 128, G. Volpi 124a,124b, M. Volpi 88, H. von der Schmitt 101, H. von Radziewski 48, E. von Toerne 21, V. Vorobel 129, K. Vorobev 98, M. Vos 169, R. Voss 30, J.H. Vossebeld 74, N. Vranjes 138, M. Vranjes Milosavljevic 13a, V. Vrba 127, M. Vreeswijk 107, T. Vu Anh 48, R. Vuillermet 30, I. Vukotic 31, Z. Vykydal 128, P. Wagner 21, W. Wagner 177, H. Wahlberg 71, S. Wahrmund 44, J. Wakabayashi 103, J. Walder 72, R. Walker 100, W. Walkowiak 143, R. Wall 178, P. Waller 74, B. Walsh 178, C. Wang 153,ai, C. Wang 45, F. Wang 175, H. Wang 15, H. Wang 40, J. Wang 42, J. Wang 33a, K. Wang 87, R. Wang 105, S.M. Wang 153, T. Wang 21, X. Wang 178, C. Wanotayaroj 116, A. Warburton 87, C.P. Ward 28, D.R. Wardrope 78, M. Warsinsky 48, A. Washbrook 46, C. Wasicki 42, P.M. Watkins 18, A.T. Watson 18, I.J. Watson 152, M.F. Watson 18, G. Watts 140, S. Watts 84, B.M. Waugh 78, S. Webb 84, M.S. Weber 17, S.W. Weber 176, J.S. Webster 31, A.R. Weidberg 120, P. Weigell 101, B. Weinert 61, J. Weingarten 54, C. Weiser 48, H. Weits 107, P.S. Wells 30, T. Wenaus 25, D. Wendland 16, Z. Weng 153,ad, T. Wengler 30, S. Wenig 30, N. Wermes 21, M. Werner 48, P. Werner 30, M. Wessels 58a, J. Wetter 163, K. Whalen 29, A. White 8, M.J. White 1, R. White 32b, S. White 124a,124b, D. Whiteson 165, D. Wicke 177, F.J. Wickens 131, W. Wiedenmann 175, M. Wielers 131, P. Wienemann 21, C. Wiglesworth 36, L.A.M. Wiik-Fuchs 21, P.A. Wijeratne 78, A. Wildauer 101, M.A. Wildt 42,aj, H.G. Wilkens 30, J.Z. Will 100, H.H. Williams 122, S. Williams 28, C. Willis 90, S. Willocq 86, A. Wilson 89, J.A. Wilson 18, I. ATLAS Collaboration Wingerter-Seez 5, F Wi kl i 116 B T Wi t 21 M Witt 145 T Witti 43 J Wittk ki 100 S J W ll t dt 83 J.P. Thomas , J. Thomas Wilsker , E.N. Thompson , P.D. Thompson , P.D. Thompson , R.J. Thompson 84, A.S. Thompson 53, L.A. Thomsen 36, E. Thomson 122, M. Thomson 28, W.M. Thong 88, R.P. Thun 89,∗, F. Tian 35, M.J. Tibbetts 15, V.O. Tikhomirov 96,af , Yu.A. Tikhonov 109,c, S. Timoshenko 98, E. Tiouchichine 85, P. Tipton 178, S. Tisserant 85, T. Todorov 5, S. Todorova-Nova 129, B. Toggerson 7, J. Tojo 70, S. Tokár 146a, K. Tokushuku 66, K. Tollefson 90, E. Tolley 57, L. Tomlinson 84, M. Tomoto 103, L. Tompkins 31, K. Toms 105, N.D. Topilin 65, E. Torrence 116, H. Torres 144, E. Torró Pastor 169, J. Toth 85,ag, F. Touchard 85, D.R. Tovey 141, H.L. Tran 117, T. Trefzger 176, L. Tremblet 30, A. Tricoli 30, I.M. Trigger 161a, S Trincaz Duvoid 80 M F Tripiana 12 W Trischuk 160 B Trocmé 55 C Troncon 91a L. Tompkins , K. Toms , N.D. Topilin , E. Torrence , H. Torres , E. Torró Pastor , J. Toth , F. Touchard 85, D.R. Tovey 141, H.L. Tran 117, T. Trefzger 176, L. Tremblet 30, A. Tricoli 30, I.M. Trigger 161a, S. Trincaz-Duvoid 80, M.F. Tripiana 12, W. Trischuk 160, B. Trocmé 55, C. Troncon 91a, M. Trottier-McDonald 15, M. Trovatelli 136a,136b, P. True 90, M. Trzebinski 39, A. Trzupek 39, C. Tsarouchas 30, J.C-L. Tseng 120, P.V. Tsiareshka 92, D. Tsionou 138, G. Tsipolitis 10, N. Tsirintanis 9, S. Tsiskaridze 12, V. Tsiskaridze 48, E.G. Tskhadadze 51a, I.I. Tsukerman 97, V. Tsulaia 15, S. Tsuno 66, D. Tsybychev 150, A. Tudorache 26a, V. Tudorache 26a, A.N. Tuna 122, S.A. Tupputi 20a,20b, S. Turchikhin 99,ae, D. Turecek 128, I. Turk Cakir 4c, R. Turra 91a,91b, A.J. Turvey 40, P.M. Tuts 35, A. Tykhonov 49, M. Tylmad 148a,148b, M. Tyndel 131, K. Uchida 21, I. Ueda 157, R. Ueno 29, M. Ughetto 85, M. Ugland 14, M. Uhlenbrock 21, F. Ukegawa 162, G. Unal 30, A. Undrus 25, G. Unel 165, F.C. Ungaro 48, Y. Unno 66, C. Unverdorben 100, D. Urbaniec 35, P. Urquijo 88, G. Usai 8, A. Usanova 62, L. Vacavant 85, V. Vacek 128, B. Vachon 87, N. Valencic 107, S. Valentinetti 20a,20b, A. ATLAS Collaboration Valero 169, L. Valery 34, S. Valkar 129, E. Valladolid Gallego 169, S. Vallecorsa 49, J.A. Valls Ferrer 169, W. Van Den Wollenberg 107, P.C. Van Der Deijl 107, R. van der Geer 107, H. van der Graaf 107, R. Van Der Leeuw 107, D. van der Ster 30, N. van Eldik 30, P. van Gemmeren 6, J. Van Nieuwkoop 144, I. van Vulpen 107, M.C. van Woerden 30, M. Vanadia 134a,134b, W. Vandelli 30, R. Vanguri 122, A. Vaniachine 6, P. Vankov 42, F. Vannucci 80, G. Vardanyan 179, R. Vari 134a, E.W. Varnes 7, T. Varol 86, D. Varouchas 80, A. Vartapetian 8, K.E. Varvell 152, F. Vazeille 34, T. Vazquez Schroeder 54, J. Veatch 7, F. Veloso 126a,126c, S. Veneziano 134a, A. Ventura 73a,73b, D. Ventura 86, M. Venturi 171, N. Venturi 160, A. Venturini 23, V. Vercesi 121a, M. Verducci 134a,134b, W. Verkerke 107, J.C. Vermeulen 107, A. Vest 44, M.C. Vetterli 144,e, O. Viazlo 81, I. Vichou 167, T. Vickey 147c,ah, O.E. Vickey Boeriu 147c, G.H.A. Viehhauser 120, S. Viel 170, R. Vigne 30, M. Villa 20a,20b, M. Villaplana Perez 91a,91b, E. Vilucchi 47, M.G. Vincter 29, V.B. Vinogradov 65, J. Virzi 15, I. Vivarelli 151, F. Vives Vaque 3, S. Vlachos 10, D. Vladoiu 100, M. Vlasak 128, A. Vogel 21, M. Vogel 32a, P. Vokac 128, G. Volpi 124a,124b, M. Volpi 88, H. von der Schmitt 101, H. von Radziewski 48, E. von Toerne 21, V. Vorobel 129, K. Vorobev 98, M. Vos 169, R. Voss 30, J.H. Vossebeld 74, N. Vranjes 138, M. Vranjes Milosavljevic 13a, V. Vrba 127, M. Vreeswijk 107, T. Vu Anh 48, R. Vuillermet 30, I. Vukotic 31, Z. Vykydal 128, P. Wagner 21, W. Wagner 177, H. Wahlberg 71, S. Wahrmund 44, J. Wakabayashi 103, J. Walder 72, R. Walker 100, W. Walkowiak 143, R. Wall 178, P. Waller 74, B. Walsh 178, C. Wang 153,ai, C. Wang 45, F. Wang 175, H. Wang 15, H. Wang 40, J. Wang 42, J. Wang 33a, K. Wang 87, R. Wang 105, S.M. Wang 153, T. Wang 21, X. 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Zaitsev 130,z, A. Zaman 150, S. Zambito 23, L. Zanello 134a,134b, D. Zanzi 88, C. Zeitnitz 177, M. Zeman 128, A. Zemla 38a, K. Zengel 23, O. Zenin 130, T. Ženiš 146a, D. Zerwas 117, G. Zevi della Porta 57, D. Zhang 89, F. Zhang 175, H. Zhang 90, J. Zhang 6, L. Zhang 153, X. Zhang 33d, Z. Zhang 117, Z. Zhao 33b, A. Zhemchugov 65, J. Zhong 120, B. Zhou 89, L. Zhou 35, N. Zhou 165, C.G. Zhu 33d, H. Zhu 33a, J. Zhu 89, Y. Zhu 33b, X. Zhuang 33 K. Zhukov 96, A. Zibell 176, D. Zieminska 61, N.I. Zimine 65, C. Zimmermann 83, R. Zimmermann 21, S. Zimmermann 21, S. Zimmermann 48, Z. Zinonos 54, M. Ziolkowski 143, G. Zobernig 175, A. Zoccoli 20a,20b M. zur Nedden 16, G. Zurzolo 104a,104b, V. Zutshi 108, L. Poland 39 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland 9 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Acade 40 Physics Department, Southern Methodist University, Dallas, TX, United States 40 Physics Department, Southern Methodist University, Dallas, TX, United States 41 Physics Department, University of Texas at Dallas, Richardson, TX, United States 41 Physics Department, University of Texas at Dallas, Richardson, TX 42 DESY, Hamburg and Zeuthen, Germany g y 43 Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 43 Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 43 Institut für Experimentelle Physik IV, Technische Universität Dortm 43 Institut für Experimentelle Physik IV, Tech 44 Institut für Kern- und Teilchenphysik, Technische Universität Dresde 44 Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Ge 45 Department of Physics, Duke University, Durham, NC, United States 45 Department of Physics, Duke University, Durham, NC, United States p f y y 46 SUPA – School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 46 SUPA – School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 46 SUPA – School of Physics and Astronomy, University of Edinburgh, f y y y f g g 47 INFN Laboratori Nazionali di Frascati, Frascati, Italy 47 INFN Laboratori Nazionali di Frascati, Frascati, Italy y 48 Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany 48 Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany 48 Fakultät für Mathematik und Physik, Albert-Ludwig 49 Section de Physique, Université de Genève, Geneva, Switzerland y q 50 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Università 50 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Università di Genova, Genova, Italy 50 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Università di Genova, Genova, Italy b ; p , , , y vili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi; (b) High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia stitut, Justus-Liebig-Universität Giessen, Giessen, Germany p y 51 (a) E. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi; (b) High Energy Physics Institute, Tbilisi Sta 1 (a) E. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State 51 (a) E. Andronikashvili Institute of Physics, Iv. ATLAS Collaboration Zwalinski 30 1 Department of Physics, University of Adelaide, Adelaide, Australia 1 Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany, NY, United States 3 Department of Physics, University of Alberta, Edmonton, AB, Canada Department of Physics, University of Alberta, Edmonton, AB, Canada 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Istanbul Aydin University, Istanbul; (d) Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey p f y y f 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ank p f y y f 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Istanbul Aydin University, Economics and Technology, Ankara, Turkey 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Istanb Economics and Technology Ankara Turkey 4 (a) Department of Physics, Ankara University, Ankara; (b) Departmen 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Istanbul Aydin University, Istanbul; (d) Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey Economics and Technology, Ankara, Turkey 5 LAPP, CNRS/IN2P3 and Université de Savoie, Annecy-le-Vieux, France 5 LAPP, CNRS/IN2P3 and Université de Savoie, Annecy-le-Vieux, France 6 High Energy Physics Division, Argonne National Laboratory, Argonne, IL, United States 7 6 High Energy Physics Division, Argonne National Laboratory, Argonne, IL, United Stat 7 Department of Physics, University of Arizona, Tucson, AZ, United States 7 Department of Physics, University of Arizona, Tucson, AZ, United States 8 Department of Physics, The University of Texas at Arlington, 9 Physics Department, University of Athens, Athens, Greece 10 Physics Department, National Technical University of Athens, Zog 10 Physics Department, National Technical University of Athens, Zografou, Greece 10 Physics Department, National Technical University of Athens, Zografou, Greece 11 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaija f y , j y f , , j 12 Institut de Física d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona, Barcelona, Spain 13 ( ) (b) f y j y f j 12 Institut de Física d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona, Barcelona, 12 Institut de Física d’Altes Energies and Departament de Física de la U 13 (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institute of Nuclear Sciences, University of Belgrade, Belgrade, Se 3 (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Insti lgrade; (b) Vinca Institute of Nuclear Sciences, University of Belgrade, Be f y y f g g f 14 Department for Physics and Technology, University of Bergen, Bergen, Norway 14 Department for Physics and Technology, University of Bergen, Bergen, Norway p f y gy y f g g y 15 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, CA, United States 15 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, CA, United States 15 Physics Division, Lawrence Berkeley National Laboratory and University of Califor 16 Department of Physics, Humboldt University, Berlin, Germany p f y , y, , y 17 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 18 17 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, Univ 17 Albert Einstein Center for Fundamental Physics and Laboratory 18 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 19 ( ) (b) 18 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom b f y y y f g g g 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics, Dogus University, Istanbul; (c) Department of Physics Engineering, Gaziantep University, Gaziantep, Turkey 20 ( ) (b) 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics, Dogus University, Istanbul; (c) Department of Physics E Turkey 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics, Dogus University, Istanbul; (c) Department of Physics Engineering, Gaziantep University, Gaziantep, Turkey y 20 (a) INFN Sezione di Bologna; (b) Dipartimento di Fisica e Astronomia, Università di Bologna, Bologna, Italy 21 20 (a) INFN Sezione di Bologna; (b) Dipartimento di Fisica e Astronomia, Università di Bologna, Bologna, Italy 21 Physikalisches Institut, University of Bonn, Bonn, Germany 22 22 Department of Physics, Boston University, Boston, MA, United States 22 Department of Physics, Boston University, Boston, MA, United States 23 Department of Physics, Brandeis University, Waltham, MA, United States 23 Department of Physics, Brandeis University, Waltham, MA, United States 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil 25 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Fe 4 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao Joao del Rei (UFSJ), Sao Joao d l (d) d d d d l l l 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil 25 Physics Department, Brookhaven National Laboratory, Upton, NY, United States ry, Upton, NY, United States ring, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, arest; (d) West University in Timisoara, Timisoara, Romania y p , y, p , , 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania 27 y p y p 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, l (c) l h h h (d) a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) N 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) N ( ) (d) f y g g f p f p Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania 27 Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania 7 Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Ar 28 Cavendish Laboratory, University of Cambridge, Cambridge, United 28 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingd 29 Department of Physics, Carleton University, Ottawa, ON, Canada 29 Department of Physics, Carleton University, Ottawa, ON, Canada 30 CERN, Geneva, Switzerland 31 Enrico Fermi Institute, University of Chicago, Chicago, IL, United States 31 Enrico Fermi Institute, University of Chicago, Chicago, IL, United States Enrico Fermi Institute, University of Chicago, Chicago, IL, United States 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, University of Science and Technology of China, Anhui; (c) Department of Physics, Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Physics Department, Shanghai Jiao Tong University, Shanghai; (f) Physics Department, T i h U i it B iji 100084 Chi 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Univers b 2 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Sa Physics, Nanjing University, Jiangsu; (d) S Tsinghua University, Beijing 100084, China rsité and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, Fra 34 Laboratoire de Physique Corpusculaire, Clermont Université and U 35 Nevis Laboratory, Columbia University, Irvington, NY, United States 35 Nevis Laboratory, Columbia University, Irvington, NY, United States 36 Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 36 Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 37 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frasca 37 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di Fisica, Università della Calabria, Rende, Italy 38 (a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow; (b) Marian Smoluchowski Institu Poland 38 (a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow; Poland 38 (a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow; (b) Marian Smoluchows Poland Poland Javakhishvili Tbilisi State University, Tbilisi; (b) 52 II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 52 II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 3 SUPA – School of Physics and Astronomy, University of Glasgow, Glasgo 53 SUPA – School of Physics and Astronomy, University of Glasgow, Glasgow, United King 54 II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany 54 II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany 55 Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS/IN2P3, Grenoble, France 55 Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS/IN2P3, Grenoble, France ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 Dodge Department of Physics and Astronomy, Univ 114 Department of Physics, Oklahoma State University, Stillwater, OK, United States 114 Department of Physics, Oklahoma State University, Stillwater, OK, United States p f y y 115 Palacký University, RCPTM, Olomouc, Czech Republic 115 Palacký University, RCPTM, Olomouc, Czech Republic 115 Palacký University, RCPTM, Olomouc, Czech Republic 116 Center for High Energy Physics, University of Oregon, Eugene, OR, United States 117 116 Center for High Energy Physics, University of Oregon, Eugene, OR, United States 117 LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France 118 Graduate School of Science, Osaka University, Osaka, Japan 119 Department of Physics, University of Oslo, Oslo, Norway 119 Department of Physics, University of Oslo, Oslo, Norway 0 Department of Physics, Oxford University, Oxford, United Kingdom 120 Department of Physics, Oxford University, Oxford, United Kingdom p f y f y f g 121 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università di Pavia, Pavia, Italy 121 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università di Pavia, Pavia, Italy 122 Department of Physics, University of Pennsylvania, Philadelphia, PA, United States 122 Department of Physics, University of Pennsylvania, Philadelphia, PA, United States 123 Petersburg Nuclear Physics Institute, Gatchina, Russia 124 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy 124 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy 125 124 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 Lebedev Institute of Physics, Academy of Sciences, Moscow, Russia 96 P.N. Lebedev Institute of Physics, Academy of Sciences, Moscow, Russia 97 Institute for Theoretical and Experimental Physics (ITEP), Moscow 98 National Research Nuclear University MEPhI, Moscow, Russia 99 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia 99 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Mosco 99 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov M 100 Fakultät für Physik, Ludwig-Maximilians-Universität München, Mü 100 Fakultät für Physik, Ludwig-Maximilians-Universität München, München f y g y 101 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 102 Nagasaki Institute of Applied Science, Nagasaki, Japan f y g y y g y J p 104 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Università di Napoli, Napoli, Italy 105 Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, United States 5 Department of Physics and Astronomy, University of New Mexico, Albu 106 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands 106 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, 107 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherla 107 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 108 Department of Physics, Northern Illinois University, DeKalb, IL, United States 108 Department of Physics, Northern Illinois University, DeKalb, IL, United States 109 Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 9 Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 110 Department of Physics, New York University, New York, NY, United States 110 Department of Physics, New York University, New York, NY, United States 111 Ohio State University, Columbus, OH, United States y 112 Faculty of Science, Okayama University, Okayama, Japan 112 Faculty of Science, Okayama University, Okayama, Japan 112 Faculty of Science, Okayama University, Okayama, Japan 113 y f , y y, y , J p 113 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK, United States 114 113 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK, United Stat 113 Homer L. ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 251 56 Department of Physics, Hampton University, Hampton, VA, United States 57 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, MA, United States 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c) ZITI Institut für technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany 59 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 60 (a) Department of Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong; (b) Department of Physics, The University of Hong Kong, Hong Kong; (c) Department of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 61 Department of Physics, Indiana University, Bloomington, IN, United States 62 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 63 f d 56 Department of Physics, Hampton University, Hampton, VA, United States 56 Department of Physics, Hampton University, Hampton, VA, United States 57 Laboratory for Particle Physics and Cosmology, Harvard University, Ca 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisc 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisc ff f y , p g, g technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany ff f y p g g technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany Faculty of Applied Information Science, Hiroshima Institute of Technolog 60 (a) Department of Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong; (b) Department of Phys 60 (a) Department of Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong; (b) Dep The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, epartment of Physics, Indiana University, Bloomington, IN, United States 61 Department of Physics, Indiana University, Bloomington, IN, United St 62 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 62 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Au Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, In 63 University of Iowa, Iowa City, IA, United States y f , y, , 64 Department of Physics and Astronomy, Iowa State University, Ames, IA, United States 64 Department of Physics and Astronomy, Iowa State University, Ame 65 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 65 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 66 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 66 KEK, High Energy Accelerator Research Organization, Tsukuba, 67 Graduate School of Science, Kobe University, Kobe, Ja 68 Faculty of Science, Kyoto University, Kyoto, Japan 68 Faculty of Science, Kyoto University, Kyoto, Japan 69 Kyoto University of Education, Kyoto, Japan y y f y J p 70 Department of Physics, Kyushu University, Fukuoka, Japan 70 Department of Physics, Kyushu University, Fukuoka, Japan p f y y y J p 71 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 71 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 71 Instituto de Física La Plata, Universidad Nacional de La Plat 72 Physics Department, Lancaster University, Lancaster, United Kingdom 72 Physics Department, Lancaster University, Lancaster, United Kingdom y p y g 73 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 73 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lec 74 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 75 Department of Physics, Jožef Stefan Institute and University of Ljublja 75 Department of Physics, Jožef Stefan Institute and University of Ljubljana, Ljub 76 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 76 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 77 Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 78 Department of Physics and Astronomy, University Co 79 Louisiana Tech University, Ruston, LA, United States 80 Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France 81 80 Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Par 81 Fysiska institutionen, Lunds universitet, Lund, Sweden 81 Fysiska institutionen, Lunds universitet, Lund, Sweden 82 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 3 82 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 82 Departamento de Fisica Teorica C-15, Universidad Auto 83 Institut für Physik, Universität Mainz, Mainz, Germany f y y 84 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 84 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 84 School of Physics and Astronomy, University of Manchester, Manch 85 CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 85 CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 86 Department of Physics, University of Massachusetts, Amherst, MA, 87 Department of Physics, McGill University, Montreal, QC, Canada 87 Department of Physics, McGill University, Montreal, QC, Canada 88 School of Physics, University of Melbourne, Victoria, Australia 88 School of Physics, University of Melbourne, Victoria, Australia 89 Department of Physics, The University of Michigan, Ann Arbor, M 90 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, United States 90 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, United S 91 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Università di Milano, Milano, Italy 93 National Scientiﬁc and Educational Centre for Particle and High Energy Physics, Minsk, Belaru 94 Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, United States Group of Particle Physics, University of Montreal, Montreal, QC, Canada 96 P.N. ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 252 131 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 131 Particle Physics Department, Rutherford Appleton Laboratory, Didc 132 Physics Department, University of Regina, Regina, SK, Canada 133 Ritsumeikan University, Kusatsu, Shiga, Japan y, , g , J p 134 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy 134 (a) INFN Sezione di Roma; (b) Dipartimento di F 134 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Univ 134 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy p p INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Universit b 136 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Un 136 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy INFN Sezione di Roma Tre; Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy 137 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des sciences Université Mohammed V-Agdal Rabat Morocco 137 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des 137 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques 137 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassan Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad sciences, Université Mohammed V-Agdal, Rabat, Morocco g M/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et aux Energies Alternat g 138 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France 139 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, United States 138 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay 138 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France 139 138 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France Santa Cruz Institute for Particle Physics, University of California Santa C 140 Department of Physics, University of Washington, Seattle, WA, United States 140 Department of Physics, University of Washington, Seattle, WA, United States 141 Department of Physics and Astronomy, University of Sheﬃeld, Sheﬃeld, United K 141 Department of Physics and Astronomy, University of Sheﬃel 141 Department of Physics and Astronomy, University of Sheﬃeld, Sheﬃeld, United Kingdom p f y y y f ﬃ ﬃ g 142 Department of Physics, Shinshu University, Nagano, Japan 142 Department of Physics, Shinshu University, Nagano, Japan 143 Fachbereich Physik, Universität Siegen, Siegen, Germany 144 Department of Physics, Simon Fraser University, Burnaby, BC, Cana p f y y y 145 SLAC National Accelerator Laboratory, Stanford, CA, United States SLAC National Accelerator Laboratory, Stanford, CA, United States 146 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 4 ( ) (b) ( ) 146 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 146 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Exp Sciences, Kosice, Slovak Republic 46 (a) Faculty of Mathematics, Physics & Informatics, Comenius Universi ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 Fe 125 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United States 125 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United States 126 (a) Laboratorio de Instrumentacao e Fisica Experimental de Particulas - LIP Lisboa; (b) Faculda Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United States 126 (a) Laboratorio de Instrumentacao e Fisica Experimental de Particulas - LIP, Lisboa; (b) Faculdade de Ciências, Universidade de Lisboa, Lisboa; (c) Department of Physics, University of Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Fisica, Universidade do Minho, Braga; (f) Departamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 127 Institute of Physics Academy of Sciences of the Czech Republic Praha Czech Republic p Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Fisica, Universidade do Minho, Bra Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departame p g p y Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Uni Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of F 127 Institute of Physics, Academy of Sciences of the Czech Republic, P 128 Czech Technical University in Prague, Praha, Czech Republic 128 Czech Technical University in Prague, Praha, Czech Republic 129 Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 129 Faculty of Mathematics and Physics, Charles University in Prag 129 Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 0 State Research Center Institute for High Energy Physics, Protvino, Russi 130 State Research Center Institute for High Energy Physics, Protvino, Rus Sciences, Kosice, Slovak Republic , , p 147 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, University of Johannesburg, Johannesburg; (c) Witwatersrand Johannesburg South Africa p 147 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics 147 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, University of Johannesburg 147 (a) Department of Physics, University of Cape Town, Cape Town; Witwatersrand, Johannesburg, South Africa 148 (a) Department of Physics, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Swed 148 (a) Department of Physics, Stockholm University; (b) The Oskar Klei 148 (a) Department of Physics, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Swede 148 (a) Department of Physics, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Sweden 149 Physics Department, Royal Institute of Technology, Stockholm, Sweden 149 Physics Department, Royal Institute of Technology, Stockholm, Sweden 150 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, NY, United States 150 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, NY, Unit 150 Departments of Physics & Astronomy and Chemistry, Stony Brook U p f y y y y y 151 Department of Physics and Astronomy, University of Sussex, Brighton, United 151 Department of Physics and Astronomy, University of Sussex 151 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 152 School of Physics, University of Sydney, Sydney, Australia 153 Institute of Physics, Academia Sinica, Taipei, Taiwan 154 Department of Physics, Technion: Israel Institute of Technology, Hai 154 Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel 155 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 156 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv 155 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 56 Department of Physics, Aristotle University of Thessaloniki, Thessalon p f y y f 157 International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan 157 International Center for Elementary Particle Physics and Department of Physics, The Universi 57 International Center for Elementary Particle Physics and Department 158 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 158 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 159 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 160 Department of Physics, University of Toronto, Toronto, ON, Canada p f y y f 161 (a) TRIUMF, Vancouver, BC; (b) Department of Physics and Astronomy, York University, Toronto, ON, Canada 161 (a) TRIUMF, Vancouver, BC; (b) Department of Physics and Astronomy, York University, Toronto, ON, Canada 161 (a) TRIUMF, Vancouver, BC; (b) Department of Physics and Astronom 162 Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukub 163 Department of Physics and Astronomy, Tufts University, Medford, MA, United States 163 Department of Physics and Astronomy, Tufts University, Medford, MA, United States 164 Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 65 Department of Physics and Astronomy, University of California Irvine, p f y y, y f f , , , 166 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento di Chimica, Fisica e Ambiente, Università 166 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento d 66 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP 167 Department of Physics, University of Illinois, Urbana, IL, United States 68 Department of Physics and Astronomy, University of Uppsala, Uppsala 169 Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecular y Nuclear and Departamento de Ingeniería Electrón 169 Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecular y Nuclear and Departamen 169 Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecular Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 170 Department of Physics, University of British Columbia, Vancouver, B 171 Department of Physics and Astronomy, University of Victoria, Victoria, BC, Canada 171 Department of Physics and Astronomy, University of Victoria, Victoria, BC, Canada 172 Department of Physics, University of Warwick, Coventry, United Kingdom 172 Department of Physics, University of Warwick, Coventry, Unite 172 Department of Physics, University of Warwick, Coventry, United Kingdom 173 Waseda University, Tokyo, Japan 173 Waseda University, Tokyo, Japan 174 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 174 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 175 Department of Physics, University of Wisconsin, Madison, WI, United State 75 Department of Physics, University of Wisconsin, Madison, WI, United 176 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 176 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 76 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, W 177 Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany 177 Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany 178 Department of Physics, Yale University, New Haven, CT, United States 178 Department of Physics, Yale University, New Ha 78 Department of Physics, Yale University, New Haven, CT, United States 179 Yerevan Physics Institute, Yerevan, Armenia 179 Yerevan Physics Institute, Yerevan, Armenia y 180 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France 180 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France a Also at Department of Physics, King’s College London, London, United Kingdom. a Also at Department of Physics, King’s College London, London, United Kingdom. b Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. b Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. c Also at Novosibirsk State University, Novosibirsk, Russia. c Also at Novosibirsk State University, Novosibirsk, Russia. d Also at Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom. d Also at Particle Physics Department, Rutherford Appleton d Also at Particle Physics Department, Rutherford Appleton Laboratory, Didcot, Unite d Also at Particle Physics Department, Rutherford Appleton L e Also at TRIUMF, Vancouver, BC, Canada. e Also at TRIUMF, Vancouver, BC, Canada. ment of Physics, California State University, Fresno, CA, United Stat f Also at Department of Physics, California State University, Fresn g Also at Tomsk State University, Tomsk, Russia. h Also at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. h Also at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. i Also at Università di Napoli Parthenope, Napoli, Italy. i Also at Università di Napoli Parthenope, Napoli, Italy. j Also at Institute of Particle Physics (IPP), Canada. j Also at Institute of Particle Physics (IPP), Canada. k Also at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Russia. k Also at Department of Physics, St. Petersburg State Polytechnical University, St. 173 Waseda University, Tokyo, Japan Petersburg, Russ k Also at Department of Physics, St. Petersburg Stat l Also at Department of Financial and Management Engineering, University of the Aegean, Chios, Greece. l Also at Department of Financial and Management Engineerin l Also at Department of Financial and Management Engineering, University of the Aegean, Chios, Gr m Also at Louisiana Tech University, Ruston, LA, United States. m Also at Louisiana Tech University, Ruston, LA, United States. n Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain. n Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spai o Also at Department of Physics, The University of Texas at Austin, Austin, TX, United o Also at Department of Physics, The University of Texas at Austin, Austin, TX, United States. p Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. p Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgi p Also at Institute of Theoretical Physics, Ilia State University, T q Also at CERN, Geneva, Switzerland. r Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan. r Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan. s Also at Manhattan College, New York, NY, United States. t Also at Institute of Physics, Academia Sinica, Taipei, Taiwan. t Also at Institute of Physics, Academia Sinica, Taipei, Taiwan. u Also at LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France. u Also at LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France. 253 ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 ATLAS Collaboration / Physics Letters B 738 (2014) 234–253 v Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan. v Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan. y q g x Also at School of Physical Sciences, National Institute of Science Education and Research, Bhubaneswar, India. x Also at School of Physical Sciences, National Institute of Science Education and Research, Bhuba y Also at Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy. y Also at Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy. aa Also at Section de Physique, Université de Genève, Geneva, Switzerland. aa Also at Section de Physique, Université de Genève, Geneva, Switzerland. ab Also at International School for Advanced Studies (SISSA), Trieste, Italy. ab Also at International School for Advanced Studies (SISSA), Trieste, Italy. ad Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China. ad Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China. ae Also at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia. ae Also at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia. af Also at National Research Nuclear University MEPhI, Moscow, Russia. Also at National Research Nuclear University MEPhI, Moscow, Russia. ag Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. ag Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hu ah Also at Department of Physics, Oxford University, Oxford, United Kingdom. ah Also at Department of Physics, Oxford University, Oxford, United Kingdom. ai Also at Department of Physics, Nanjing University, Jiangsu, China. ai Also at Department of Physics, Nanjing University, Jiangsu, China. aj Also at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Ger ak Also at Department of Physics, The University of Michigan, Ann Arbor, MI, United States ak Also at Department of Physics, The University of Michigan, Ann Arbor, MI, United Stat al Also at Discipline of Physics, University of KwaZulu-Natal, Durban, South Africa. al Also at Discipline of Physics, University of KwaZulu-Natal, Durban, South Africa. am Also at University of Malaya, Department of Physics, Kuala Lumpur, Malaysia. am Also at University of Malaya, Department of Physics, Kuala Lumpur, Malaysia. ∗Deceased.
https://openalex.org/W3001705452	https://www.business-inform.net/export_pdf/business-inform-2019-11_0-pages-234_240.pdf	Ukrainian	null	Planning Dividends in the Enterprise	Bìznes ìnform/Bìznes ìnform	2,019	cc-by-sa	5,066	REFERENCES Paientko, T. V., and Fedosov, V. M. “Upravlinnia finansamy: kontrolinh na makrorivni“ [Financial Management: Controlling at the Micro Level]. Finansy Ukrainy, no. 3 (2018): 80-96. Babo, A. Pribyl [Profit]. Moscow: Progress, 1993. Babo, A. Pribyl [Profit]. Moscow: Progress, 1993. Blynda, Yu. O. “Investytsiina stratehiia yak element an tykryzovoho upravlinnia pidpryiemstvom“ [Investment Stra tegy as an Element of Enterprise Crisis Management]: dys. … kand. ekon. nauk : 08.00.04, 2019. Plankett, L., and Kheyl, G. Vyrabotka i prinyatiye uprav lencheskikh resheniy [Development and Adoption of Manage ment Decisions]. Moscow: Ekonomika, 1984. Melikhova, T. O. “Mekhanizm upravlinnia ekonomichnoiu bezpekoiu pidpryiemstva: pidkhody do vyznachennia“ [Mecha- Utkin, E. A. Antikrizisnoye upravleniye [Crisis Manage ment]. Moscow: EKMOS, 1997. Utkin, E. A. Antikrizisnoye upravleniye [Crisis Manage ment]. Moscow: EKMOS, 1997. УДК 658.147 JEL: G35 УДК 658.147 JEL: G35 Ситник Г. В., Рибак Р. І. Планування дивідендів на підприємстві Метою статті є обґрунтування принципів та методичного підходу до планування дивідендів на підприємстві, які б забезпечували його збалансо ваний фінансовий розвиток, сприяли зростанню інвестиційної привабливості та ринкової вартості. У статті розглянуто основні концепції пла нування дивідендів, що сформувались у класичній економічній теорії. Прояснено позицію та бажання різних типів акціонерів щодо виплати або ка піталізації дивідендів. Виокремлено принципи планування дивідендів та запропоновано послідовність їх планування, виходячи з даних принципів. Запропоновано здійснювати планування обсягу дивідендних виплат, які може дозволити собі підприємство, виходячи з потреб операційної та інвестиційної діяльності на основі показника «рівень реінвестування чистого грошового потоку від операційної діяльності». Припустимий обсяг дивідендів обґрунтовується виходячи з цього показника та планових показників чистого грошового потоку від операційної діяльності, приросту основних засобів, нематеріальних активів, незавершених капітальних інвестицій та чистого робочого капіталу. Коефіцієнт дивідендних виплат і планову суму дивідендів пропонується визначати виходячи з необхідності підтримання запланованого обсягу діяльності підприємства на основі моделі стійкого зростання. Остаточне рішення щодо обґрунтування обсягу дивідендів приймається в межах отриманої області значень плано вих дивідендних виплат. Перспективами подальших досліджень у даному напрямі передбачається планування дивідендів на багатокритеріальній основі, що зводиться до обґрунтування їх обсягів за критеріями: максимізації задоволення фінансових інтересів власників, що віддають перевагу поточним доходам; забезпечення стійкого зростання; збереження фінансової рівноваги; мінімізації середньозваженої вартості капіталу. Також проводитиметься пошук цільового значення дивідендів шляхом імітаційного моделювання у визначених межах. ЕКОНОМІКА ПІДПРИЄМСТВА ЕКОНОМІКА ЕКОНОМІКА ПІДПРИЄМСТВА Ключові слова: дивіденди, дивідендні виплати, рівень реінвестування чистого грошового потоку, коефіцієнт дивідендних виплат, область зна чень планової суми дивідендних виплат. DOI: https://doi.org/10.32983/2222-4459-2019-11-234-240 Ситник Ганна Вікторівна – доктор економічних наук, доцент, професор кафедри економіки та фінансів підприємства, Київський національний торговельно-економічний університет (вул. Кіото, 19, Київ, 02156, Україна) E-mail: anna_sytnik@bigmir.net Рибак Руслана Іванівна – старший викладач кафедри економіки та фінансів підприємства, Київський національний торговельно-економічний університет (вул. Кіото, 19, Київ, 02156, Україна) ЕКОНОМІКА 6. Бабо А. Прибыль / пер. с франц. М. : Прогресс, 1993. 176 с. 6. Бабо А. Прибыль / пер. с франц. М. : Прогресс, 1993. 176 с. nism of Economic Security Management at the Enterprise: Ap proaches to Definition]. Ahrosvit, no. 3 (2018): 61-67. Oleshko, A. A. “Mekhanizm antykryzovoho upravlinnia fi nansovymy korporatsiiamy“ [The Mechanism of Crisis Manage ment of Financial Corporations]. Efektyvna ekonomika. 2018. http://www.economy.nayka.com.ua/pdf/2_2018/12.pdf 7. Олешко А. А. Механізм антикризового управління фінансовими корпораціями. Ефективна економіка. 2018. № 2. URL: http://www.economy.nayka.com.ua/pdf/2_2018/12.pdf 8. Блинда Ю. О. Інвестиційна стратегія як елемент ан тикризового управління підприємством : дис. … канд. екон. наук : 08.00.04. Львів, 2019. 207 с. Oleshko, A. A. “Prohramno-tsilyovyi pidkhid do antykry zovoho upravlinnia finansovymy korporatsiiamy“ [Program- Target Approach to Crisis Management of Financial Corpora tions]. Ekonomika ta derzhava, no. 6 (2018): 25-27. Sytnyk H. V., Rybak R. I. Planning Dividends in the Enterprise y y , y g p The article is aimed at substantiating the principles and a methodical ap proach to planning dividends in the enterprise, which would provide ability to ensure the enterprise’s balanced financial development, would facilitate a growth of investment attractiveness and market value. The article considers the basic concepts of planning dividends, which were formed in the classical economic theory. The position and desire of different types of sharehold УДК 658.147 JEL: G35 Sytnyk H. V., Rybak R. I. Planning Dividends in the Enterpriseiii Сытник А. В., Рыбак Р. И. Планирование дивидендов на предприятии Целью статьи является обоснование принципов и методического под хода к планированию дивидендов на предприятии, которые смогли бы обеспечить его сбалансированное финансовое развитие, способство вали бы росту инвестиционной привлекательности и рыночной сто имости. В статье рассмотрены основные концепции планирования дивидендов, которые сформировались в классической экономической теории. Прояснена позиция и желание разных типов акционеров от БІЗНЕСІНФОРМ № 11 ’2019 Перспективами последующих исследований в данном направлении являются углубленное планирова ние дивидендов на многокритериальной основе, которая сводится к обоснованию их объемов по критериям: максимизации удовлетворения финансовых интересов владельцев, которые отдают преимущество текущим доходам; обеспечение стойкого роста; сохранение финансо вого равновесия; минимизации средневзвешенной стоимости капита ла. Также предусмотрен поиск целевого значения дивидендов путем имитационного моделирования в определенных пределах. Keywords: dividends, dividend payments, level of reinvestment of net money flow, ratio of dividend payments, area of values of the planned amount of dividend payments. Ключевые слова: дивиденды, дивидендные выплаты, уровень реинве стирования чистого денежного потока, коэффициент дивидендных выплат, область значений плановой суммы дивидендных выплат. Рис.: 1. Табл.: 1. Формул: 5. Библ.: 13. Fig.: 1. Tabl.: 1. Formulae: 5. Bibl.: 13. Fig.: 1. Tabl.: 1. Formulae: 5. Bibl.: 13. Сытник Анна Викторовна – доктор экономических наук, доцент, про фессор кафедры экономики и финансов предприятия, Киевский наци ональный торгово-экономический университет (ул. Киото, 19, Киев, 02156, Украина) Sytnyk Hanna V. – D. Sc. (Economics), Associate Professor, Professor of the Department of Economics and Finance of Company, Kyiv National University of Trade and Economics (19 Kіoto Str., Kyiv, 02156, Ukraine) E-mail: anna_sytnik@bigmir.net Rybak Ruslana I. – Senior Lecturer of the Department of Economics and Fi nance of Company, Kyiv National University of Trade and Economics (19 Kіoto Str., Kyiv, 02156, Ukraine) E il l b k25@ il Рыбак Руслана Ивановна – старший преподаватель кафедры эконо мики и финансов предприятия, Киевский национальный торгово-эко номический университет (ул. Киото, 19, Киев, 02156, Украина) E-mail: ruslanarybak25@gmail.com E-mail: ruslanarybak25@gmail.com ЕКОНОМІКА ЕКОНОМІКА ПІДПРИЄМСТВА ЕКОНОМІКА ПІДПРИЄМСТВА МІКА ЕКОНОМІКА ПІДПРИЄМСТВА Е крема імплементації плану BEPS [2], підвищення рів ня інвестиційної активності підприємств, а з іншого – розроблення дієвого механізму та виваженої диві дендної політики окремих підприємств. Е кономічний розвиток будь-якої економічної системи потребує нагромадження капіталу та його ефективного інвестування. Це вимагає раціонального розподілу прибутку між споживаною та капіталізованою частинами як на рівні підприєм ства, так і в масштабах економіки. Однією з проблем економічного розвитку України, його низьких темпів є «проїдання» капіталу, високі обсяги споживання та низький рівень нагромадження. Так, за даними ста тистичних спостережень, впродовж 2016–2018 рр. рівень капіталізації прибутку в основних галузях еко номіки становив у середньому 2,4% [1], що свідчить про хронічну нестачу власних фінансових ресурсів для повноцінного фінансування діяльності підпри ємств реального сектора економіки. Це призводить до зростання позик і, як наслідок, – відпливу ресурсів за каналом доходів від власності. БІЗНЕСІНФОРМ № 11 ’2019 БІЗНЕСІНФОРМ № 11 ’2019 234 www.business-inform.net www.business-inform.net ers regarding the payments or capitalization of dividends are clarified. The principles of planning dividends are allocated and a coherence of planning dividends is proposed based on these principles. It is proposed to plan the amount of dividend payments that an enterprise can afford according to the needs of operating and investment activities on the basis of the indicator of the «level of reinvestment of the net money flow from operating activi ties». A substantiation of the allowable amount of dividends is based on this indicator and the planned indicators of the net money flow from operating activities, growth of fixed assets, non-material assets, unfinished capital in vestments and net working capital. The dividend payments ratio and the planned amount of dividends are proposed to define on the basis of the need to ensure the planned volume of the enterprise’s activities on the basis of a sustainable growth model. The final decision as to the substantiation of the amount of dividends is made within the obtained area of the values of planned dividend payments. Prospects for further researches in this direc tion are in-depth planning of dividends on a multi-criteria basis, which is reduced to substantiating their volumes according to the following criteria: maximizing the satisfaction of the financial interests of owners that give an advantage to the current income; ensuring the sustainable growth; preserv ing the financial equilibrium; minimizing the weighted average cost of capi tal. Also it is provisioned to find the target value of dividends by carrying out simulation modeling within certain limits. носительно выплаты или капитализации дивидендов. Выделены прин ципы планирования дивидендов и предложена последовательность их планирования, исходя из данных принципов. Предложено осуществлять планирование объема дивидендных выплат, которые может позво лить себе предприятие, исходя из потребностей операционной и инве стиционной деятельности на основе показателя «уровень реинвести рования чистого денежного потока от операционной деятельности». Допустимый объем дивидендов обосновывается исходя из этого пока зателя и плановых показателей чистого денежного потока от опера ционной деятельности, прироста основных средств, невещественных активов, незавершенных капитальных инвестиций и чистого рабочего капитала. Коэффициент дивидендных выплат и плановую сумму диви дендов предлагается определять исходя из необходимости обеспечения запланированного объема деятельности предприятия на основе моде ли устойчивого роста. Окончательное решение относительно обосно вания объема дивидендов принимается в пределах полученной области значений плановых дивидендных выплат. Дослідження проведено в межах НДР «Формування системи економічної безпеки підприємництва в Україні» (Державний реє страційний № 0117U000504), що виконується на замовлення МОН України. БІЗНЕСІНФОРМ № 11 ’2019 Вирішення пробле ми потребує, з одного боку, формування державної політики стимулювання нагромадження капіталу, зо Формування дивідендної політики є однією з центральних проблем фінансового менеджменту. Її вивченню приділяла увагу ціла низка закордон них дослідників, у роботах яких проаналізовано взаємозв'язок рівня дохідності власного капіталу та рівня дивідендних виплат [3], вплив дивідендної полі тики на ринкову ціну підприємства [4], взаємозв'язок дивідендної політики та політики формування капі талу [5]. Сформульовані ними висновки вважаються такими, що становлять сучасну парадигму фінансо вого менеджменту і, безумовно, служитимуть вихід ним базисом нашого дослідження. ЕКОНОМІКА У роботах вітчизняних дослідників: І. О. Бланка, Н. С. Акімової, О. О. Терещенка, А. Б. Педька, Л. Є. Дов- ганя, Л. В. Фролової, Н. В. Харченка, Н. В. Юрчук, Л. М. Янчевої, К. О. Горової, І. В. Спільник, Л. О. Ме ренкової, Ю. М. Великого та інших розглядаються пи тання оптимізації дивідендної політики з урахуван ням етапу економічного розвитку, галузевих особли востей функціонування суб’єктів господарювання. Дослідження проведено в межах НДР «Формування системи економічної безпеки підприємництва в Україні» (Державний реє страційний № 0117U000504), що виконується на замовлення МОН України. БІЗНЕСІНФОРМ № 11 ’2019 www.business-inform.net 235 www.business-inform.net ємства і, відповідно, впливати на динаміку інвести ційної діяльності – найбільш активного інструменту нарощення ринкової вартості підприємства. Таким чином, очевидним є оптимізаційний характер рішень щодо дивідендних виплат. Такі рішення повинні за безпечити узгодженість фінансових інтересів власни ків, які зацікавлені в поточних доходах, і власників, що орієнтуються переважно на приріст ринкової вар тості підприємства. Значна увага приділяється визначенню терміна «ди віденди», його видів та типів дивідендної політики [6; 7], Проводять аналіз співвідношення «дивіденди – нерозподілений прибуток» на провідних корпораціях [8], розглядають основні фактори, що впливають на формування дивідендної політики [9]. Проте проблема планування дивідендних ви плат у частині обґрунтування методологічних прин ципів та чітких методичних підходів наразі залиша ється недостатньо дослідженою та потребує подаль шого розроблення. Так, для багатьох акціонерів важливими є саме поточні доходи від дивідендів. Отже, для таких інвес торів привабливість і ціна акції залежатиме не лише від фінансових показників розвитку підприємства, а від сплачуваних дивідендів. З огляду на те, що роз мір та динаміка дивідендів впливає на курс акції, на практиці це сприяє підтриманню стабільних дивіден дів або їх нарощенню деякими підприємствами. Так, окремі публічні компанії нарощують дивіденди, коли відчувають спроможність і в перспективі сплачувати дивіденди ще в більших обсягах. Тому аналітики та інвестори розглядають зростання дивідендів як пози тивний сигнал про довгострокове зростання. БІЗНЕСІНФОРМ № 11 ’2019 Прин ципові зміни дивідендної політики можуть призво дити до появи у акціонерів додаткових трансакційних витрат. Ці аргументи стимулюють компанії дотриму ватись стабільності у своїй дивідендній політиці. Метою статті є обґрунтування принципів та методичного підходу до планування дивідендів під приємства, які б забезпечували його збалансований фінансовий розвиток, сприяли зростанню інвестицій ної привабливості та ринкової вартості підприємства. Дослідження ґрунтується на принципах сис темного підходу та здійснювалось із застосуванням методів наукової абстракції, аналізу й синтезу, систе матизації й узагальнення. У У сучасній теорії фінансового менеджменту сформовано декілька класичних підходів до планування дивідендних виплат, коротка ха рактеристика яких наведена в табл. 1. Незважаючи на те, що зазначені підходи вважа ються класичними і висвітлюються у базових підруч никах з фінансового менеджменту, переважна біль шість науковців сходяться на думці, що в чистому ви гляді ці моделі майже не застосовуються на практиці. Обумовлюється це мінливістю умов зовнішнього та внутрішнього середовища підприємства, що робить процес обґрунтування дивідендних виплат більш складним, ніж це описується зазначеними моделями. Іншим власникам вигідний дохід у формі при росту капіталу, у тому числі з податкових мотивів, що спонукає підприємства проводити активну ефектив ну інвестиційну діяльність, спрямовану на таке зрос тання. ЕКОНОМІКА ПІДПРИЄМСТВА ЕКОНОМІКА ЕКОНОМІКА ПІДПРИЄМСТВА Т Т аким чином, пошук оптимальної пропорції між прибутком, що капіталізується та спла чується у вигляді дивідендів, чинить вплив на розвиток підприємства і повинен здійснюватися на багатокритеріальній основі. Виходячи із сучасної парадигми фінансового менеджменту, в межах якої визначається, що осно вною метою функціонування підприємства, а, отже, і фінансового управління є зростання добробуту власників, формування дивідендної політики також підпорядковується цій меті. На наш погляд, можна виокремити такі принци пи планування дивідендів. 1. Принцип врахування кон’юнктури фінансо вого ринку. Так, рішення щодо обсягів, рівня диві дендних виплат має враховувати рівень дохідності акцій аналогічних підприємств на ринку, а також склад і структуру власників підприємства, їх фінан сові інтереси. 1. Принцип врахування кон’юнктури фінансо вого ринку. Так, рішення щодо обсягів, рівня диві дендних виплат має враховувати рівень дохідності акцій аналогічних підприємств на ринку, а також склад і структуру власників підприємства, їх фінан сові інтереси. Добробут власників підприємства формується з двох частин: поточних дивідендів та приросту рин кової вартості підприємства. Тому можна вважати, що дивідендна політика має забезпечувати максимі зацію сумарної вигоди для власників підприємства. 2. Принцип забезпечення зростання підпри ємства. Сприятлива кон’юнктура товарного ринку, наявність реальних можливостей для нарощування обсягів діяльності потребує інвестицій у розвиток, що пов’язано з необхідністю акумуляції достатнього обсягу фінансових ресурсів. БІЗНЕСІНФОРМ № 11 ’2019 Частина цих ресурсів забезпечується шляхом реінвестування чистого при бутку, що необхідно враховувати при плануванні об сягу дивідендних виплат. Так, максимізація поточних доходів власників забезпечується максимізацією дивідендних виплат. Проте приріст ринкової вартості підприємства, окрім заходів з оптимізації поточних витрат, удосконален ня управління окремими параметрами діяльності потребує реалізації активної та ефективної інвести ційної політики, реалізація якої пов’язана з пошуком і забезпеченням необхідного фінансування. Таким чином, дивідендна політика є невід’ємною частиною рішень щодо фінансування діяльності. Коефіцієнт дивідендних виплат визначатиме частину прибутку, що може бути реінвестована в діяльність підпри ЕКОНОМІКА 3. Принцип дотримання фінансової рівноваги підприємства. Основним джерелом виплати дивіден дів є чистий прибуток підприємства, який необхідно БІЗНЕСІНФОРМ № 11 ’2019 БІЗНЕСІНФОРМ № 11 ’2019 236 www.business-inform.net www.business-inform.net Таблиця 1 Концепції планування дивідендних виплат підприємства Концепція Коротка характеристика 1 2 Модель Лінтнера (1956) Дж. Лінтнер на основі дослідження виявив, що дивіденди коригуються відповідно до дина міки прибутку, але із запізненням, і першим провів аналіз моделі часткової адаптації для по яснення дивідендної політики. Ця модель передбачає встановлення коефіцієнта дивіденд них виплат, що дозволяє пов’язати обсяги дивідендних виплат з фінансовими результатами. На основі дослідження динаміки прибутку та коефіцієнта дивідендних виплат і застосування регресійного моделювання обчислюється поправочний коефіцієнт, на основі якого визнача ється коефіцієнт дивідендних виплат планового періоду, що дозволяє оцінити планову суму дивідендів Модель Майєрса (1984) С. Майєрс (1984) запропонував для пояснення дивідендної політики теорію послідовності дій. Логіка фінансування компанії полягає у такому: – підприємство віддає перевагу внутрішньому фінансуванню; – підприємства коригують плановий рівень дивідендів відповідно до інвестиційних можли востей. Рівень дивідендів поступово адаптується до змін інвестиційної активності; – результатом інертності дивідендної політики та непередбачуваних змін прибутковості та інвестиційних можливостей є те, що власних коштів може виявитися більше або менше інвестиційних потреб. У першому випадку надлишкові кошти спрямовуються у фонд нероз поділеного прибутку для майбутніх інвестиційних проектів, у іншому – підприємство вико ристовує накопичений нерозподілений прибуток; – коли необхідно залучати зовнішнє фінансування, компанії починають з емісії безпечних і дешевих цінних паперів – облігацій Модель стабільних дивідендів Дивідендна політика – спосіб інформування інвесторів; дивіденд – не просто виплата, а й інформаційний контент. Стабільність дивідендів – позитивний сигнал для ринку. Зменшення обсягів (або невиплата) дивідендів може сприйматись як погіршення фінансового стану під приємства, а частина інвесторів, що орієнтуються на поточний дохід (пенсіонери, пенсійні фонди) негативно сприймає подібну політику, що може призводити до падіння ціни акцій. БІЗНЕСІНФОРМ № 11 ’2019 Таким чином, цей підхід передбачає виплату стабільних обсягів дивідендів Модель екстрадивідендів Збільшити поточні доходи акціонерів в окремі періоди можна за допомогою додаткових ди відендів (екстрадивідендів). Проте, оголошуючи виплату екстра-дивідендів, підприємство не змінює обсяг регулярних дивідендів. Екстра-дивіденди сплачуються в окремі періоди, коли спостерігаються високі фінансові результати діяльності та відсутність цікавих інвестиційних проектів Джерело: складено за [9–11]. ЕКОНОМІКА ПІДПРИЄМСТВА ОНОМІКА ЕКОНОМІКА ПІДПРИЄМСТВА Джерело: складено за [9–11]. неприйнятним для підприємства, що необхідно вра ховувати в процесі обґрунтування обсягів дивіденд них виплат. «монетизувати» у вигляді чистого грошового потоку. Таким чином, обсяги виплачуваних дивідендів мають узгоджуватися з обсягом чистого грошового потоку від операційної діяльності. Його повинно вистачати як для виплати дивідендів, так і для фінансування по точних потреб операційної діяльності, підтримання необхідного обсягу та стану необоротних активів для ведення основної діяльності. В иходячи із зазначених принципів, процес пла нування дивідендів пропонуємо здійснювати в такій послідовності. В На першому етапі обґрунтовується бажана сума дивідендних виплат, виходячи з фінансових інтересів власників, що віддають перевагу поточним доходам і необхідності підтримки інвестиційної привабливості акцій на фондовому ринку (для публічних компаній) серед таких інвесторів. Планування бажаних обсягів дивідендних виплат здійснюється на основі: 4. Принцип дотримання цільової структури та вартості капіталу. Необхідність реалізації ін вестиційної програми та її фінансування може забез печуватись шляхом вибору альтернативних джерел залучення капіталу. Так, цільова потреба у власному капіталі може покриватися за рахунок чистого при бутку та додаткових внесків у статутний капітал (до даткової емісії акцій). Ці джерела можуть заміщати одне одного. Проте емісія акцій пов’язана з додат ковими витратами, що призводить до здорожчання власного капіталу і, в цілому, до зростання середньо зваженої вартості капіталу. Таке зростання може бути 4. Принцип дотримання цільової структури та вартості капіталу. Необхідність реалізації ін вестиційної програми та її фінансування може забез печуватись шляхом вибору альтернативних джерел залучення капіталу. Так, цільова потреба у власному капіталі може покриватися за рахунок чистого при бутку та додаткових внесків у статутний капітал (до даткової емісії акцій). Ці джерела можуть заміщати одне одного. Проте емісія акцій пов’язана з додат ковими витратами, що призводить до здорожчання власного капіталу і, в цілому, до зростання середньо зваженої вартості капіталу. Таке зростання може бути ЕКОНОМІКА а) дослідження динаміки обсягів та рівня диві дендних виплат у попередні періоди; б) дослідження динаміки чистого прибутку під приємства та його прогнозного значення; в) вивчення динаміки дивідендних виплат під приємствами-конкурентами. БІЗНЕСІНФОРМ № 11 ’2019 237 БІЗНЕСІНФОРМ № 11 2019 www.business-inform.net www.business-inform.net У межах цього етапу підприємство може вико ристовувати метод екстраполяції (виходячи із серед нього темпу зростання чистого прибутку), розрахун ково-аналітичний метод (з урахуванням можливих напрямів зростання доходів та скорочення витрат), метод експоненційного згладжування (виходячи з динаміки чистого прибутку), метод пропорційно го зростання (прогнозні обсяги дивідендних виплат визначаються пропорційно до обраного показника, серед яких можуть бути важливі макроіндикатори, показники фондового ринку тощо), методи кореля ційно-регресійного аналізу. Так, у межах цього етапу може використовуватися модель Лінтнера (1956) [10], який на основі дослідження виявив, що дивіденди ко ригуються відповідно до динаміки прибутку, але із запізненням, і першим провів аналіз моделі часткової адаптації для пояснення дивідендної політики. швидкість зростання обсягу реалізації обсягу прода жу може підтримувати підприємство при заданих об меженнях на обсяг емісії акцій, обсяг запозичень та рівень дивідендних виплат. Запропонувавши модель стійкого зростання обсягів реалізації та здійснивши її емпіричну апробацію, він довів залежність фінансо вих, дивідендних та інвестиційних рішень, пояснив ши, що коефіцієнт стійкого зростання – це функція від прибутковості реалізації, коефіцієнта фінансової автономії, оборотності активів та коефіцієнта капіта лізації чистого прибутку [13] (3): , чвр чвр а а чп ТП ЧР КО КЛ КК = ⋅ ⋅ ⋅ (3) (3) де ТПчвр – темп приросту чистої виручки, що забезпе чує стійке зростання, %; ЧРчвр – чиста рентабельність обороту, %; КОа – коефіцієнт оборотності активів; КЛа – коефіцієнт левериджу активів (розраховуєть ся як співвідношення активів до власного капіталу); ККчп – коефіцієнт капіталізації чистого прибутку. На другому етапі здійснюється планування об сягу дивідендних виплат, які може дозволити собі підприємство, виходячи з потреб операційної та ін вестиційної діяльності. З цією метою пропонуємо ви користовувати показник «рівень реінвестування чи стого грошового потоку від операційної діяльності», який оцінюється таким чином (1): В и п т В иходячи з цієї моделі та запланованого темпу приросту чистої виручки, планових параме трів рентабельності активів, їх оборотності та левериджу плановий коефіцієнт капіталізації чистого прибутку оцінюється (4): , од ЧГП п п ЧГП Д КР ОЗ НА НКІ ЧРК − = + + + (1) , од ЧГП п п ЧГП Д КР ОЗ НА НКІ ЧРК − = + + + (1) (1) . БІЗНЕСІНФОРМ № 11 ’2019 чвр чп а чвр а ТП КК ЧР КО КЛ = ⋅ ⋅ (4) (4) де КРЧГП – коефіцієнт реінвестування чистого гро шового потоку; ЧПГод – реінвестування чистого гро шового потоку від операційної діяльності; Д – диві денди; ОЗп – приріст основних засобів за первісною вартістю; НАп – приріст нематеріальних активів за первісною вартістю; НКІ – приріст незавершених ка пітальних інвестицій; ЧРК – приріст чистого робочо го капіталу. Таким чином, запланований коефіцієнт диві дендних виплат (Кд) становитиме (5): Таким чином, запланований коефіцієнт диві дендних виплат (Кд) становитиме (5): д 1 . д чп К КК = − (5) (5) Використання цієї моделі та визначення з її ви користанням планового коефіцієнта дивідендних виплат передбачає пошук оптимальної з точки зору підприємства моделі розвитку підприємства шляхом цілеспрямованого впливу на окремі параметри, що визначають допустимі межі зростання. Так, у про цесі такого планування здійснюється пошук резервів зростання рентабельності обороту (шляхом оптимі зації витрат), коефіцієнта оборотності активів; роз глядаються можливості щодо зміни структури фінан сування (левериджу активів). ЕКОНОМІКА ПІДПРИЄМСТВА ЕКОНОМІКА ЕКОНОМІКА ПІДПРИЄМСТВА В в г д В важається, що рівень реінвестування чистого грошового потоку в розмірі 8–10% є достатнім для підтримування нормального фінансового стану підприємства [12]. Виходячи з цього положення та планових показників чистого грошового потоку від операційної діяльності, приросту основних засобів, нематеріальних активів, незавершених капітальних інвестицій і чистого робочого капіталу можна обґрун тувати припустимий обсяг дивідендів. Якщо припус тити, що підприємство орієнтується на нижню межу цього показника (хоча воно може варіюватися в діа пазоні 8–10% і вище за потребою підприємства), пла новий обсяг дивідендів визначається таким чином (2): 0,08 ( ). од п Д ЧГП ОЗ НА НКІ ЧРК = − ⋅ + + + (2) На четвертому етапі здійснюється планування обсягу дивідендних виплат виходячи з критерію міні мізації середньозваженої вартості капіталу. Потребу в капіталі підприємство може покривати за рахунок різних джерел і, відповідно, заміщати недостатню суму нерозподіленого прибутку додатковими внеска ми в статутний капітал (додатковою емісією акцій – для публічної компанії) або борговим фінансуванням, якщо у підприємства прийнятний рівень фінансової стійкості та кредитоспроможності. Важливим об меженням при цьому виступає вартість капіталу, яка є мірою його мінімально-необхідної прибутковості. Отже, рішення щодо вибору джерел покриття по треби в капіталі, а, відповідно, і обсягів дивідендних ЕКОНОМІКА На третьому етапі обґрунтовується коефіці єнт дивідендних виплат і планова сума дивідендів, виходячи з необхідності підтримання запланованого обсягу діяльності підприємства. Такий розрахунок пропонується здійснювати виходячи з моделі стій кого зростання. Так, Р. С. Хіггінс (1977) дослідив, яку БІЗНЕСІНФОРМ № 11 ’2019 БІЗНЕСІНФОРМ № 11 ’2019 БІЗНЕСІНФОРМ № 11 ’2019 238 www.business-inform.net www.business-inform.net виплат має прийматися виходячи з показника серед ньозваженої вартості капіталу. Це обумовлено тим, що додаткова емісія акцій пов’язана з додатковими витратами, що збільшує вартість власного капіталу, тоді як залучення позикового капіталу поряд з ефек том податкової економії тягне додаткові витрати мо ніторингу та банкрутства, що відображається на його вартості. Тому рішення щодо виплати дивідендів пе редбачає моделювання різних варіантів фінансування (при різних рівнях капіталізації чистого прибутку) та вибір варіанта, що забезпечує мінімізацію показника середньозваженої вартості капіталу (WACC). Пошук цільового значення планових дивідендів здійснюється шляхом імітаційного моделювання, яке передбачає зміну основних параметрів інвестицій ної, операційної та фінансової діяльності та дозволяє ідентифікувати той обсяг дивідендних виплат, який найбільшою мірою відповідає встановленим параме трам і стратегії розвитку підприємства. ВИСНОВКИ Дивідендна політика є важливим фактором ди наміки економічного розвитку підприємства та його ринкової вартості. Хоча цей вплив є опосередкова ним і проявляється через взаємозв'язок дивідендної політики з рішеннями щодо інвестування та фінансу вання. Зважаючи на цей складний взаємозв'язок, «не лінійність» залежності ринкової вартості від обсягів сплачуваних дивідендів, відмінності у фінансових На п’ятому етапі приймається остаточне рі шення щодо планової суми дивідендних виплат. Так, на основі проведених розрахунків підприємство отримує область значень, у межах яких обґрунтову ється сума планових дивідендних виплат (рис. 1). Область значень планової суми дивідендних виплат Д1 Д2 Д3 Д4 де Д1 (Д2; Д3; Д4) – планова сума бажаних дивідендних виплат відповідно до потреб власників, що віддають перевагу поточним доходам, що забезпечує фінансову рівновагу; підтримує стійкий темп зростання; мінімізує середньозважену вартість капіталу відповідно Область значень планової суми дивідендних виплат Д4 Д3 де Д1 (Д2; Д3; Д4) – планова сума бажаних дивідендних виплат відповідно до потреб власників, що віддають перевагу поточним доходам, що забезпечує фінансову рівновагу; підтримує стійкий темп зростання; мінімізує середньозважену вартість капіталу відповідно Рис. 1. Область вибору планового обсягу дивідендних виплат підприємства Рис. 1. Область вибору планового обсягу дивідендних виплат підприємства ЕКОНОМІКА ПІДПРИЄМСТВА В ибір планового цільового обсягу дивідендних виплат у межах розрахованих значень Д1–Д4 обумовлюється низкою факторів, основними з яких, на наш погляд є: В ЕКОНОМІКА ЕКОНОМІКА ПІДПРИЄМСТВА інтересах різних власників щодо дивідендів, обґрун тування їх планового обсягу є складним оптимізацій ним завданням. Таким чином, планування дивідендів повинне здійснюватися на багатокритеріальній осно ві, що зводиться до обґрунтування їх обсягів за кри теріями: максимізації задоволення фінансових інтер есів власників, що віддають перевагу поточним до ходам; забезпечення стійкого зростання; збереження фінансової рівноваги; мінімізації середньозваженої вартості капіталу та пошуку цільового значення ди відендів шляхом імітаційного моделювання у визна чених межах. Це дозволить сформувати виважений підхід до формування дивідендної політики підпри ємства відповідно до його стратегії розвитку.  інтересах різних власників щодо дивідендів, обґрун тування їх планового обсягу є складним оптимізацій ним завданням. Таким чином, планування дивідендів повинне здійснюватися на багатокритеріальній осно ві, що зводиться до обґрунтування їх обсягів за кри теріями: максимізації задоволення фінансових інтер есів власників, що віддають перевагу поточним до ходам; забезпечення стійкого зростання; збереження фінансової рівноваги; мінімізації середньозваженої вартості капіталу та пошуку цільового значення ди відендів шляхом імітаційного моделювання у визна чених межах. Це дозволить сформувати виважений підхід до формування дивідендної політики підпри ємства відповідно до його стратегії розвитку.  1. Фінансовий менталітет власників, їх переваж на схильність до поточних доходів чи орієнтація на приріст ринкової вартості підприємства. 2. Кон'юнктура фінансового ринку: зростання дивідендних виплат підприємствами-аналогами спо нукає нарощувати дивіденди задля забезпечення ін вестиційної привабливості акцій на ринку. 3. Інвестиційна програма підприємства: розро блена інвестиційна програма з ефективними інвести ційними проектами сприяє зменшенню обсягу диві дендних виплат за інших рівних умов. 4. Рівень фінансової стійкості, гнучкості та кре дитоспроможності підприємства. Ці фінансові ха рактеристики обумовлюють здатність підприємства залучати боргове фінансування, яке може викорис товуватись для фінансування інвестиційних проектів при збереженні обсягу дивідендних виплат. ЛІТЕРАТУРА ЕКОНОМІКА 1. Показники діяльності суб’єктів господарювання. URL: http:// ukrstat.gov.ua 1. Показники діяльності суб’єктів господарювання. URL: http:// ukrstat.gov.ua 2. Реун А. До чого готуватись бізнесу у зв’язку із запро вадженням плану BEPS. 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Ченг Ли Ф., Финнерти Дж. И. Финансы корпора ций: теория, методы и практика / пер. с англ. М. : ИНФРА-М, 2000. 686 с. 12. Бернстайн Л. А. Анализ финансовой отчетности: теория, практика и интерпретация / пер. с англ. М. : Финансы и статистика, 1996. 624 с. Myers, S. C. “Capital Structure Puzzle“. The Journal of Fi nance, vol. 39, no. 3 (1984): 575-592. “Pokaznyky diialnosti subiektiv hospodariuvannia“ [Per formance Indicators of Economic Entities]. http://ukrstat.gov.ua 13. Хиггинс Р. С. БІЗНЕСІНФОРМ № 11 ’2019 Финансовый анализ: инструменты для принятия бизнес-решений / пер. с англ. М. : ООО «И.Д. Вильямс», 2007. 464 с. Reun, A. “Do choho hotuvatys biznesu u zviazku iz za provadzhenniam planu BEPS“ [What Business to Prepare for When Implementing a BEPS Plan]. https://evris.law/uk/stattja- do-chogo-gotuvatisja-biznesu-u-zvjazku-iz-zaprovadzhen njam-planu-beps/ REFERENCES Babichenko, V. V. “Dyvidendna polityka korporatsii: vyk lyky suchasnoho etapu“ [Dividends Policy of Corporations: Challenges of Modern Stage]. Efektyvna ekonomika. 2014. http://www.economy.nayka.com.ua/?op=1&z=3227i Velykyi, Yu. M., and Velyka, O. Yu. “Osoblyvosti dyvi dendnoi polityky ukrainskykh korporatsii“ [Peculiarities of the Dividend Policy of Ukrainian Corporations]. Hlobalni ta natsion alni problemy ekonomiky. 2017. http://global-national.in.ua/ archive/17-2017/49.pdf ЕКОНОМІКА ЕКОНОМІКА ПІДПРИЄМСТВА Bernstayn, L. A. Analiz finansovoy otchetnosti: teoriya, praktika i interpretatsiya [Analysis of Financial Statements: Theo ЕКОНОМІКА БІЗНЕСІНФОРМ № 11 ’2019 240
https://openalex.org/W4391810620	https://www.nature.com/articles/s41398-024-02813-2.pdf	English	null	Correction: Attenuating human fear memory retention with minocycline: a randomized placebo-controlled trial	Translational psychiatry	2,024	cc-by	210	Translational Psychiatry Translational Psychiatry Translational Psychiatry www.nature.com/tp CORRECTION OPEN Correction: Attenuating human fear memory retention with minocycline: a randomized placebo-controlled trial © The Author(s) 2024 Translational Psychiatry (2024) 14:97 ; https://doi.org/10.1038/s41398-024-02813-2 Translational Psychiatry (2024) 14:97 ; https://doi.org/10.1038/s41398-024-02813-2 Correction to: Translational Psychiatry https://doi.org/10.1038/ s41398-024-02732-2, published online 17 January 2024 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creativecommons.org/licenses/by/4.0/. In the Abstract, the full term for “FPS” should be “fear-potentiated startle”, but not “fear-potentially startle”. The original article has been corrected. The original article has been corrected. © The Author(s) 2024
https://openalex.org/W2791260248	http://umu.diva-portal.org/smash/get/diva2:1204659/FULLTEXT01	English	null	Autonomic Neuropathy—a Prospective Cohort Study of Symptoms and E/I Ratio in Normal Glucose Tolerance, Impaired Glucose Tolerance, and Type 2 Diabetes	Frontiers in neurology	2,018	cc-by	5,932	http://www.diva-portal.org http://www.diva-portal.org http://www.diva-portal.org This is the published version of a paper published in Frontiers in Neurology. Citation for the original published paper (version of record): Zimmerman, M., Pourhamidi, K., Rolandsson, O., Dahlin, L B. (2018) Autonomic Neuropathy: a Prospective Cohort Study of Symptoms and E/I Ratio in Normal Glucose Tolerance, Impaired Glucose Tolerance, and Type 2 Diabetes Frontiers in Neurology, 9: 154 https://doi.org/10.3389/fneur.2018.00154 Access to the published version may require subscription. N.B. When citing this work, cite the original published paper. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-146440 Original Research published: 14 March 2018 doi: 10.3389/fneur.2018.00154 Citation: Zimmerman M, Pourhamidi K, Rolandsson O and Dahlin LB (2018) Autonomic Neuropathy—a Prospective Cohort Study of Symptoms and E/I Ratio in Normal Glucose Tolerance, Impaired Glucose Tolerance, and Type 2 Diabetes. Front. Neurol. 9:154. doi: 10.3389/fneur.2018.00154 A Malin Zimmerman1,2, Kaveh Pourhamidi 3, Olov Rolandsson3 and Lars B. Dahlin1,2 1 Hand Surgery, Department of Translational Medicine, Lund University, Skåne University Hospital, Malmö, Sweden, 2 Department of Hand Surgery, Skåne University Hospital, Malmö, Sweden, 3 Family Medicine, Department of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden 1 Hand Surgery, Department of Translational Medicine, Lund University, Skåne University Hospital, Malmö, Sweden, 2 Department of Hand Surgery, Skåne University Hospital, Malmö, Sweden, 3 Family Medicine, Department of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden Background: Autonomic neuropathy in diabetes, in addition to causing a range of symptoms originating from the autonomic nervous system, may increase cardiovas cular morbidity. Our aim was to study the progression of autonomic neuropathy, based on symptom score and evaluation of an autonomic test, in persons with normal and impaired glucose tolerance and in patients with type 2 diabetes (T2D). Edited by: Mathias Baumert, University of Adelaide, Australia Methods: Participants were recruited in 2003/2004 with a followup in 2014. The participants’ glucose tolerance was categorized using oral glucose tolerance tests. Symptoms were evaluated using an autonomic symptom score (ASS), ECG was used to test cardiac autonomic function based on the expiration/inspiration ratio (E/I ratio), and blood samples were taken on both occasions. Reviewed by: Csaba Lengyel, University of Szeged, Hungary Phyllis Kravet Stein, Washington University in St. Louis, United States results: ASSs were higher at followup in the T2D patients than in the normal glucose tolerance group (mean 1.21 ± 1.30 vs. 0.79 ± 0.7; p < 0.05). E/I ratio did not deteriorate more than could be expected as an aging effect in wellcontrolled T2D. No relationship was found between E/I ratio and HbA1c or ASS. Correspondence: Malin Zimmerman malin.zimmerman@med.lu.se Specialty section: This article was submitted to Autonomic Neuroscience, a section of the journal Frontiers in Neurology conclusion: The presence of autonomic symptoms increased over time in T2D patients, but the symptoms did not correlate with the E/I ratio in this metabolically wellcontrolled cohort. ASSs can be a useful clinical tool when assessing the progression of autonomic dysfunction in patients with abnormal glucose metabolism. Received: 29 November 2017 Accepted: 01 March 2018 Published: 14 March 2018 Keywords: diabetes mellitus type 2, glucose intolerance, autonomic nervous system diseases, autonomic nervous system, diabetic neuropathies, glycosylated hemoglobin A, glucose tolerance test, disease progression Statistics Statistical calculations were performed using IBM SPSS Statistics, version 22 (SPSS Inc., Chicago, IL, USA). Variables normally dis- tributed are presented as mean ± SD. Non-normally distributed variables are presented as median [interquartile range].f Symptoms of Autonomic Nerve Function— Autonomic Symptom Score (ASS) Symptoms of Autonomic Nerve Function— Autonomic Symptom Score (ASS) A 7-item questionnaire was used to assess whether the partici- pants had autonomic symptoms (14). The items were as follows: postural hypotension, urinary incontinence, nocturnal diarrhea, gustatory sweating, gastric atony, hypoglycemic unawareness, and erectile dysfunction. The questions were scored according to how often the patient experienced the symptoms: 0 = never, 1 = sometimes, or 2 = often. The scores were then added to give a total ASS. There are conflicting data regarding whether or not metabolic factors other than diabetes influence the development of auto- nomic neuropathy. Body mass index (BMI) has been associated with CAN in T2D in some studies (7, 8), but not in all (9). INTRODUCTION The rising global incidence of diabetes mellitus means that an increasing number of people are living with complications from diabetes (1). Neuropathy, known to occur in 30–40% of patients with diabetes, can be divided into peripheral and autonomic neuropathy. Autonomic neuropathy causes difficulties in adapting to changes in posture or activity level, erectile dysfunction, incontinence, March 2018 \| Volume 9 \| Article 154 Frontiers in Neurology \| www.frontiersin.org 1 Autonomic Neuropathy—a Prospective Study Zimmerman et al. intervals, was used to calculate the expiration: inspiration ratio (E:I ratio) (13). gastrointestinal disturbances, and an increased risk of cardiovas- cular morbidity (2). At present, available treatments are aimed at secondary prevention, including strict glucose control and lifestyle modifications (3). Blood Samples Fasting blood samples were drawn both at baseline and at follow- up, and included HbA1c, S-cholesterol, triglycerides, high- density lipoprotein (HDL), and low-density lipoprotein (LDL). In this study, we aimed to investigate the prevalence of auto- nomic neuropathy and its temporal development, measured as E/I ratio and patient-reported symptoms, in persons with NGT and IGT, and in patients with T2D. Study Populationh Wilcoxon signed-ranks test was used to compare differences over time. McNemar was used to analyze difference in propor- tions over time. To compare groups, we used Kruskal–Wallis and the Bonferroni correction for multiple testing. Linear regression analysis was used to calculate the effect of individual variables on E/I ratio. Pearson’s correlation test was used to calculate correla- tion and we considered a R2 of >±0.3 significant. A p-value of <0.05 was considered statistically significant. The study population has been described in an earlier publication (10). Briefly, participants were recruited from the Västerbotten Intervention Programme (11) in Skellefteå, Sweden, during 2003 and 2004. Two standardized oral glucose tolerance tests (OGTT) were performed to determine whether the individuals had NGT or IGT. T2D patients were recruited from their respective pri- mary care centers in the same area during the same time period. At follow-up, two OGTT were performed on both the participants with NGT and those with IGT. Study Participants Initially, 129 participants were included. Of these, six were excluded (three had vitamin B12 or folate deficiency and three had a neurological disease) and four declined to participate. Thus, 119 participants could be included in the study. There were 39 with NGT, 29 with IGT, and 51 with T2D. In 2014, 6 were deceased and 31 chose to withdraw from the study, leaving 87 participants to be included in the follow-up (Figure 1). E/I Ratio Expiration/inspiration ratio (E/I) as a measure of autonomic function was tested in order to detect any abnormalities. R–R variation, which is used to calculate the E/I ratio, is the meas- urement of sinus arrhythmia (5). The sinus arrhythmia that is seen in healthy individuals when breathing (and is exaggerated in deep breathing) is a result of cyclic variations in sympathetic and parasympathetic tone that modulate the rate of the sinoatrial node (12). In patients with diabetes and autonomic dysfunction, the heart rate variability is reduced or absent (13). Using standard ECG recording, R–R intervals were analyzed during deep inspira- tion and expiration (over 1 min at a frequency of 6 breaths/min). The mean value of inspirations and expirations, in terms of R–R RESULTS All participants gave their signed, informed consent, and the study was approved by the Regional Ethics Board in Umeå, Sweden. Abnormal E/I Ratioh i In a small recent study on type 1 diabetes (T1D), an increase in HbA1c was related to impaired cardiovascular autonomic func- tion (3), which correlates well with the results of a number of large studies that have shown beneficial effects from strict glucose control on cardiac autonomic neuropathy (CAN) in patients with T1D (4). In the DCCT trial, intensive glucose control prevented the development of abnormal heart rate variability (5). One previous study has shown abnormal expiration/inspiration ratio (E/I ratio) in 35% of the studied population of patients with type 2 diabetes (T2D) (6), and a large trial demonstrated that CAN was present in 15% of newly diagnosed T2D cases (7). However, less is known about the progression over time of autonomic neuropathy in individuals with normal glucose tolerance (NGT) and impaired glucose tolerance (IGT) and in patients with T2D diabetes.hl The 2004 NGT group was chosen as reference, with a mean E/I ratio of 1.27 (95% CI 1.21–1.32). An E/I ratio of <1.5 SD of the mean for the NGT group was considered abnormal (14). Frontiers in Neurology \| www.frontiersin.org Population Characteristicsh The characteristics of the study group (n = 87) are presented in Table 1. HbA1c rose over time in those with NGT and IGT, but not in patients with T2D. S-cholesterol, S-HDL, and S-LDL, as well as triglycerides, were higher in 2014 than in 2004 in the total population. The NGT group did not gain weight over the 10 years. More NGT individuals were treated with anti-hypertension March 2018 \| Volume 9 \| Article 154 Frontiers in Neurology \| www.frontiersin.org 2 Autonomic Neuropathy—a Prospective Study Zimmerman et al. Table 1 \| Characteristics of the population comprising healthy persons and patients with impaired glucose tolerance (IGT) and type 2 diabetes (T2D) 2004 and 2014. Normal glucose tolerance (NGT) 2004 NGT 2014 IGT 2004 IGT 2014 T2D 2004 T2D 2014 Total 2004 Total 2014 Total (n) 33 36 20 9 34 42 87 87 Female, n (%) 18 (55) 19 (53) 9 (45) 4 (44) 15 (44) 19 (45) 42 (47) 42 (47) Body mass index (kg/m2)a 25.8 (±3.6) 25.5 (±3.6) 27.1 (±5.4) 27.4 (±6.3) 29.7 (±4.3) 29.4 (±4.3) 27.6 (±4.6) 27.5 (±4.6) HbA1c (mmol/mol)a 35 [33–37] 38 [36–40]* 36.5 [34–38] 40 [38–42]* 55 [47–63]€ 52 [46–64]€,* 38 [34–52] 42 [38–52]* S-cholesterol (mmol/L) 5.9 [5.2–6.4] 5.9 [5.1–7.0]* 5.1 [4.3–5.9] 5.9 [4.9–6.05] 4.7 [4.3–5.1] 4.6 [4.2–5.33]€ 5.1 [4.4–5.9] 5.2 [4.5–6.2]* S-triglycerides (mmol/L) 1.09 [0.82–1.52] 1.35 [0.84–1.78]* 1.35 [0.85–1.65] 1.41 [1.05–2.51] 1.47 [1.08–1.99] 2.2 [1.58–2.82]€,* 1.34 [0.95–1.70] 1.73 [1.09–2.37]* S-high-density lipoprotein (mmol/L) 1.38 [1.2–1.66] 1.41 [1.27–1.70] 1.15 [1.01–1.54] 1.17 [0.88–1.69] 1.10 [0.81–1.37] 1.28 [1.0–1.43]€,* 1.22 [1.04–1.47] 1.33 [1.09–1.53]* S-low-density lipoprotein (mmol/L) 3.9 [3.3–4.4] 3.9 [3.2–4.7]* 3.1 [2.08–3.85] 3.5 [2.9–3.95] 2.80 [2.38–3.25] 2.5 [2.03–2.98]€ 3.2 [2.5–4.0] 3.2 [2.4–4.3]ns Oral hypoglycemic agent,bn (%) 0 (0) 0 (0) 0 (0) 0 (0) 19 (56) 26 (62) 19 (22) 26 (30) Insulin treatment, n (%) 0 (0) 0 (0) 0 (0) 0 (0) 9 (26) 14 (33) 9 (10) 14 (16) Beta blocker treatment, n (%) 7 (21) 3 (8) 4 (20) 4 (44) 6 (18) 11 (26) 17 (20) 18 (21) Anti-hypertension agents, n (%) 11 (33) 16 (44) 7 (35) 4 (44) 19 (56) 31 (74) 37 (43) 51 (59) Lipid-lowering agents, n (%) 3 (9) 6 (17) 3 (15) 1 (11) 17 (50) 22 (52) 23 (26) 29 (33) aData already published (29). bData missing in one patient. p < 0.05; 2004 vs. 2014, Wilcoxon. Population Characteristicsh €p < 0.05, comparison between groups, Kruskal–Wallis adjusted by the Bonferroni correction for multiple tests. The significant differences were found between NGT and T2D. ns, non-significant. V l SD di [25 h 75 h il i IQR] March 2018 \| Vol me 9 \| Artic Figure 1 \| Flowchart of patients over time. NGT, normal glucose tolerance; IGT, impaired glucose tolerance; T2D, type 2 diabetes mellitus. Figure 1 \| Flowchart of patients over time. NGT, normal glucose tolerance; IGT, impaired glucose tolerance; T2D, type 2 diabetes mellitus. rsons and patients with impaired glucose tolerance (IGT) and type 2 diabetes (T2D) 2004 and 2014. IGT 2014 Figure 1 \| Flowchart of patients over time. NGT, normal glucose tolerance; IGT, impaired glucose tolerance; T2D, type 2 diabetes mellitus. agents and lipid-lowering agents in 2014 than in 2004 (Table 1). In patients with T2D, triglycerides and HDL were higher in 2014, whereas LDL cholesterol was lower in 2014 compared to 2004 (Table 1). Baseline characteristics of the study population can be reviewed in Table S5 in Supplementary Material. Frontiers in Neurology \| www.frontiersin.org E/I Ratio and HbA1c No correlation was found between change in E/I ratio and change in HbA1c. Furthermore, no correlation was found between E/I ratio and HbA1c. Figure 2 shows the relationship between E/I and HbA1c based on groups. In the regression analysis, HbA1c levels were not associated with E/I ratio. Autonomic Testing—E/I Ratioh The rate of abnormal E/I ratio in the T2D group was 12% (5/42). No differences were found in HbA1c, BMI, ASS, duration of diabetes, or age between patients with abnormal E/I ratios and those with normal E/I ratios (data not shown). The E/I ratio for the population as a whole deteriorated over the study period (Table 2). No statistically significant differences were found in E/I ratios between groups in 2004 or in 2014. The change in E/I ratio (i.e., delta E/I) did not differ between groups. The E/I ratio did not correlate with the ASS. Two individuals with abnormal E/I ratios in 2014 were treated with beta blockers (one in the NGT group and one in the T2D group). Autonomic Symptom Scoreh 2014, Wilcoxon. #p < 0.05; 2004 vs. 2014, McNemar. €p < 0.05, comparison between groups, Kruskal–Wallis adjusted using the Bonferroni correction for multiple tests. A significant difference was found between NGT and T2D. ns non-significant Table 3 \| Presence of autonomic symptoms in the study population at follow- up (2014). NGT (n = 36) Impaired glucose tolerance (n = 9) Type 2 diabetes (T2D) (n = 42) p-Value Postural hypotension, n (%) 15 (42) 4 (44) 25 (60) ns Urinary incontinence, n (%) 1 (3) 1 (11) 7 (28) ns Nocturnal diarrhea, n (%) 1 (3) 0 (0) 0 (0) ns Gustatory sweating, n (%) 0 (0) 0 (0) 6 (14) <0.05 Gastric atony, n (%) 2 (6) 0 (0) 5 (12) ns Hypoglycemic unawareness, n (%) 3 (8) 1 (11) 13 (31) <0.05 Erectile dysfunction, n (%) 6 (17) 3 (33) 11 (26) ns Chi-square was used to calculate statistical significance. ns, non-significant. Table 3 \| Presence of autonomic symptoms in the study population at follow- up (2014). ASS 0.79 ± 0.70 0.83 ± 1.12 0.60 ± 0.68 1.11 ± 0.93 1.21 ± 1.30 1.98 ± 1.59 0.91 ± 1.0 1.42 ± 1.45 Delta ASS 0.06 ± 0.80 0.33 ± 1.12 0.90 ± 1.53 0.5 ± 1.29€ E/I ratio 1.24 [1.17–1.34] 1.17 [1.07–1.24] 1.17 [1.09–1.30] 1.24 [1.10–1.74] 1.19 [1.11–1.32] 1.13 [1.07–1.22]* 1.21 [1.13–1.32] 1.14 [1.07–1.24]* Abnormal E/I ratio 0 3 0 0 0 5 0 8# Delta E/I −0.12 [−0.2 to 0.0] 0.02 [−0.10 to 0.48] −0.06 [−0.18 to 0.0] −0.07 [−0.18 to 0.0]ns p < 0.05; 2004 vs. 2014, Wilcoxon. #p < 0.05; 2004 vs. 2014, McNemar. €p < 0.05, comparison between groups, Kruskal–Wallis adjusted using the Bonferroni correction for multiple tests. A significant difference was found between NGT and T2D. ns, non-significant. E/I ratio (Table 3). Nocturnal diarrhea was not analyzed further, since it was only present in one patient with NGT.hf There were no differences in blood pressure levels between individuals who reported postural hypotension and those who did not. Body Mass Index In the regression analysis, we found that BMI was the only factor that influenced the E/I ratio in the T2D patients (Table 4). We did not see any effect of BMI on E/I ratio in the NGT group (Table 4). Duration of Diabetesh There was no correlation between the duration of diabetes and E/I ratio, nor between duration of diabetes and ASS symptoms. Frontiers in Neurology \| www.frontiersin.org Autonomic Symptom Scoreh The ASS rose over time in the whole population (Table 2). The NGT group showed almost no difference at all in their ASS (mean difference between 2004 and 2014 = 0.06), while the difference was higher in the T2D patients (mean difference between 2004 and 2014 = 0.9; p < 0.05) between 2004 and 2014 (Table 2). Results from the individual questions are shown in Table 3. Thirteen out of 42 (31%) patients with T2D reported hypo- glycemia unawareness (Table 3). Of these, nine received insulin treatment, one was treated with two oral agents, two patients did not have any anti-diabetic medication and data were missing for one patient. Two of the 13 (15%) T2D patients who reported hypoglycemia unawareness had an abnormal E/I ratio. Six (14%) T2D patients reported gustatory sweating. Of these, one had an abnormal E/I ratio. Urinary incontinence was reported in 7 (28%) T2D patients, and one of these had an abnor- mal E/I ratio. Erectile dysfunction was reported in 11 (26%) T2D patients, and 1 of these had an abnormal E/I ratio. Gastric atony was reported in five (12%) T2D patients, one had an abnormal March 2018 \| Volume 9 \| Article 154 Frontiers in Neurology \| www.frontiersin.org March 2018 \| Volume 9 \| Article 154 3 Zimmerman et al. Autonomic Neuropathy—a Prospective Study Table 2 \| Autonomic symptom score (ASS) and expiration/inspiration ratio at baseline (2004) and follow-up (2014). Normal glucose tolerance (NGT) 2004 NGT 2014 Impaired glucose tolerance (IGT) 2004 IGT 2014 Type 2 diabetes (T2D) 2004 T2D 2014 Total 2004 Total 2014 ASS 0.79 ± 0.70 0.83 ± 1.12 0.60 ± 0.68 1.11 ± 0.93 1.21 ± 1.30 1.98 ± 1.59€ 0.91 ± 1.0 1.42 ± 1.45 Delta ASS 0.06 ± 0.80 0.33 ± 1.12 0.90 ± 1.53 0.5 ± 1.29€ E/I ratio 1.24 [1.17–1.34] 1.17 [1.07–1.24]* 1.17 [1.09–1.30] 1.24 [1.10–1.74] 1.19 [1.11–1.32] 1.13 [1.07–1.22]* 1.21 [1.13–1.32] 1.14 [1.07–1.24]* Abnormal E/I ratio 0 3 0 0 0 5 0 8# Delta E/I −0.12 [−0.2 to 0.0] 0.02 [−0.10 to 0.48] −0.06 [−0.18 to 0.0] −0.07 [−0.18 to 0.0]ns p < 0.05; 2004 vs. 2014, Wilcoxon. #p < 0.05; 2004 vs. 2014, McNemar. €p < 0.05, comparison between groups, Kruskal–Wallis adjusted using the Bonferroni correction for multiple tests. A significant difference was found between NGT and T2D. i ifi t [ ] [ ] [ ] [ ] p < 0.05; 2004 vs. DISCUSSION Our study shows that the patients with T2D, drawn from well- defined populations of T2D and individuals with normal and IGT, had higher ASSs both at baseline and at the long-term March 2018 \| Volume 9 \| Article 154 Frontiers in Neurology \| www.frontiersin.org 4 Autonomic Neuropathy—a Prospective Study Zimmerman et al. Figure 2 \| E/I ratio and HbA1c in (A) 2004 and (B) 2014 and (C) the change in E/I ratio over time vs. the change in HbA1c over time. Cases labeled by groups [normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and type 2 diabetes (T2D)]. Figure 2 \| E/I ratio and HbA1c in (A) 2004 and (B) 2014 and (C) the change in E/I ratio over time vs. the change in HbA1c over time. Cases labeled by groups [normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and type 2 diabetes (T2D)]. Table 4 \| Linear regression analysis. Model 1 T2D Model 1 NGT Age −0.002 (−0.053 to 0.049) −0.003 (−0.128 to 0.122) Male gender −0.02 (−0.140 to 0.099) 0.105 (−0.067 to 0.277) BMI 0.02* (0.006 to 0.033) 0.016 (−0.002 to 0.034) HbA1c −0.003 (−0.008 to 0.001) −0.009 (−0.038 to 0.021) Beta blocker treatment −0.064 (−0.202 to 0.074) −0.098 (−0.364 to 0.169) Duration of diabetes 0.002 (−0.009 to 0.014) N 33 42 The effect of individual variables on E/I ratio in 2014 in patients with T2D and individuals with NGT. B-coefficient, 95% confidence interval in brackets. *p < 0.05. T2D, type 2 diabetes; NGT, normal glucose tolerance. Table 4 \| Linear regression analysis. Erectile dysfunction is an unreliable marker of autonomic neuropathy since the pathogenesis of such symptoms is multi factorial and may be related to aging (19). Hypoglycemia una- wareness and gustatory sweating are considered more reliable symptoms in the diagnosis of autonomic dysfunction. They probably appear later in the disease trajectory and indicate more severe nerve damage (20). However, as Vinik et al. conclude in their review from 2003 (19), there is conflicting evidence regarding the association between autonomic dysfunction and hypoglycemia unawareness. In our data, very few of the patients who reported autonomic symptoms actually had an abnormal E/I ratio, which suggests that the correlation is weak between the E/I ratio and symptom manifestations. We do, however, see that the T2D patients experience more autonomic symptoms over time, whereas the individuals with NGT did not increase their ASS. DISCUSSION As we have no data on individual parameters of the ASS from baseline, these figures are not presented.h follow-up after 10 years. Furthermore, the increase in ASSs over time was higher in T2D patients. This was in spite of strict glucose control, since HbA1c values did not change significantly in the patients with T2D during the study period. During the time frame of 10 years, the E/I ratio deteriorated in the population as a whole, but there were no significant differences between the three groups, indicating that the deterioration was most probably an effect of aging (15, 16).i There is an ongoing debate about how to measure and diag- nose CAN. Ewing et al. suggested the Ewing battery of tests in 1985, which included blood pressure response to standing and sustained handgrip as well as heart rate response to standing (30:15 ratio), deep breathing (E/I ratio) and Valsalva maneuver (21). Since then, several suggestions for simplifying the tests have been published. Some authors advocate the use of the 30:15 ratio as the most simple and accurate test (22) while others promote the deep breathing test (23). The Toronto Expert Group recom- mends that at least two tests should be used in order to reach a definitive diagnosis of CAN, but argues that the presence of one abnormal test can identify early CAN (15). We used the E/I ratio to detect the presence of autonomic neuropathy, since it is easily reproduced and can be used in a primary care setting as no advanced equipment is needed. Only 12% of our T2D patients had an abnormal E/I ratio at follow-up, which is slightly lower than previously reported in patients with long-standing T2D (6, 24). This might be because the T2D patients in this study are well-defined and intensively treated, as can be seen from their low and stable HbA1c levels. There is probably also both a healthy volunteer bias and a survival bias in our study.h f We did not find any correlation between the reported auto- nomic symptoms, as expressed by the autonomic score, and the E/I ratio, which is in line with previous findings in T2D (17, 18). Autonomic symptoms in T2D seem to be unspecific and not correlated to an abnormal E/I ratio. Many of our participants reported symptoms of postural hypotension regardless of glyce- mic status. Frontiers in Neurology \| www.frontiersin.org AUTHOR CONTRIBUTIONS OR initiated the study. MZ made the calculations and wrote the manuscript. All authors contributed to the discussion and to revising the manuscript. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at http://www.frontiersin.org/articles/10.3389/fneur.2018.00154/ full#supplementary-material. Strengths and Limitations The study group is well defined and the follow-up time of 10 years is long. All study participants were evaluated using the same methods, carried out by the same examiner (the same physi- cian both at baseline and at follow-up), in order to ensure that each participant experienced exactly the same test conditions, thereby reducing potential confounding factors (15). Our study is probably prone to survival bias and healthy participant bias, ETHICS STATEMENT All participants gave their signed informed consent, and the study was approved by the Regional Ethics Board in Umeå, Sweden. All participants gave their signed informed consent, and the study was approved by the Regional Ethics Board in Umeå, Sweden. CONCLUSION The presence of autonomic symptoms rises over time in T2D patients, but such symptoms do not correlate with the E/I ratio in a well-defined cohort. Autonomic symptom scorings can be a useful clinical tool when assessing the progression of autonomic dysfunction in patients with abnormal glucose metabolism. p p p ( ) In future prospective studies, it would probably be wise to use more than one method for measuring autonomic neuropathy, in order to increase sensitivity in identifying affected individuals. We would like to suggest blood pressure response to standing as an additional test, since it is simple, non-invasive, and can be used in basically any setting. We would also like to argue that a symptom-scoring system should be used and that self-reported symptoms might be of more use to the clinician than the results of the autonomic tests in providing the best possible treatment for diabetes patients. When designing future symptom-scoring questionnaires, we suggest that instead of asking about erectile dysfunction a more gender neutral question could be used to evaluate the prevalence of both male and female sexual dysfunc- tion. Complex questions have been used in earlier studies (28). We suggest asking whether the responder is experiencing any sexual dysfunction, whether it started recently and whether they would like to specify the dysfunction. DISCUSSION The explanation for this might be that many factors, other than autonomic dysfunction, influence the blood pressure response to standing up, such as anti-hypertension medication, which was present in many of our patients. Hypoglycemia unawareness was reported by some individuals in the NGT group—the most plausible explanation for this is that there might be a misinterpretation of hypoglycemia unawareness. In the DCCT trial, the number of patients with T1D reporting hypoglycemia unawareness was higher in the intensive treat- ment group (5). Furthermore, in this study, the majority of T2D patients reporting hypoglycemia unawareness were on insulin treatment. The E/I ratio in our study had a wide distribution, independent of glycemic status. This suggests that there is a natural variation in E/I ratio even in a healthy population. Perhaps the best measure March 2018 \| Volume 9 \| Article 154 Frontiers in Neurology \| www.frontiersin.org 5 Autonomic Neuropathy—a Prospective Study Zimmerman et al. of autonomic deterioration is the drop in E/I ratio over time. However, there was no significant difference between groups when comparing the change in E/I ratio, perhaps because the changes demonstrated were very small, even after 10 years. since 4 of the T2D patients (7%) passed away during the study period and 31 patients (25%) withdrew from the study; many of them probably due to poor health. This is a population with an expected high mortality due to age and co-morbidity. Moreover, the questionnaire did not include a question regarding female sexual dysfunction, so only half of the population could report on symptoms of sexual dysfunction. t Traditionally, the E/I ratio has been thought of as a measure of primary parasympathetic function (13). However, newer research suggests a delicate sympathovagal balance behind the variability in heart rate during deep breathing (25, 26). The discussion concerning these delicate mechanisms is outside the scope of the present study, but has been highlighted by other authors with more sophisticated methods than used in the present paper (27). ACKNOWLEDGMENTS Our sincerest gratitude to Dr Sigbritt Rasmark, RN Karin Nilsson, and biomedical scientist Anette Broberg for their skillful assess- ment of the participants. We are indebted to the late Professor Göran Sundqvist who was one of the initiators of the study. We would also like to thank all the participants who made this study possible. Regarding metabolic factors, we found a positive association between E/I ratio and BMI in the T2D patients. This contrasts with previous studies where the association has been in the other direction (7, 8). The effect in this study is, however, very small and may be a chance effect, rather than a true effect, and thus of no clinical significance. i In the study design, we included patients with IGT. At follow- up, this group was very small, and we have therefore chosen not to focus on these individuals. FUNDING The study was funded by Västerbottens läns landsting and Umeå University (to OR), Skåne University Hospital, Region Skåne, Lund University and the Swedish Diabetes Foundation (to LD). 4. Ang L, Jaiswal M, Martin C, Pop-Busui R. Glucose control and diabetic neurop- athy: lessons from recent large clinical trials. Curr Diab Rep (2014) 14(9):528. doi:10.1007/s11892-014-0528-7 hhf 4. Ang L, Jaiswal M, Martin C, Pop-Busui R. Glucose control and diabetic neurop- athy: lessons from recent large clinical trials. Curr Diab Rep (2014) 14(9):528. doi:10.1007/s11892-014-0528-7 5. The Diabetes Control and Complications Trial Research Group. The effect of intensive diabetes therapy on measures of autonomic nervous system function in the Diabetes Control and Complications Trial (DCCT). Diabetologia (1998) 41(4):416–23. doi:10.1007/s001250050924 6. Bergstrom B, Lilja B, Osterlin S, Sundkvist G. Autonomic neuropathy in non- insulin dependent (type II) diabetes mellitus. Possible influence of obesity. J Intern Med (1990) 227(1):57–63. doi:10.1111/j.1365-2796.1990.tb00119.x 1. International Diabetes Federation. IDF Diabetes Atlas. 7th ed. (2015). REFERENCES Pafili K, Trypsianis G, Papazoglou D, Maltezos E, Papanas N. Simplified diagnosis of cardiovascular autonomic neuropathy in type 2 diabetes using Ewing’s battery. Rev Diabet Stud (2015) 12(1–2):213–9. doi:10.1900/ RDS.2015.12.213 23. Stranieri A, Abawajy J, Kelarev A, Huda S, Chowdhury M, Jelinek HF. An approach for Ewing test selection to support the clinical assessment of car- diac autonomic neuropathy. 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Autonomic neuropathy in type I diabetes: influence of duration and other diabetic complications. Acta Med Scand (1987) 222(2):147–54. doi:10.1111/j.0954-6820.1987.tb10652.x 28. Hotaling JM, Sarma AV, Patel DP, Braffett BH, Cleary PA, Feldman E, et al. Cardiovascular autonomic neuropathy, sexual dysfunction, and urinary incon- tinence in women with type 1 diabetes. Diabetes Care (2016) 39(9):1587–93. doi:10.2337/dc16-0059 15. Spallone V, Ziegler D, Freeman R, Bernardi L, Frontoni S, Pop-Busui R, et al. Cardiovascular autonomic neuropathy in diabetes: clinical impact, assessment, diagnosis, and management. REFERENCES 4. Ang L, Jaiswal M, Martin C, Pop-Busui R. Glucose control and diabetic neurop- athy: lessons from recent large clinical trials. Curr Diab Rep (2014) 14(9):528. doi:10.1007/s11892-014-0528-7 hhf 1. International Diabetes Federation. IDF Diabetes Atlas. 7th ed. (2015). 1. International Diabetes Federation. IDF Diabetes Atlas. 7th ed. (2015). 5. 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Factors in development of diabetic neuropathy. Baseline analysis of neuropathy in feasibility phase of Diabetes Control and Complications Trial (DCCT). The DCCT Research Group. Diabetes (1988) 37(4):476–81. doi:10.2337/diab.37.4.476 Conflict of Interest Statement: The research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest. 17. Low PA, Benrud-Larson LM, Sletten DM, Opfer-Gehrking TL, Weigand SD, O’Brien PC, et al. Autonomic symptoms and diabetic neuropathy: a population-based study. Diabetes Care (2004) 27(12):2942–7. doi:10.2337/ diacare.27.12.2942 Copyright © 2018 Zimmerman, Pourhamidi, Rolandsson and Dahlin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2018 Zimmerman, Pourhamidi, Rolandsson and Dahlin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 18. Nilsson H, Bergstrom B, Lilja B, Juul-Moller S, Carlsson J, Sundkvist G. Prospective study of autonomic nerve function in type 1 and type 2 diabetic patients: 24 hour heart rate variation and plasma motilin levels disturbed in parasympathetic neuropathy. Diabet Med (1995) 12(11):1015–21. doi:10.1111/ j.1464-5491.1995.tb00415.x 19. Vinik AI, Maser RE, Mitchell BD, Freeman R. Diabetic autonomic neuropathy. Diabetes Care (2003) 26(5):1553–79. doi:10.2337/diacare.26.5.1553 March 2018 \| Volume 9 \| Article 154 Frontiers in Neurology \| www.frontiersin.org
https://openalex.org/W4385391172	https://etj.uotechnology.edu.iq/article_102340_417eededca6bae45b7f201e5e1c8c6c8.pdf	Chinese	null	Effect of some Physical Elicitors on some Secondary Metabolite Induction of Hypercom Triquetrifolium in Vitro	Maǧallaẗ al-handasaẗ wa-al-tiknūlūǧiyā	2,014	cc-by	5,805	ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 Received on: 8/11/2012 & Accepted on: 4/4/2013 Received on: 8/11/2012 Effect of some Physical Elicitors on some Secondary Metabolite Induction of Hypercom Triquetrifolium in Vitro Baan M.Abdulrazzak College of Science, Al-Mustansiriyah University/\|Baghdad Email:Baan_twaij@yahoo.com Dr.Saadia H.Mahmood College of Science, Al-Mustansiriyah University/\|Baghdad Dr.Kadhim M.Ibrahim College of Science, Al-Nahrian University/ Baghdad Received on: 8/11/2012 & Accepted on: 4/4/2013 ﺗﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypericum triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ ﺗﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypericum triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ 1 https://doi.org/10.30684/etj.32.2B.20 2412-0758/University of Technology-Iraq, Baghdad, Iraq This is an open access article under the CC BY 4.0 license http://creativecommons.org/licenses/by/4.0 ABSTRACT وﺑﻐﯿ ﺔ زﯾ ﺎدة إﻧﺘ ﺎج اﻟﻤﺮﻛﺒ ﺎت https://doi.org/10.30684/etj.32.2B.20 2412-0758/University of Technology-Iraq, Baghdad, Iraq This is an open access article under the CC BY 4.0 license http://creativecommons.org/licenses/by/4.0 ﺗﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypericum triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ اﻟﺨﻼﺻﺔ اﺟﺮي اﻟﺒﺤﺚ اﻟﺤ ﺎﻟﻲ ﺑﮭ ﺪف زﯾ ﺎدة إﻧﺘ ﺎج ﺑﻌ ﺾ ﻣﺮﻛﺒ ﺎت اﻻﯾ ﺾ اﻟ ﺜ ﺎﻧﻮي ﻓ ﻲ اﻟﻤ ﺰارع اﻟﻨﺴ ﯿﺠﯿﺔ ﻟﻨﺒ ﺎت اﻟﺮوﺟ ﺔ Hypericum triquetrifolium ,ﻗ ﺪرت ﻣﺮﻛﺒ ﺎت اﻻﯾ ﺾ اﻟﺜ ﺎﻧﻮي ﺑﺎﻟﺘﺤﻠﯿ ﻞ اﻟﻜﻤ ﻲ واﻟﻨ ﻮﻋﻲ ﺑﺎﺳﺘﻌﻤﺎل ﺟﮭﺎز HPLC وﻗﻮرﻧ ﺖ ﻣ ﻊ اﻟﻤﺴ ﺘﺨﻠﺺ اﻟﻤﯿﺜ ﺎﻧﻮﻟﻲ ﻟ ﻸوراق اﻟﺠﺎﻓ ﺔ ﻟﻠﻨﺒ ﺎت اﻟﻜﺎﻣ ﻞ . ﺗ ﻢ اﻟﺤﺼ ﻮل ﻋﻠ ﻰ اﻟﻜ ﺎﻟﺲ ﺑﺄﺧ ﺬ أﺟ ﺰاء ﻣ ﻦ أوراق اﻟﻨﺒ ﺎت وزراﻋﺘﮭ ﺎ ﻋﻠ ﻰ اﻟﻮﺳ ﻂ اﻟﻐ ﺬاﺋﻲ ( MS) Murashige and Skoog اﻟﺤ ﺎوي ﻋﻠ ﻰ ﻣﻨﻈﻤ ﺎت اﻟﻨﻤ ﻮ ﺷ ﻤﻠﺖ2,4-Dichlorophenoxy acetic acid ) (2,4-D وBenzyl adinenine ) BA (ﺑﺎﻟﺘﺮ ﻛﯿﺰ 0.1 و0.5 ﻣﻠﻐﻢ / ﻟﺘﺮ ﻋﻠﻰ اﻟﺘ ﻮاﻟﻲ . وﺑﻐﯿ ﺔ زﯾ ﺎدة إﻧﺘ ﺎج اﻟﻤﺮﻛﺒ ﺎت ABSTRACT This project aimed to increase the production of some secondary metabolites using physical and chemical elicitors in tissue cultures of Hypercom triquetrifolium L.. The quality and quantity of photochemical were estimated using methanolic extracts of dried leaves and callus were analyses using HPLC. Callus was initiated on leaf discs cultured on Murashig and Skoog (MS) medium supplemented with 2,4-Dichlorophenoxy acetic acid (2,4-D) and Benzyl adenosine (BA) at concentrations of 0,0.1, 0.5, 2.0 or 5.0 mg/l for H. triquetrifolium callus. Results showed that the combination of 2,4-D at 0.1 mg/l with BA at 0.5 mg/l was the most effective for callus induction percentage reached 90%. The highest mean fresh weight reached 64.33 mg for H. triquetrfolium. The same combination was used for callus maintenance for plants. Results also showed an increase in the concentration of secondary metabolites in methanol extracts induced on leaves. Callus cultures induced on leaf discs were treated with some physical stimuli such as light, ultraviolet, the different exposure to photoperiod (dark for 24 hrs, 12 hrs light, 16 hrs light or 24 hrs light), the uv exposure time was 10 or 20 minutes. Result showed that there are significant differences between the various treatmeant, The best light exposure time caused an increase in the production of secondary metabolism was 12 hrs light for calli in both plants. Both exposure times (10 or 20 mint) induced the yield of secondary metabolites in callus cultures of H. triquetrifolium equally. 1 ﺗﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypericum triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ اﻟﺨﻼﺻﺔ اﺟﺮي اﻟﺒﺤﺚ اﻟﺤ ﺎﻟﻲ ﺑﮭ ﺪف زﯾ ﺎدة إﻧﺘ ﺎج ﺑﻌ ﺾ ﻣﺮﻛﺒ ﺎت اﻻﯾ ﺾ اﻟ ﺜ ﺎﻧﻮي ﻓ ﻲ اﻟﻤ ﺰارع اﻟﻨﺴ ﯿﺠﯿﺔ ﻟﻨﺒ ﺎت اﻟﺮوﺟ ﺔ Hypericum triquetrifolium ,ﻗ ﺪرت ﻣﺮﻛﺒ ﺎت اﻻﯾ ﺾ اﻟﺜ ﺎﻧﻮي ﺑﺎﻟﺘﺤﻠﯿ ﻞ اﻟﻜﻤ ﻲ واﻟﻨ ﻮﻋﻲ ﺑﺎﺳﺘﻌﻤﺎل ﺟﮭﺎز HPLC وﻗﻮرﻧ ﺖ ﻣ ﻊ اﻟﻤﺴ ﺘﺨﻠﺺ اﻟﻤﯿﺜ ﺎﻧﻮﻟﻲ ﻟ ﻸوراق اﻟﺠﺎﻓ ﺔ ﻟﻠﻨﺒ ﺎت اﻟﻜﺎﻣ ﻞ . ﺗ ﻢ اﻟﺤﺼ ﻮل ﻋﻠ ﻰ اﻟﻜ ﺎﻟﺲ ﺑﺄﺧ ﺬ أﺟ ﺰاء ﻣ ﻦ أوراق اﻟﻨﺒ ﺎت وزراﻋﺘﮭ ﺎ ﻋﻠ ﻰ اﻟﻮﺳ ﻂ اﻟﻐ ﺬاﺋﻲ ( MS) Murashige and Skoog اﻟﺤ ﺎوي ﻋﻠ ﻰ ﻣﻨﻈﻤ ﺎت اﻟﻨﻤ ﻮ ﺷ ﻤﻠﺖ2,4-Dichlorophenoxy acetic acid ) (2,4-D وBenzyl adinenine ) BA (ﺑﺎﻟﺘﺮ ﻛﯿﺰ 0.1 و0.5 ﻣﻠﻐﻢ / ﻟﺘﺮ ﻋﻠﻰ اﻟﺘ ﻮاﻟﻲ . اﻟﻤﻘﺪﻣﺔ وﺟﺪ ﺄنﺑ اﻟﻀﻮء ﯾﻌﻤﻞ ﻋﻠﻰ زﯾﺎدة اﻟﻤﺮﻛﺒﺎت اﻟﻔﯿﻨﻮﻟﯿﺔ داﺧﻞ اﻷﻧﺴ ﺠﺔ اﻟﻤﺰروﻋ ﺔ ﻓ ﻲ اﻟﻤ ﺰارع اﻟﻨﺴ ﯿﺠﯿﺔ ﻟﻠﻨﺒﺎت ﻛﻮﻧﮫ ﯾﺰﯾﺪ ﻣﻦ اﻟﻨﺸﺎط اﻷﻧﺰﯾﻤﻲ ﻋﺸﺮة أﺿﻌﺎف ) ﻣﻘﺎرﻧﺔ ﺑﺎﻟﻈﻼم ( ﻟﻠﺪور اﻟﺬي ﯾﺆدﯾﮫ اﻟﻀﻮء ﻓﻲ ﻋﻤﻠﯿ ﺔ اﻟﺒﻨﺎء اﻟﻀﻮﺋﻲ واﻟﻨﻤﻮ وﺣﺴﺐ اﺧﺘﻼف ﺷﺪﺗﮫ وطﻮل اﻟﻔﺘﺮة اﻟﻀﻮﺋﯿﮫ [1] . وﻗﺪ أدرك اﻟﻜﺜﯿﺮ ﻣﻦ اﻟﺒﺎﺣﺜﯿﻦ ﺗﺄﺛﯿﺮ اﻷﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ ﺳ ﻮاء ﻛﺎﻧ ﺖ ﻣﻔﯿ ﺪة أو ﻣﻀ ﺮة ﻟﻠﻨﺒﺎﺗ ﺎت إذ . إﻧﮭ ﺎ ﻗ ﺪ ﺗﺘﻠ ﻒ اﻟﻤ ﺎدة اﻟﻮراﺛﯿ ﺔ داﺧ ﻞ اﻟﺨﻠﯿ ﺔ اﻟﻨﺒﺎﺗﯿﺔ، ﻓﯿﺘﺎﺛﺮ ﻣﺤﺘﻮى اﻟﺨﻠﯿﺔ ﻣﻦ اﻟﺸﻔﺮات اﻟﻮراﺛﯿﺔ اﻟﺘﻲ ﺗﻨﻈﻢ ﻋﻤﻠﯿﺎﺗﮫ اﻟﺤﯿﻮﯾﺔ [2] . ﺑ ﺎﻟﻨﻈﺮ ﻟﻸھﻤﯿ ﺔ اﻟﻄﺒﯿ ﺔ ﻟﻨﺒﺎت اﻟﺮوﺟﺔ وﻻﺣﺘﻮاﺋﮫ ﻋﻠﻰ ﻣﺮﻛﺒﺎت ﺛﺎﻧﻮﯾﺔ ﻣﮭﻤﺔ ﺗ ﺪﺧﻞ ﻓ ﻲ اﻟﻌﻤﻠﯿ ﺎت اﻟﺼ ﯿﺪﻻﻧﯿﺔ وﻟﻜ ﻦ إﻧﺘﺎﺟ ﮫ ﻣﻨﮭ ﺎ ﻗﻠﯿ ﻞ ﻣﻘﺎرﻧﺔ ﺑﺎﻟﺤ ﺎﺟﺔ اﻟﻔﻌﻠﯿﺔ ﻟﮭ ﺬه اﻟﻤﺮﻛﺒ ﺎت . ﻟ ﺬا أ ﺳ ﺘﻮﺟﺐ اﻷﻣ ﺮ إﻛﺜ ﺎر اﻟﻨﺒ ﺎت ﻧﺴ ﯿﺠﯿﺎ وإﺿ ﺎﻓﺔ ﺑﻌ ﺾ اﻟﻤﺤﻔ ﺰات اﻟﺘﻲ ﻗﺪ ﺗﺰﯾﺪ ﻣﻦ إﻧﺘﺎج اﻟﻨﺒﺎت ﻟﻠﻤﺮﻛﺒﺎت اﻟﻔﻌﺎﻟﺔ . ﻋﻨﺪھﺎ ﯾﻤﻜﻦ ﺗﺒﻨﻲ ﺧﻄ ﻮط إﻧﺘﺎﺟﯿ ﺔ ﻓ ﻲ ﻣﻌﺎﻣ ﻞ اﻷدوﯾ ﺔ ﻹﻧﺘ ﺎج ھﺬه اﻟﻤﺮﻛﺒﺎت ﺑﻜﻤﯿﺎت ﻛﺒﯿﺮة وﺑﺎﺳﺘﺨﺪام اﻟﻤﻔﺎﻋﻼ ت اﻟﺤﯿﻮﯾﺔ . وﺑﻨ ﺎءا ﻋﻠ ﻰ ﻣﺎﺳ ﺒﻖ ﻓﻘ ﺪ ھ ﺪﻓﺖ اﻟﺪراﺳ ﺔ اﻟﺤﺎﻟﯿ ﺔ إﻟﻰ :- أﻣﻜﺎﻧﯿﺔ زﯾﺎدة إﻧﺘﺎج اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ ﻓﻲ أﻧﺴﺠﺔ اﻟﻨﺒﺎت ﺑﺎﻻﺳﺘﻔﺎدة ﻣﻦ ﺗﻘﺎﻧﺎت اﻟﺰراﻋﺔ اﻟﻨﺴﯿﺠﯿﺔ . اﻟﺘﺤﺮي ﻋﻦ ﻣﻘﺪار اﻟﺰﯾﺎدة اﻟﺤﺎﺻﻠﺔ ﻓﻲ ﺑﻌﺾ اﻟﻤﺮﻛﺒﺎت اﻟﻔﻌﺎﻟﺔ واﻟﻤﮭﻤﺔ ﺻﯿﺪﻻﻧﯿﺎ ﻧﺘﯿﺠ ﺔ ﻻﺳ ﺘﻌﻤﺎل ﺑﻌ ﺾ اﻟﻤﺤﻔ ﺰات ا ﻟﻔﯿﺰﯾﺎﺋﯿ ﺔ وﻣﻘﺎرﻧﺘﮭ ﺎ ﻣ ﻊ اﻟﻤﺮﻛﺒ ﺎت اﻟﻨﺒﺎﺗﯿ ﺔ ﻏﯿ ﺮ اﻟﻤﻌﺎﻣﻠ ﺔ ﻣﻨﮭ ﺎ . و اﻟﻜﺸ ﻒ ﻛﻤ ﺎ وﻧﻮﻋ ﺎ ﻋ ﻦ ﻣﺮﻛﺒ ﺎت اﻻﯾ ﺾ اﻟﺜﺎﻧﻮي اﻟﻤﻨﺘﺠﺔ ﻋﻦ طﺮﯾﻖ اﻟﺘﺤﻠﯿﻞ اﻟﻜﺮوﻣﺎﺗﻮﻛﺮاﻓﻲ ﺑﺎﺳﺘﺨﺪام ﺟﮭﺎز ﻛﺮوﻣﺎﺗﻮﻛﺮاﻓﯿﺎ اﻟﺴ ﺎﺋﻞ ذو اﻷداء اﻟﻌ ﺎﻟﻲ )HPLC .( اﻟﻤﻮاد وطﺮاﺋﻖ اﻟﻌﻤﻞ ﺗﺤﻀﯿﺮ اﻟﻤﺤﻠﻮل اﻟﺨﺰﯾﻦ : ﺣﻀ ﺮ اﻟﻤﺤﻠ ﻮل اﻟ ﺨ ﺰﯾﻦ ﻟﺠﻤﯿ ﻊ ﻣﻨﻈﻤ ﺎت اﻟﻨﻤ ﻮ اﻟﻨﺒﺎﺗﯿ ﺔ ﺑﻮاﻗ ﻊ 100 ﻣﻠﻐ ﻢ / ﻟﺘ ﺮ . وﺣﻔﻆ ﻓﻲ ﺣﺎﺿﻨﺔ ﻋﻠﻰ درﺟﺔ ﺣﺮارة 25 °م واﺳﺘﺒﺪل ﺷﮭﺮﯾﺎ ﺑﺘﺤﻀﯿﺮ ﻣﺤﻠﻮل ﺧﺰﯾﻦ ﺟﺪﯾﺪ . اﻟﻮﺳ ﻂ اﻟﻐ ﺬاﺋﻲ MS : ﺣﻀ ﺮ وﺳ ﻂMS [16] ﻣﺨﺘﺒﺮﯾ ﺎ ﻣ ﻦ ﻣﺠﻤﻮﻋ ﺔ اﻟﻌﻨﺎﺻ ﺮ اﻟﻜﺒ ﺮى واﻟﺼ ﻐﺮى وﻣﺼﺪر اﻟﺤﺪﯾﺪ وﻓﯿﺘﺎﻣﯿﻨﺎت وﺳﻜﺮوز , وأﺿﯿﻒ إﻟﯿﮫ ﻣﻨﻈﻤﺎت اﻟﻨﻤﻮ اﻟﻨﺒﺎﺗﯿ ﺔ وﺣﺴ ﺐ اﻟﺘﺮﻛﯿ ﺰ اﻟﻤﻄﻠ ﻮب . https://doi.org/10.30684/etj.32.2B.20 https://doi.org/10.30684/etj.32.2B.20 2412-0758/University of Technology-Iraq, Baghdad, Iraq This is an open access article under the CC BY 4.0 license http://creativecommons.org/licenses/by/4.0 ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 ﻧ ﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypercom triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ Ғƶ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 اﻟﺜﺎﻧﻮﯾﺔ وظﻔﺖ ﻋﻮاﻣﻞ ﺗﺤﻔﯿﺰ ﻓﯿﺰﯾﺎﺋﯿﺔ وﺗﻀﻤﻨﺖ اﻟﻀﻮء )Light ( واﻷﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ )Ultraviolet (, ﻣﺪد ﺗﻌ ﺮض اﻟﺰروﻋ ﺎت ﻟﻺﺿ ﺎءة واﻷﺷ ﻌﺔ ﻓ ﻮق اﻟ ﺒﻨﻔﺴ ﺠﯿﺔ ﻓﻜﺎﻧ ﺖ 24 ﺳ ﺎﻋﺔ ظ ﻼم , 12 ﺳ ﺎﻋﺔ ﺿ ﻮء , 16 ﺳﺎﻋﺔ ﺿﻮء , 24 ﺳﺎﻋﺔ ﺿﻮء إﻣﺎ اﻷﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ ﻛﺎﻧﺖ ﻣ ﺪة اﻟﺘﻌ ﺮﯾﺾ10 أو20 دﻗﯿﻘ ﺔ . أﻓﻀ ﻞ ﻣ ﺪة ﺗﻌﺮﯾﺾ ﻟﻺﺿﺎءة ﻟﺰﯾﺎدة إﻧﺘﺎج اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ 12 ﺳ ﺎﻋﺔ ﺿ ﻮء ﻟﻜ ﺎﻟﺲ اﻟﻨﺒ ﺎت . أﻣ ﺎ ﺑﺎﻟﻨﺴ ﺒﺔ ﻟﻸﺷ ﻌﺔ ﻓ ﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ ﻓﻘﺪ أﻋﻄﺖ ﻛﻼ اﻟﻤﺪﺗ ﯿﻦ ﺗﺤﻔﯿﺰ ﻟﻜﺎﻟﺲ ﻧﺒﺎت اﻟﺮوﺟﺔ ﻋﻠﻰ إﻧﺘﺎج اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ ﺑﺎﻟﺘﺴﺎوي . اﻟﻜﻠﻤﺎت اﻟﻤﻔﺘﺎﺣﯿﺔ : زراﻋﺔ اﻻﻧﺴﺠﺔ , ﻧﺒﺎت اﻟﺮوﺟﺔ . Hypericum triquetrifolium , ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي , HPLC اﻟﻤﻘﺪﻣﺔ اﻟﺘﻘﺪﯾﺮ اﻟﻜﻤﻲ واﻟﻨﻮﻋﻲ ﻟﻠﻨ ﻮاﺗﺞ اﻟﺜﺎﻧﻮﯾ ﺔ : اﺳ ﺘﻌﻤﻞ ﺟﮭ ﺎز ﻛﺮوﻣﺎﺗﻮﻏﺮاﻓﯿ ﺎ اﻟﺴ ﺎﺋﻞ ذي اﻷداء اﻟﻌ ﺎﻟﻲ HPLC ﻧ ﻮعSpectrophysics / UV-visible detector ﻓ ﻲ ﺗﻘ ﺪﯾﺮ ﻛﻤﯿ ﺔ وﻧﻮﻋﯿ ﺔ اﻟﻨ ﻮاﺗﺞ اﻟ ﺜﺎﻧﻮﯾ ﺔ ﻓ ﻲ ﻣﺴﺘﺨﻠﺼﺎت اﻷوراق واﻟﻜﺎﻟﺲ [6] وﻗﻮرﻧﺖ ﺑﺎﻟﻌﯿﻨﺎت اﻟﻘﯿﺎﺳﯿﺔ . ﻓﺼ ﻠﺖ اﻟﻤﻜﻮﻧ ﺎت اﻟﻔﻌﺎﻟ ﺔ ﻟﻨﺒ ﺎت اﻟﺮوﺟ ﺔ ﺗﺤ ﺖ اﻟﻈ ﺮوف اﻟﻘﯿﺎﺳ ﯿﺔ اﻟﺜﺎﺑﺘ ﺔ وﺣﺴ ﺐ [15] , إذ ﺣﻘﻨ ﺖ اﻟﻌﯿﻨﺎت ﻓﻲ ﻋﻤﻮد اﻟﻄﻮر اﻟﻤﻌﻜﻮس ﻧﻮع Reversed phase suspelcosil C-180D ذي أﺑﻌﺎد )50 × 4.6 mm. I.D (وﺣﺠﻢ اﻟ ﺪﻗ ﺎﺋﻖ µm 3 . ودرﺟﺔ ﺣﺮارة 30°C [11] وﻗ ﺪرت اﻟﻨ ﻮاﺗﺞ اﻟﺜﺎﻧﻮﯾ ﺔ ﻟﻤﺴ ﺘﺨﻠﺺ اﻷوراق واﻟﻜ ﺎﻟﺲ ﺑﺤﻘ ﻦ20 ﻣ ﺎﯾﻜﺮوﻟﯿﺘﺮ ﻓ ﻲ اﻟﻌﻤ ﻮد وﺗﺤ ﺖ اﻟﻈ ﺮوف اﻟﺘﺎﻟﯿ ﺔ :- اﻟﻄ ﻮر اﻟﻤﺘﺤ ﺮك : M 0.01 ﺧ ﻼت اﻻﻣﻮﻧﯿ ﻮمAmmonium acetate ﺑﻨﺴ ﺒﺔV/V) 40 : 60 ( ,ﺳ ﺮﻋﺔ اﻟﺠﺮﯾ ﺎن : 0.8 ﻣ ﻞ / دﻗﯿﻘ ﺔ , اﻟﻄ ﻮل أﻟﻤ ﻮﺟﻲ : nm 285 ﻧﺎﻧﻮﻣﯿﺘﺮ , درﺟﺔ ﺣﺮارة : 30 درﺟﺔ ﻣﺌﻮﯾﺔ , اﻟﻮﻗﺖ اﻟﻤﺴﺘﻐﺮق : 10 دﻗﺎﺋﻖ . ﺳ ﺠﻠﺖ اﻟﻘ ﺮاءات ﻋﻠ ﻰ اﻷط ﻮال اﻟﻤﻮﺟﯿ ﺔ وﺣﺴ ﺐ زﻣ ﻦ اﻻﺣﺘﺠ ﺎز Rt ﻟﻠﻤﺤﺎﻟﯿ ﻞ اﻟﻘﯿﺎﺳ ﯿﺔ واﻟﻌﯿﻨ ﺎت ﻤ اﻟ ﺪروﺳﺔ . ﻗﺪرت ﺗﺮاﻛﯿﺰ اﻟﻤﻮاد اﻟﻔﻌﺎﻟﺔ ﻛﻤﯿﺎ ﺑﻤﻘﺎرﻧﺔ ﻣﺴﺎﺣﺔ ﺣﺰﻣﺔ اﻟﻤﺎدة اﻟﻘﯿﺎﺳﯿﺔ ﻣﻊ ﻣﺴﺎﺣﺔ ﺣﺰﻣﺔ اﻟﻨ ﻤ ﻮذج ﺗﺤﺖ ﻧﻔﺲ اﻟﻈﺮوف ﺑﺎﺳﺘﺨﺪام اﻟﻘﺎﻧﻮن اﻟﺘﺎﻟﻲ :- ﻣﺴﺎﺣﺔ ﺣﺰﻣﺔ اﻟﻨﻤﻮذج Reversed phase suspelcosil C-180D ذي أﺑﻌﺎد )50 × 4.6 mm. I.D (وﺣﺠﻢ اﻟ ﺪﻗ ﺎﺋﻖ µm 3. ودرﺟﺔ ﺣﺮارة 30°C [11] وﻗ ﺪرت اﻟﻨ ﻮاﺗﺞ اﻟﺜﺎﻧﻮﯾ ﺔ ﻟﻤﺴ ﺘﺨﻠﺺ اﻷوراق واﻟﻜ ﺎﻟﺲ ﺑﺤﻘ ﻦ20 ﻣ ﺎﯾﻜﺮوﻟﯿﺘﺮ ﻓ ﻲ اﻟﻌﻤ ﻮد وﺗﺤ ﺖ اﻟﻈ ﺮوف اﻟﺘﺎﻟﯿ ﺔ :- اﻟﻄ ﻮر اﻟﻤﺘﺤ ﺮك : M 0.01 ﺧ ﻼت اﻻﻣﻮﻧﯿ ﻮمAmmonium acetate ﺑﻨﺴ ﺒﺔV/V) 40 : 60 ( ,ﺳ ﺮﻋﺔ اﻟﺠﺮﯾ ﺎن : 0.8 ﻣ ﻞ / دﻗﯿﻘ ﺔ , اﻟﻄ ﻮل أﻟﻤ ﻮﺟﻲ : nm 285 ﻧﺎﻧﻮﻣﯿﺘﺮ , درﺟﺔ ﺣﺮارة : 30 درﺟﺔ ﻣﺌﻮﯾﺔ , اﻟﻮﻗﺖ اﻟﻤﺴﺘﻐﺮق : 10 دﻗﺎﺋﻖ . اﻟﻤﻘﺪﻣﺔ ت ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﻤﺪروﺳﺔ زﻣﻦ اﻻﺣﺘﺠﺎز ) دﻗﯿﻘﺔ ( ﻣﺴﺎﺣﺔ اﻟﻤﺮﻛﺒﺎت اﻟﻘﯿﺎﺳﯿﺔ ﺗﺮﻛﯿﺰ اﻟﻤﺤﻠﻮل اﻟﻘﯿﺎﺳﻲ ) ﻣﺎﯾﻜﺮوﻏﺮام (ﻣﻞ \ 1 Catchin 1.01 14183 25 2 Hypersoid 1.82 20299 25 ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 ﻧ ﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypercom triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ ﻧ ﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypercom triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 اﺳﺘﺨ ﻼص اﻷوراق وﻛﺎﻟﺲ ﻧﺒﺎت اﻟﺮوﺟﮫ [12] :- وزن10 ﻣﻠﻐ ﻢ ﻣ ﻦ اﻟﻌﯿﻨ ﺎت ) اﻷوراق اﻟﺠﺎﻓ ﺔ , واﻟﻜ ﺎﻟﺲ ( ﻟﻨﺒﺎت ﻟﺮوﺟﮫ وأﺿﯿﻒ إﻟﯿﮭﺎ 10 ﻣﻞ ﻣﻦ اﻟﻤﯿﺜﺎﻧﻮل ﺗﺮﻛﯿﺰ 95 % ﻧﻮعHPLC grade . ﺣﻤﺾ ﺑﺈﺿﺎﻓﺔ ﺑﻀﻊ ﻗﻄﺮات ﻣﻦ ﺣ ﺎﻣﺾ أﻟﺨﻠﯿ ﻚ ﺗﺮﻛﯿ ﺰ 1 .% و ﺣ ﺮك اﻟﻨﻤ ﻮذج ﺑﻮاﺳ ﻄﺔ ﺟﮭ ﺎز اﻷﻣ ﻮاج ﻓ ﻮق اﻟﺼ ﻮﺗﯿﺔ ﻟﻤ ﺪة 10 دﻗﺎﺋﻖ . ﺑﻌﺪھﺎ رﻛﺰ اﻟﻤﺬﯾﺐ اﻟﺤﺎوي ﻋﻠﻰ اﻟﻤﻮاد اﻟﻔﻌﺎﻟﺔ ﺑﻮﺳﺎطﺔ ﺗﯿﺎر ﻣﻦ اﻟﻨﯿﺘﺮوﺟﯿﻦ ) (N2 ﻟﻠﻮﺻﻮل ﺑﺎﻟﺤﺠﻢ إﻟﻰ 0.5 .ﻣﻞ ﺗﻢ زﯾﺎدة اﻟﺘﺮﻛﯿﺰ ﻟﻠﻤﺬﯾﺐ اﻟﻤﺤﺘﻮي ﻋﻠﻰ اﻟﻤ ﻮاد اﻟﻔﻌﺎﻟ ﺔ ﺑﺈﺿ ﺎﻓﺔ ﺧ ﻼت اﻻﻣﻮ ﻧﯿ ﻮم وﺻ ﻮﻻ اﻟ ﻰ ﺣﺠ ﻢ 1 ﻣ ﻞ . ﺑﻌ ﺪھﺎ رﺷ ﺢ اﻟﺤﺠ ﻢ اﻷﺧﯿ ﺮ ﺑﻮﺳ ﺎطﺔ ورق ﺗﺮﺷ ﯿﺢ ﻗﯿ ﺎس 0.25 ﻣ ﺎﯾ ﻜﺮوﻣﯿﺘﺮ . و ﺣﻘ ﻦ 20 ﻣﺎﯾﻜﺮوﻟﯿﺘﺮ ﻓﻲ ﺟﮭﺎزHPLC ﺗﺤﺖ ظﺮوف اﻟﻔﺼﻞ اﻟﻤﺜﺒﺘﮫ ﻣﻦ ﻗﺒﻞ اﻟﻤﺼﻨﻊ . اﺳﺘﺨ ﻼص اﻷوراق وﻛﺎﻟﺲ ﻧﺒﺎت اﻟﺮوﺟﮫ [12] :- وزن10 ﻣﻠﻐ ﻢ ﻣ ﻦ اﻟﻌﯿﻨ ﺎت ) اﻷوراق اﻟﺠﺎﻓ ﺔ , واﻟﻜ ﺎﻟﺲ ( ﻟﻨﺒﺎت ﻟﺮوﺟﮫ وأﺿﯿﻒ إﻟﯿﮭﺎ 10 ﻣﻞ ﻣﻦ اﻟﻤﯿﺜﺎﻧﻮل ﺗﺮﻛﯿﺰ 95 % ﻧﻮعHPLC grade . ﺣﻤﺾ ﺑﺈﺿﺎﻓﺔ ﺑﻀﻊ ﻗﻄﺮات ﻣﻦ ﺣ ﺎﻣﺾ أﻟﺨﻠﯿ ﻚ ﺗﺮﻛﯿ ﺰ 1 .% و ﺣ ﺮك اﻟﻨﻤ ﻮذج ﺑﻮاﺳ ﻄﺔ ﺟﮭ ﺎز اﻷﻣ ﻮاج ﻓ ﻮق اﻟﺼ ﻮﺗﯿﺔ ﻟﻤ ﺪة 10 دﻗﺎﺋﻖ . ﺑﻌﺪھﺎ رﻛﺰ اﻟﻤﺬﯾﺐ اﻟﺤﺎوي ﻋﻠﻰ اﻟﻤﻮاد اﻟﻔﻌﺎﻟﺔ ﺑﻮﺳﺎطﺔ ﺗﯿﺎر ﻣﻦ اﻟﻨﯿﺘﺮوﺟﯿﻦ ) (N2 ﻟﻠﻮﺻﻮل ﺑﺎﻟﺤﺠﻢ إﻟﻰ 0.5 .ﻣﻞ ﺗﻢ زﯾﺎدة اﻟﺘﺮﻛﯿﺰ ﻟﻠﻤﺬﯾﺐ اﻟﻤﺤﺘﻮي ﻋﻠﻰ اﻟﻤ ﻮاد اﻟﻔﻌﺎﻟ ﺔ ﺑﺈﺿ ﺎﻓﺔ ﺧ ﻼت اﻻﻣﻮ ﻧﯿ ﻮم وﺻ ﻮﻻ اﻟ ﻰ ﺣﺠ ﻢ 1 ﻣ ﻞ . ﺑﻌ ﺪھﺎ رﺷ ﺢ اﻟﺤﺠ ﻢ اﻷﺧﯿ ﺮ ﺑﻮﺳ ﺎطﺔ ورق ﺗﺮﺷ ﯿﺢ ﻗﯿ ﺎس 0.25 ﻣ ﺎﯾ ﻜﺮوﻣﯿﺘﺮ . و ﺣﻘ ﻦ 20 ﻣﺎﯾﻜﺮوﻟﯿﺘﺮ ﻓﻲ ﺟﮭﺎزHPLC ﺗﺤﺖ ظﺮوف اﻟﻔﺼﻞ اﻟﻤﺜﺒﺘﮫ ﻣﻦ ﻗﺒﻞ اﻟﻤﺼﻨﻊ . اﻟﻤﻘﺪﻣﺔ ﻗﺪرت ﺗﺮاﻛﯿﺰ اﻟﻤﻮاد اﻟﻔﻌﺎﻟﺔ ﻛﻤﯿﺎ ﺑﻤﻘﺎرﻧﺔ ﻣﺴﺎﺣﺔ ﺣﺰﻣﺔ اﻟﻤﺎدة اﻟﻘﯿﺎﺳﯿﺔ ﻣﻊ ﻣﺴﺎﺣﺔ ﺣﺰﻣﺔ اﻟﻨ ﻤ ﻮذج ﺗﺤﺖ ﻧﻔﺲ اﻟﻈﺮوف ﺑﺎﺳﺘﺨﺪام اﻟﻘﺎﻧﻮن اﻟﺘﺎﻟﻲ :- ﻣﺴﺎﺣﺔ ﺣﺰﻣﺔ اﻟﻨﻤﻮذج ﺗﺮﻛﯿﺰ اﻟﻤﺎدة اﻟﻤﺠﮭﻮﻟﺔ = ------------------------ Χ ﺗﺮﻛﯿﺰ اﻟﻘﯿﺎﺳﻲΧ ﻋﺪد ﻣﺮات اﻟﺘﺨﻔﯿﻒ ﻣﺴﺎﺣﺔ ﺣﺰﻣﺔ اﻟﻘﯿﺎﺳﻲ اﻟﻨﺘﺎﺋﺞ واﻟﻤﻨﺎﻗﺸﺔ اﻟﺘﻘ ﺪﯾﺮ اﻟﻜﻤ ﻲ واﻟﻨ ﻮﻋﻲ ﻟ ﺒﻌﺾ اﻟﻤﺮﻛ ﺒ ﺎت اﻟﻜﯿﻤﯿﺎﺋﯿ ﺔ ﻓ ﻲ ﻣﺴﺘﺨﻠﺼ ﺎت أوراق ﻧﺒ ﺎت اﻟﺮوﺟ ﺔ : ﻋﻨ ﺪ ﺣﺴ ﺎب ﺗﺮﻛﯿﺰ اﻟﻤﺮﻛﺒﺎت اﻟﻘﯿﺎﺳﯿﺔ ﻛﻤﺎ ﻣﺒﯿﻦ ﺑﺎﻟﺠﺪوﻟﯿﻦ 1 و2 ﻛﺎﻧﺖ ﻣﻘﺎدﯾﺮھﺎ ﻓﻲ اﻷوراق إذ ﺳﺠﻞ Hypericin أﻋﻠﻰ ﻣﻌﺪل ﻟﻠﻤﺮﻛﺒﺎت ﻓﻲ اﻷوراق ﺑﻠﻎ 0.81174 ﻣﻠﻐﻢ ﻏﻢ / وﻓﻲ اﻟﻜﺎﻟﺲ ﺳﺠﻞ0.6899 ﻣﻠﻐﻢ .ﻏﻢ / [19] وﺟﺪا ﺑﺄن ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي وﺑﺎﻷﺧﺺ اﻟﮭﺎﯾﺒﺮﺳﯿﻦ ﻗﺪ اﺧﺘﻠﻒ ﺗﺮﻛﯿﺰھﺎ ﻓﻲ اﻟﻜﺎﻟﺲ اﻟﻤﺴﺘﺤﺚ ﻣﻨﮭﺎ ﻓ ﻲ اﻟﻨﺒ ﺎت اﻷم وﻋﺰﯾﺎ ذﻟﻚ إﻟﻰ أن اﺧﺘﻼف ظﺮوف زراﻋﺔ اﻷﻧﺴﺠﺔ ﻋﻦ اﻟﻈﺮوف اﻟﻄﺒﯿﻌﯿﺔ ﻓﻲ اﻟﺤﻘﻞ أدت إﻟﻰ ﺗﺤﻔﯿ ﺰ ﺧﻼﯾ ﺎ اﻟﻜ ﺎﻟﺲ ﻋﻠ ﻰ زﯾ ﺎدة إﻧﺘ ﺎج ﻣﺮﻛﺒ ﺎت اﻻﯾ ﺾ اﻟﺜ ﺎﻧﻮي ﻟﻠﻨﺒﺎﺗ ﺎت أو ﻗ ﺪ ﯾﺮﺟ ﻊ اﻟﺴ ﺒﺐ إﻟ ﻰ ﻋﻮاﻣ ﻞ إﺿ ﺎﻓﯿﺔ ﻣﺜ ﻞ ) اﻟﻀﻮء , اﻟﺮطﻮﺑﺔ , ﻧﻮع اﻟﺠﺰء اﻟﻨﺒﺎﺗﻲ , ﻣﺮﺣﻠﺔ ﻧﻤﻮ اﻟﻨﺒﺎت , واﻟﻌﻮاﻣﻞ اﻟﻮراﺛﯿﺔ (. [9] ﻋﻠﻞ اﻟﺴﺒﺐ إﻟﻰ ﻋﻮاﻣ ﻞ أﺧﺮى ﻣﺜﻞ ﻣﻜﺎن ﺗﻮاﺟﺪ اﻟﻨﺒﺎت وﺣﺎﻟﺔ اﻟﻨﺒﺎت اﻟﻔﺴﻠﺠﯿﺔ ﻗﺒﻞ اﻻﺳﺘﺨﻼص . أﻣﺎ [13] أﻛﺪوا أن ﻣﺮﻛﺒ ﺎت اﻻﯾ ﺾ اﻟﺜﺎﻧﻮي ﻛﺎﻧﺖ أﻋﻠﻰ ﻧﺴﺒﺔ ﻓﻲ ﻛﺎﻟﺲ اﻷوراق اﻟﻔﺘﯿﺔ ﻟﻨﺒﺎت اﻟﺮوﺟﮫ ﻣﻨﮭ ﺎ ﻓ ﻲ اﻟﻨﺒ ﺎت اﻷم وﻋﻠﻠ ﻮا اﻟﺴ ﺒﺐ إﻟ ﻰ أن ﻣﺮﺣﻠﺔ ﻧﺸﻮء اﻟﺰروﻋﺎت ﻣﻦ اﺳﺘﺤﺜﺎث وأداﻣ ﮫ وﺗﻀ ﻤﯿﻦ ﻟﻤﻨﻈﻤ ﺎت اﻟﻨﻤ ﻮ رﺑﻤ ﺎ أدت إﻟ ﻰ ﺗﺤﻔﯿ ﺰ اﻟﺨﻼﯾ ﺎ ﻋﻠ ﻰ زﯾﺎدة إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي . ﺟﺪول )1 (زﻣﻦ اﻻﺣﺘﺠﺎز ا , ﻟﻤﺴﺎﺣﺔ , ﺗﺮاﻛﯿﺰ اﻟﻤﺮﻛﺒﺎت اﻟﻘﯿﺎﺳﯿﺔ ﻟﻤﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي اﻟﺘﻲ ظﮭﺮت ﻣﻦ ﺗﺤﻠﯿﻞ HPLC . اﻟﻤﻘﺪﻣﺔ ﻋ ﺪل اﻟ ﺮﻗﻢ اﻟﮭﯿ ﺪروﺟﯿﻨﻲ إﻟ ﻰ 5.7 ﺑﺈﺿ ﺎﻓﺔ ﻗﻄ ﺮات ﻣ ﻦ ھﯿﺪروﻛﺴ ﯿﺪ اﻟﺼ ﻮدﯾﻮم ﻋﯿ ﺎري0.1 أو ﺣ ﺎﻣﺾ اﻟﮭﺎﯾﺪروﻛﻠﻮرﯾﻚ ﻋﯿﺎري 0.1 ﺛﻢ أﺿﯿﻒ إﻟﯿﮫ اﻻﻛﺎر (Agar-Agar) وﺣﺴﺐ اﻟﺘﺮﻛﯿﺰ اﻟﻤﻄﻠ ﻮب , ووزﻋ ﺖ اﻷوﺳﺎط ﻓﻲ ﻗﻨﺎﻧﻲ زﺟﺎﺟﯿﺔ (Universal tubes) . اﻟﻤﻮاد وطﺮاﺋﻖ اﻟﻌﻤﻞ ﺗﺤﻀﯿﺮ اﻟﻤﺤﻠﻮل اﻟﺨﺰﯾﻦ : ﺣﻀ ﺮ اﻟﻤﺤﻠ ﻮل اﻟ ﺨ ﺰﯾﻦ ﻟﺠﻤﯿ ﻊ ﻣﻨﻈﻤ ﺎت اﻟﻨﻤ ﻮ اﻟﻨﺒﺎﺗﯿ ﺔ ﺑﻮاﻗ ﻊ 100 ﻣﻠﻐ ﻢ / ﻟﺘ ﺮ . وﺣﻔﻆ ﻓﻲ ﺣﺎﺿﻨﺔ ﻋﻠﻰ درﺟﺔ ﺣﺮارة 25 °م واﺳﺘﺒﺪل ﺷﮭﺮﯾﺎ ﺑﺘﺤﻀﯿﺮ ﻣﺤﻠﻮل ﺧﺰﯾﻦ ﺟﺪﯾﺪ . ﺬ ﻲ م اﻟﻮﺳ ﻂ اﻟﻐ ﺬاﺋﻲ MS : ﺣﻀ ﺮ وﺳ ﻂMS [16] ﻣﺨﺘﺒﺮﯾ ﺎ ﻣ ﻦ ﻣﺠﻤﻮﻋ ﺔ اﻟﻌﻨﺎﺻ ﺮ اﻟﻜﺒ ﺮى واﻟﺼ ﻐﺮى وﻣﺼﺪر اﻟﺤﺪﯾﺪ وﻓﯿﺘﺎﻣﯿﻨﺎت وﺳﻜﺮوز , وأﺿﯿﻒ إﻟﯿﮫ ﻣﻨﻈﻤﺎت اﻟﻨﻤﻮ اﻟﻨﺒﺎﺗﯿ ﺔ وﺣﺴ ﺐ اﻟﺘﺮﻛﯿ ﺰ اﻟﻤﻄﻠ ﻮب . ﻋ ﺪل اﻟ ﺮﻗﻢ اﻟﮭﯿ ﺪروﺟﯿﻨﻲ إﻟ ﻰ 5.7 ﺑﺈﺿ ﺎﻓﺔ ﻗﻄ ﺮات ﻣ ﻦ ھﯿﺪروﻛﺴ ﯿﺪ اﻟﺼ ﻮدﯾﻮم ﻋﯿ ﺎري0.1 أو ﺣ ﺎﻣﺾ اﻟﮭﺎﯾﺪروﻛﻠﻮرﯾﻚ ﻋﯿﺎري 0.1 ﺛﻢ أﺿﯿﻒ إﻟﯿﮫ اﻻﻛﺎر (Agar-Agar) وﺣﺴﺐ اﻟﺘﺮﻛﯿﺰ اﻟﻤﻄﻠ ﻮب , ووزﻋ ﺖ اﻷوﺳﺎط ﻓﻲ ﻗﻨﺎﻧﻲ زﺟﺎﺟﯿﺔ (Universal tubes) . ﻲ ﻲ () ا ﺳﺘﺤﺜﺎث اﻟﻜﺎﻟﺲ : اﻋﺘﻤ ﺪت اﻟﺘﻮﻟﯿﻔ ﺔ BA ﺑﺘﺮﻛﯿ ﺰ 0.5 ﻣﻠﻐ ﻢ / ﻟﺘ ﺮ و 2,4-D ﺑﺘﺮﻛﯿ ﺰ0.1 ﻣﻠﻐ ﻢ / ﻟﺘ ﺮ واﻟﺘﻲ ﻛﺎﻧﺖ اﻷﻓﻀ ﻞ ﻓ ﻲ إﻋﻄ ﺎء أﻋﻠ ﻰ ﻧﺴ ﺒﺔ ﻣ ﻦ ﻛ ﺎﻟﺲ ﻧﺒ ﺎت اﻟﺮوﺟ ﮫ وﻛ ﺮرت ﻋﻤﻠﯿ ﺔ إﻋ ﺎدة اﻟﺰراﻋ ﺔ Re culture) ( ﻛﻞ8 أﺳﺎﺑﯿﻊ واﻋﺘﻤﺪ ھﺬا اﻟﻮﺳﻂ ﻛﻮﺳﻂ إداﻣﺔ . ﻊ اﻟﺘﻌﺮﯾﺾ ﻟﻔﺘﺮات إﺿﺎءة ﻣﺨﺘﻠ : ﻔﺔ وزﻋ ﺖ أوزان ﻣﻘ ﺪارھﺎ 60 ﻣﻠﻐ ﻢ ﻣ ﻦ ﻛ ﺎﻟﺲ ﻧﺒ ﺎت اﻟﺮوﺟ ﺔ , ﻋﻠ ﻰ أوﺳ ﺎط ﺟﺪﯾﺪة اﺣﺘﻮت ﻋﻠﻰ ﻧﻔﺲ ﻣﻜﻮﻧﺎت وﺳﻂ إداﻣﺔ اﻟﻜﺎﻟﺲ , وﻗﺴﻤﺖ إﻟ ﻰ 4 ﻣﺠ ﺎﻣﯿﻊ وﻛ ﻞ ﻣﺠﻤﻮﻋ ﺔ ﺣﻀ ﻨﺖ ﺗﺤ ﺖ اﻟﻈﺮوف اﻋﻼه ﻣﻊ اﺧﺘﻼف زﻣﻦ اﻟﺘﻌﺮض ﻟﻺﺿﺎءة ﺣﯿﺚ ﻛﺎﻧﺖ 24 ﺳﺎﻋﺔ ظﻼم أو 12 ﺳﺎﻋﺔ ﺿ ﻮء أو 16 ﺳﺎﻋﺔ ﺿﻮء أو 24 ﺳﺎﻋﺔ ﺿﻮء ﻣﺴﺘﻤﺮ . اﻟﺘﻌﺮض ﻟﻸﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ : اﺳﺘﺨﺪﻣﺖ ﻧﻔﺲ ﻣﻜﻮﻧﺎت وﺳﻂ إداﻣﺔ اﻟﻜﺎﻟﺲ وﻗﺴﻤﺖ إﻟﻰ ﻣﺠﻤﻮﻋﺘﯿﻦ وﻛ ﻞ ﻣﺠﻤﻮﻋﺔ ﻋﺮﺿﺖ إﻟﻰ اﻷﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ ﻧﻮع UV–c ذات اﻷﻣ ﻮاج اﻟﻘﺼ ﯿﺮة ﺑﻄ ﻮل ﻣ ﻮﺟﻲ 100 – 280 ﻧﺎﻧﻮﻣﯿﺘﺮ وﺑﻄﺎﻗﺔ ) 4.43 – 12.4 (ﻓﻮﻟﺖ , وﺑﻔﺘﺮات ﺗﻌﺮﯾﺾ 10 – 20 دﻗﯿﻘﺔ . اﻟﻤﻘﺪﻣﺔ 2 ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 ﻧ ﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypercom triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ 3 اﺳﺘﺨ ﻼص اﻷوراق وﻛﺎﻟﺲ ﻧﺒﺎت اﻟﺮوﺟﮫ [12] :- وزن10 ﻣﻠﻐ ﻢ ﻣ ﻦ اﻟﻌﯿﻨ ﺎت ) اﻷوراق اﻟﺠﺎﻓ ﺔ , واﻟﻜ ﺎﻟﺲ ( ﻟﻨﺒﺎت ﻟﺮوﺟﮫ وأﺿﯿﻒ إﻟﯿﮭﺎ 10 ﻣﻞ ﻣﻦ اﻟﻤﯿﺜﺎﻧﻮل ﺗﺮﻛﯿﺰ 95 % ﻧﻮعHPLC grade . ﺣﻤﺾ ﺑﺈﺿﺎﻓﺔ ﺑﻀﻊ ﻗﻄﺮات ﻣﻦ ﺣ ﺎﻣﺾ أﻟﺨﻠﯿ ﻚ ﺗﺮﻛﯿ ﺰ 1 .% و ﺣ ﺮك اﻟﻨﻤ ﻮذج ﺑﻮاﺳ ﻄﺔ ﺟﮭ ﺎز اﻷﻣ ﻮاج ﻓ ﻮق اﻟﺼ ﻮﺗﯿﺔ ﻟﻤ ﺪة 10 دﻗﺎﺋﻖ . ﺑﻌﺪھﺎ رﻛﺰ اﻟﻤﺬﯾﺐ اﻟﺤﺎوي ﻋﻠﻰ اﻟﻤﻮاد اﻟﻔﻌﺎﻟﺔ ﺑﻮﺳﺎطﺔ ﺗﯿﺎر ﻣﻦ اﻟﻨﯿﺘﺮوﺟﯿﻦ ) (N2 ﻟﻠﻮﺻﻮل ﺑﺎﻟﺤﺠﻢ إﻟﻰ 0.5 .ﻣﻞ ﺗﻢ زﯾﺎدة اﻟﺘﺮﻛﯿﺰ ﻟﻠﻤﺬﯾﺐ اﻟﻤﺤﺘﻮي ﻋﻠﻰ اﻟﻤ ﻮاد اﻟﻔﻌﺎﻟ ﺔ ﺑﺈﺿ ﺎﻓﺔ ﺧ ﻼت اﻻﻣﻮ ﻧﯿ ﻮم وﺻ ﻮﻻ اﻟ ﻰ ﺣﺠ ﻢ 1 ﻣ ﻞ . ﺑﻌ ﺪھﺎ رﺷ ﺢ اﻟﺤﺠ ﻢ اﻷﺧﯿ ﺮ ﺑﻮﺳ ﺎطﺔ ورق ﺗﺮﺷ ﯿﺢ ﻗﯿ ﺎس 0.25 ﻣ ﺎﯾ ﻜﺮوﻣﯿﺘﺮ . و ﺣﻘ ﻦ 20 ﻣﺎﯾﻜﺮوﻟﯿﺘﺮ ﻓﻲ ﺟﮭﺎزHPLC ﺗﺤﺖ ظﺮوف اﻟﻔﺼﻞ اﻟﻤﺜﺒﺘﮫ ﻣﻦ ﻗﺒﻞ اﻟﻤﺼﻨﻊ . اﻟﺘﻘﺪﯾﺮ اﻟﻜﻤﻲ واﻟﻨﻮﻋﻲ ﻟﻠﻨ ﻮاﺗﺞ اﻟﺜﺎﻧﻮﯾ ﺔ : اﺳ ﺘﻌﻤﻞ ﺟﮭ ﺎز ﻛﺮوﻣﺎﺗﻮﻏﺮاﻓﯿ ﺎ اﻟﺴ ﺎﺋﻞ ذي اﻷداء اﻟﻌ ﺎﻟﻲ HPLC ﻧ ﻮعSpectrophysics / UV-visible detector ﻓ ﻲ ﺗﻘ ﺪﯾﺮ ﻛﻤﯿ ﺔ وﻧﻮﻋﯿ ﺔ اﻟﻨ ﻮاﺗﺞ اﻟ ﺜﺎﻧﻮﯾ ﺔ ﻓ ﻲ ﻣﺴﺘﺨﻠﺼﺎت اﻷوراق واﻟﻜﺎﻟﺲ [6] وﻗﻮرﻧﺖ ﺑﺎﻟﻌﯿﻨﺎت اﻟﻘﯿﺎﺳﯿﺔ . ﻓﺼ ﻠﺖ اﻟﻤﻜﻮﻧ ﺎت اﻟﻔﻌﺎﻟ ﺔ ﻟﻨﺒ ﺎت اﻟﺮوﺟ ﺔ ﺗﺤ ﺖ اﻟﻈ ﺮوف اﻟﻘﯿﺎﺳ ﯿﺔ اﻟﺜﺎﺑﺘ ﺔ وﺣﺴ ﺐ [15] , إذ ﺣﻘﻨ ﺖ اﻟﻌﯿﻨﺎت ﻓﻲ ﻋﻤﻮد اﻟﻄﻮر اﻟﻤﻌﻜﻮس ﻧﻮع Reversed phase suspelcosil C-180D ذي أﺑﻌﺎد )50 × 4.6 mm. I.D (وﺣﺠﻢ اﻟ ﺪﻗ ﺎﺋﻖ µm 3 . ودرﺟﺔ ﺣﺮارة 30°C [11] وﻗ ﺪرت اﻟﻨ ﻮاﺗﺞ اﻟﺜﺎﻧﻮﯾ ﺔ ﻟﻤﺴ ﺘﺨﻠﺺ اﻷوراق واﻟﻜ ﺎﻟﺲ ﺑﺤﻘ ﻦ20 ﻣ ﺎﯾﻜﺮوﻟﯿﺘﺮ ﻓ ﻲ اﻟﻌﻤ ﻮد وﺗﺤ ﺖ اﻟﻈ ﺮوف اﻟﺘﺎﻟﯿ ﺔ :- اﻟﻄ ﻮر اﻟﻤﺘﺤ ﺮك : M 0.01 ﺧ ﻼت اﻻﻣﻮﻧﯿ ﻮمAmmonium acetate ﺑﻨﺴ ﺒﺔV/V) 40 : 60 ( ,ﺳ ﺮﻋﺔ اﻟﺠﺮﯾ ﺎن : 0.8 ﻣ ﻞ / دﻗﯿﻘ ﺔ , اﻟﻄ ﻮل أﻟﻤ ﻮﺟﻲ : nm 285 ﻧﺎﻧﻮﻣﯿﺘﺮ , درﺟﺔ ﺣﺮارة : 30 درﺟﺔ ﻣﺌﻮﯾﺔ , اﻟﻮﻗﺖ اﻟﻤﺴﺘﻐﺮق : 10 دﻗﺎﺋﻖ . ﺳ ﺠﻠﺖ اﻟﻘ ﺮاءات ﻋﻠ ﻰ اﻷط ﻮال اﻟﻤﻮﺟﯿ ﺔ وﺣﺴ ﺐ زﻣ ﻦ اﻻﺣﺘﺠ ﺎز Rt ﻟﻠﻤﺤﺎﻟﯿ ﻞ اﻟﻘﯿﺎﺳ ﯿﺔ واﻟﻌﯿﻨ ﺎت ﻤ اﻟ ﺪروﺳﺔ . اﻟﻤﻘﺪﻣﺔ ت ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﻤﺪروﺳﺔ زﻣﻦ اﻻﺣﺘﺠﺎز ) دﻗﯿﻘﺔ ( ﻣﺴﺎﺣﺔ اﻟﻤﺮﻛﺒﺎت اﻟﻘﯿﺎﺳﯿﺔ ﺗﺮﻛﯿﺰ اﻟﻤﺤﻠﻮل اﻟﻘﯿﺎﺳﻲ ) ﻣﺎﯾﻜﺮوﻏﺮام (ﻣﻞ \ 1 Catchin 1.01 14183 25 2 Hypersoid 1.82 20299 25 3 2 ﻧ ﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypercom triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 ﻧ ﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypercom triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ 3 Hypericin 2.99 18011 25 4 Pseudohyperi cin 3.97 16950 25 5 Hyperforin 4.67 15646 25 6 Prenylated phlorglucin 5.52 14279 25 ﺟﺪول )2 ( اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ ) ﻣﻠﻐﻢ ( ﻏﻢ / ﻓﻲ اﻷوراق اﻟﻤﺠﻔﻔﺔ وﻛﺎﻟﺲ ﻧﺒﺎت اﻟﺮوﺟﺔ n=3 . اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ اﻷوراق اﻟﺠﺎﻓﺔ اﻟﻜﺎﻟﺲT ﻗﯿﻤﺔ P≤0.05 Catchin 0.03065 0.16570 0.018 Hypersoid 0.08385 0.09482 0.004 Hypericin 0.81174 0.6899 0.025 Pseudohypericin 0.13581 0.1785 0.0092 Hyperforin 0.01917 0.1796 0.037 Prenylated phlorglucin 0.11583 0.2275 0.014 ﺗﺄﺛﯿﺮ ﻣﺪة اﻹﺿﺎءة ) ﺳﺎﻋﺔ ( ﻓﻲ إﻧﺘﺎج اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ ﻣﻦ ﻛﺎﻟﺲ ﻧﺒﺎت اﻟﺮوﺟ ﺔ واﻟﺘﻘ ﺪﯾﺮ اﻟﻜﻤ ﻲ وا ﻟﻨ ﻮﻋﻲ ﻟﮭﺎ : ﻋﻨﺪ ﺣﺴﺎب ﺗﺮﻛﯿﺰ اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ اﻋﺘﻤ ﺎدا ﻋﻠ ﻰ ﻣ ﺪة اﻹﺿ ﺎءة وﻣﻼﺣﻈ ﺔ اﻟﺠ ﺪول 3 وﻟ ﻮﺣﻆ ھﻨ ﺎك ﻓﺮوﻗﺎ ﻣﻌﻨﻮﯾﺔ ﺑﯿﻦ ﺗﺮاﻛﯿﺰ اﻟﻤﺮﻛﺐ اﻟﻮاﺣﺪ ﻣﻘﺎرﻧﺔ ﺑ ﻤﻌﺎﻣﻠﺔ اﻟﺴ ﯿﻄﺮة , إذ وﺟ ﺪ ارﺗﻔ ﺎع ﺗﺮﻛﯿ ﺰ اﻏﻠ ﺐ اﻟﻤﺮﻛﺒ ﺎت اﻟﺜﺎﻧﻮ ﯾ ﺔ ﻋﻨ ﺪ ﺗﻌ ﺮﯾﺾ اﻟﻜ ﺎﻟﺲ إﻟ ﻰ اﻟﻀ ﻮء ﻟﻤ ﺪة 12 ﺳ ﺎﻋﺔ , ﺑﻠ ﻎ أوزان اﻟﻤﺮﻛﺒ ﺎت 0.14156, 1.1025, 0.3278, 0.9242 ﻣﻠﻐ ﻢ / ﻏ ﻢ ﻟﻠﻤﺮﻛﺒ ﺎت Hypersoid, Hypericin, Pseudohypericin, Hyperforin ﻋﻠﻰ اﻟﺘﻮاﻟﻲ , ارﺗﻔﻊ ﺗﺮﻛﯿﺰ اﻟﻤﺮﻛﺐ Catchin إﻟﻰ0.19482 ﻣﻠﻐﻢ / ﻏﻢ ﻋﻨﺪ ﺗﻌ ﺮﯾﺾ اﻟﻜ ﺎﻟﺲ ﻟﻠﻀ ﻮء ﻟﻤ ﺪة 24 ﺳ ﺎﻋ , ﺔ أﻣ ﺎ ﻣﺮﻛ ﺐ Prenylated phlorglucin وﺻ ﻞ ﺗﺮﻛﯿ ﺰه إﻟ ﻰ0.2275 ﻣﻠﻐ ﻢ / ﻏ ﻢ ﻋﻨﺪﻣﺎ ﺣﻀﻦ ﻓﻲ اﻟﻈﻼم . وﯾﻼﺣﻆ أن أﻓﻀﻞ ﻣﺪة ﺗﻌﺮﯾﺾ ﻟﻺﺿﺎءة ھﻲ 12 ﺳ ﺎﻋﺔ ﺿ ﻮء ﻟﺰﯾ ﺎدة إﻧﺘ ﺎج اﻏﻠ ﺐ اﻟﻤﺮﻛﺒ ﺎت اﻟﺜﺎﻧﻮﯾ ﺔ ﻓ ﻲ ﻛ ﺎﻟﺲ اﻟﻨﺒ ﺎت . اﻟﻤﻘﺪﻣﺔ اﻟﻨﺘﺎﺋﺞ واﻟﻤﻨﺎﻗﺸﺔ اﻟﺘﻘ ﺪﯾﺮ اﻟﻜﻤ ﻲ واﻟﻨ ﻮﻋﻲ ﻟ ﺒﻌﺾ اﻟﻤﺮﻛ ﺒ ﺎت اﻟﻜﯿﻤﯿﺎﺋﯿ ﺔ ﻓ ﻲ ﻣﺴﺘﺨﻠﺼ ﺎت أوراق ﻧﺒ ﺎت اﻟﺮوﺟ ﺔ : ﻋﻨ ﺪ ﺣﺴ ﺎب ﺗﺮﻛﯿﺰ اﻟﻤﺮﻛﺒﺎت اﻟﻘﯿﺎﺳﯿﺔ ﻛﻤﺎ ﻣﺒﯿﻦ ﺑﺎﻟﺠﺪوﻟﯿﻦ 1 و2 ﻛﺎﻧﺖ ﻣﻘﺎدﯾﺮھﺎ ﻓﻲ اﻷوراق إذ ﺳﺠﻞ Hypericin أﻋﻠﻰ ﻣﻌﺪل ﻟﻠﻤﺮﻛﺒﺎت ﻓﻲ اﻷوراق ﺑﻠﻎ 0.81174 ﻣﻠﻐﻢ ﻏﻢ / وﻓﻲ اﻟﻜﺎﻟﺲ ﺳﺠﻞ0.6899 ﻣﻠﻐﻢ .ﻏﻢ / [19] وﺟﺪا ﺑﺄن ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي وﺑﺎﻷﺧﺺ اﻟﮭﺎﯾﺒﺮﺳﯿﻦ ﻗﺪ اﺧﺘﻠﻒ ﺗﺮﻛﯿﺰھﺎ ﻓﻲ اﻟﻜﺎﻟﺲ اﻟﻤﺴﺘﺤﺚ ﻣﻨﮭﺎ ﻓ ﻲ اﻟﻨﺒ ﺎت اﻷم وﻋﺰﯾﺎ ذﻟﻚ إﻟﻰ أن اﺧﺘﻼف ظﺮوف زراﻋﺔ اﻷﻧﺴﺠﺔ ﻋﻦ اﻟﻈﺮوف اﻟﻄﺒﯿﻌﯿﺔ ﻓﻲ اﻟﺤﻘﻞ أدت إﻟﻰ ﺗﺤﻔﯿ ﺰ ﺧﻼﯾ ﺎ اﻟﻜ ﺎﻟﺲ ﻋﻠ ﻰ زﯾ ﺎدة إﻧﺘ ﺎج ﻣﺮﻛﺒ ﺎت اﻻﯾ ﺾ اﻟﺜ ﺎﻧﻮي ﻟﻠﻨﺒﺎﺗ ﺎت أو ﻗ ﺪ ﯾﺮﺟ ﻊ اﻟﺴ ﺒﺐ إﻟ ﻰ ﻋﻮاﻣ ﻞ إﺿ ﺎﻓﯿﺔ ﻣﺜ ﻞ ) اﻟﻀﻮء , اﻟﺮطﻮﺑﺔ , ﻧﻮع اﻟﺠﺰء اﻟﻨﺒﺎﺗﻲ , ﻣﺮﺣﻠﺔ ﻧﻤﻮ اﻟﻨﺒﺎت , واﻟﻌﻮاﻣﻞ اﻟﻮراﺛﯿﺔ (. [9] ﻋﻠﻞ اﻟﺴﺒﺐ إﻟﻰ ﻋﻮاﻣ ﻞ أﺧﺮى ﻣﺜﻞ ﻣﻜﺎن ﺗﻮاﺟﺪ اﻟﻨﺒﺎت وﺣﺎﻟﺔ اﻟﻨﺒﺎت اﻟﻔﺴﻠﺠﯿﺔ ﻗﺒﻞ اﻻﺳﺘﺨﻼص . أﻣﺎ [13] أﻛﺪوا أن ﻣﺮﻛﺒ ﺎت اﻻﯾ ﺾ اﻟﺜﺎﻧﻮي ﻛﺎﻧﺖ أﻋﻠﻰ ﻧﺴﺒﺔ ﻓﻲ ﻛﺎﻟﺲ اﻷوراق اﻟﻔﺘﯿﺔ ﻟﻨﺒﺎت اﻟﺮوﺟﮫ ﻣﻨﮭ ﺎ ﻓ ﻲ اﻟﻨﺒ ﺎت اﻷم وﻋﻠﻠ ﻮا اﻟﺴ ﺒﺐ إﻟ ﻰ أن ﻣﺮﺣﻠﺔ ﻧﺸﻮء اﻟﺰروﻋﺎت ﻣﻦ اﺳﺘﺤﺜﺎث وأداﻣ ﮫ وﺗﻀ ﻤﯿﻦ ﻟﻤﻨﻈﻤ ﺎت اﻟﻨﻤ ﻮ رﺑﻤ ﺎ أدت إﻟ ﻰ ﺗﺤﻔﯿ ﺰ اﻟﺨﻼﯾ ﺎ ﻋﻠ ﻰ زﯾﺎدة إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي . ﺟﺪول )1 (زﻣﻦ اﻻﺣﺘﺠﺎز ا , ﻟﻤﺴﺎﺣﺔ , ﺗﺮاﻛﯿﺰ اﻟﻤﺮﻛﺒﺎت اﻟﻘﯿﺎﺳﯿﺔ ﻟﻤﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي اﻟﺘﻲ ظﮭﺮت ﻣﻦ ﺗﺤﻠﯿﻞ HPLC . ت ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﻤﺪروﺳﺔ زﻣﻦ اﻻﺣﺘﺠﺎز ) دﻗﯿﻘﺔ ( ﻣﺴﺎﺣﺔ اﻟﻤﺮﻛﺒﺎت اﻟﻘﯿﺎﺳﯿﺔ ﺗﺮﻛﯿﺰ اﻟﻤﺤﻠﻮل اﻟﻘﯿﺎﺳﻲ ) ﻣﺎﯾﻜﺮوﻏﺮام (ﻣﻞ \ 1 Catchin 1.01 14183 25 2 Hypersoid 1.82 20299 25 ﺟﺪول )1 (زﻣﻦ اﻻﺣﺘﺠﺎز ا , ﻟﻤﺴﺎﺣﺔ , ﺗﺮاﻛﯿﺰ اﻟﻤﺮﻛﺒﺎت اﻟﻘﯿﺎﺳﯿﺔ ﻟﻤﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي اﻟﺘﻲ ظﮭﺮت ﻣﻦ ﺗﺤﻠﯿﻞ HPLC . اﻟﻤﻘﺪﻣﺔ وﻣ ﻦ ھ ﺬا ﻧﺠ ﺪ أن ﻟﻄ ﻮل اﻟﻔﺘ ﺮة اﻟﻀ ﻮﺋﯿﺔ ﻗﺎﺑﻠﯿ ﺔ ﻋﻠ ﻰ ﺗﺤﻔﯿ ﺰ وإﻧﺘ ﺎج اﻟﻤﺮﻛﺒﺎت ا ﻟﺜﺎﻧﻮﯾﺔ ﻓﻲ ﻧﺒﺎت اﻟﺮوﺟﺔ وﺑﺎﻷﺧﺺ ﻟﻤﺮﻛﺐ Hypericin وھﺬا ﻣﺎ أﺷ ﺎر إﻟﯿ ﮫ ]10 ; 14 ; 18 [ ﺣﯿﺚ ﯾﻌﺪ اﻟﻀﻮء ﻋﺎﻣﻞ أوﻟﻲ ﯾﺤﻔﺰ ﻗﺎﺑﻠﯿﺔ اﻟﻤﺰارع اﻟﻨﺴﯿﺠﯿﺔ ﻟﺘﻜ ﻮﯾﻦ اﻟﻜﻠﻮروﻓﯿ ﻞ واﻟﺼ ﺒﻐﺎت اﻷﺧ ﺮى ] 8 [ . وذﻛﺮ ] 3 [ ﺑ ﺎن ﻣﺴ ﺘﻮى Hypericin ﺗﻀ ﺎﻋﻔﺖ ﻓ ﻲ اﻟﻈ ﻼم ﻣﻘﺎرﻧ ﺔ ﺑﺎﻟﻀ ﻮء ﻟﻜ ﻦ ﺳ ﺠﻞ ﺑﻌ ﺾ اﻟﺒ ﺎﺣﺜﯿﻦ ﺗﺄﺛﯿﺮات ﻣﻐﺎﯾﺮة ﺣﯿﺚ وﺟﺪ أن ﺗﺄﺛﯿﺮ ﻣﺪة اﻹﺿﺎءة أدى إﻟﻰ زﯾﺎدة ﻣﺴﺘﻮى Hypericin ﻓﻲ ﻧﺒﺎت اﻟﺮوﺟﺔ ] 5 ; 4 ; 7 ; 17 [ إذ أﻛ ﺪوا ﺑ ﺎن ﻣ ﺪة اﻹﺿ ﺎءة ﻛ ﺎن ﻟﮭ ﺎ اﻟﺤ ﺎﻓﺰ اﻷﻛﺒ ﺮ ﻋﻠ ﻰ زﯾ ﺎدة إﻧﺘ ﺎج ﻣﺮﻛﺒ ﺎت اﻻﯾ ﺾ اﻟﺜﺎﻧﻮي ﻓﻲ ﻧﺒﺎت اﻟﺮوﺟﺔ إذ ﯾﻌﻤﻞ اﻟﻀﻮء ﻋﻠﻰ زﯾﺎدة إﻧﺘﺎج ﺻﺒﻐﺔ اﻟﮭ ﺎﯾﺒﺮﺳﯿﻦ ﻓﻲ اﻟﻨﺒﺎت . ﺗﺄﺛﯿﺮ ﻣﺪة اﻹﺿﺎءة ) ﺳﺎﻋﺔ ( ﻓﻲ إﻧﺘﺎج اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ ﻣﻦ ﻛﺎﻟﺲ ﻧﺒﺎت اﻟﺮوﺟ ﺔ واﻟﺘﻘ ﺪﯾﺮ اﻟﻜﻤ ﻲ وا ﻟﻨ ﻮﻋﻲ ﻟﮭﺎ : ﻋﻨﺪ ﺣﺴﺎب ﺗﺮﻛﯿﺰ اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ اﻋﺘﻤ ﺎدا ﻋﻠ ﻰ ﻣ ﺪة اﻹﺿ ﺎءة وﻣﻼﺣﻈ ﺔ اﻟﺠ ﺪول 3 وﻟ ﻮﺣﻆ ھﻨ ﺎك ﻓﺮوﻗﺎ ﻣﻌﻨﻮﯾﺔ ﺑﯿﻦ ﺗﺮاﻛﯿﺰ اﻟﻤﺮﻛﺐ اﻟﻮاﺣﺪ ﻣﻘﺎرﻧﺔ ﺑ ﻤﻌﺎﻣﻠﺔ اﻟﺴ ﯿﻄﺮة , إذ وﺟ ﺪ ارﺗﻔ ﺎع ﺗﺮﻛﯿ ﺰ اﻏﻠ ﺐ اﻟﻤﺮﻛﺒ ﺎت اﻟﺜﺎﻧﻮ ﯾ ﺔ ﻋﻨ ﺪ ﺗﻌ ﺮﯾﺾ اﻟﻜ ﺎﻟﺲ إﻟ ﻰ اﻟﻀ ﻮء ﻟﻤ ﺪة 12 ﺳ ﺎﻋﺔ , ﺑﻠ ﻎ أوزان اﻟﻤﺮﻛﺒ ﺎت 0.14156, 1.1025, 0.3278, 0.9242 ﻣﻠﻐ ﻢ / ﻏ ﻢ ﻟﻠﻤﺮﻛﺒ ﺎت Hypersoid, Hypericin, Pseudohypericin, Hyperforin ﻋﻠﻰ اﻟﺘﻮاﻟﻲ , ارﺗﻔﻊ ﺗﺮﻛﯿﺰ اﻟﻤﺮﻛﺐ Catchin إﻟﻰ0.19482 ﻣﻠﻐﻢ / ﻏﻢ ﻋﻨﺪ ﺗﻌ ﺮﯾﺾ اﻟﻜ ﺎﻟﺲ ﻟﻠﻀ ﻮء ﻟﻤ ﺪة 24 ﺳ ﺎﻋ , ﺔ أﻣ ﺎ ﻣﺮﻛ ﺐ Prenylated phlorglucin وﺻ ﻞ ﺗﺮﻛﯿ ﺰه إﻟ ﻰ0.2275 ﻣﻠﻐ ﻢ / ﻏ ﻢ ﻋﻨﺪﻣﺎ ﺣﻀﻦ ﻓﻲ اﻟﻈﻼم . وﯾﻼﺣﻆ أن أﻓﻀﻞ ﻣﺪة ﺗﻌﺮﯾﺾ ﻟﻺﺿﺎءة ھﻲ 12 ﺳ ﺎﻋﺔ ﺿ ﻮء ﻟﺰﯾ ﺎدة إﻧﺘ ﺎج اﻏﻠ ﺐ اﻟﻤﺮﻛﺒ ﺎت اﻟﺜﺎﻧﻮﯾ ﺔ ﻓ ﻲ ﻛ ﺎﻟﺲ اﻟﻨﺒ ﺎت . اﻟﻤﻘﺪﻣﺔ وﻣ ﻦ ھ ﺬا ﻧﺠ ﺪ أن ﻟﻄ ﻮل اﻟﻔﺘ ﺮة اﻟﻀ ﻮﺋﯿﺔ ﻗﺎﺑﻠﯿ ﺔ ﻋﻠ ﻰ ﺗﺤﻔﯿ ﺰ وإﻧﺘ ﺎج اﻟﻤﺮﻛﺒﺎت ا ﻟﺜﺎﻧﻮﯾﺔ ﻓﻲ ﻧﺒﺎت اﻟﺮوﺟﺔ وﺑﺎﻷﺧﺺ ﻟﻤﺮﻛﺐ Hypericin وھﺬا ﻣﺎ أﺷ ﺎر إﻟﯿ ﮫ ]10 ; 14 ; 18 [ ﺣﯿﺚ ﯾﻌﺪ اﻟﻀﻮء ﻋﺎﻣﻞ أوﻟﻲ ﯾﺤﻔﺰ ﻗﺎﺑﻠﯿﺔ اﻟﻤﺰارع اﻟﻨﺴﯿﺠﯿﺔ ﻟﺘﻜ ﻮﯾﻦ اﻟﻜﻠﻮروﻓﯿ ﻞ واﻟﺼ ﺒﻐﺎت اﻷﺧ ﺮى ] 8 [ . وذﻛﺮ ] 3 [ ﺑ ﺎن ﻣﺴ ﺘﻮى Hypericin ﺗﻀ ﺎﻋﻔﺖ ﻓ ﻲ اﻟﻈ ﻼم ﻣﻘﺎرﻧ ﺔ ﺑﺎﻟﻀ ﻮء ﻟﻜ ﻦ ﺳ ﺠﻞ ﺑﻌ ﺾ اﻟﺒ ﺎﺣﺜﯿﻦ ﺗﺄﺛﯿﺮات ﻣﻐﺎﯾﺮة ﺣﯿﺚ وﺟﺪ أن ﺗﺄﺛﯿﺮ ﻣﺪة اﻹﺿﺎءة أدى إﻟﻰ زﯾﺎدة ﻣﺴﺘﻮى Hypericin ﻓﻲ ﻧﺒﺎت اﻟﺮوﺟﺔ ] 5 ; 4 ; 7 ; 17 [ إذ أﻛ ﺪوا ﺑ ﺎن ﻣ ﺪة اﻹﺿ ﺎءة ﻛ ﺎن ﻟﮭ ﺎ اﻟﺤ ﺎﻓﺰ اﻷﻛﺒ ﺮ ﻋﻠ ﻰ زﯾ ﺎدة إﻧﺘ ﺎج ﻣﺮﻛﺒ ﺎت اﻻﯾ ﺾ اﻟﺜﺎﻧﻮي ﻓﻲ ﻧﺒﺎت اﻟﺮوﺟﺔ إذ ﯾﻌﻤﻞ اﻟﻀﻮء ﻋﻠﻰ زﯾﺎدة إﻧﺘﺎج ﺻﺒﻐﺔ اﻟﮭ ﺎﯾﺒﺮﺳﯿﻦ ﻓﻲ اﻟﻨﺒﺎت . 4 2 ﻧ ﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypercom triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 ﻧ ﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypercom triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 5 ﺟﺪول )3 ( ﺗﺄﺛﯿﺮ ﻣﺪد اﻹﺿﺎءة ) ﺳﺎﻋﺔ ( ﻓﻲ إﻧﺘﺎج اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ ) ﻣﻠﻐﻢ ( ﻏﻢ / ﻣﻦ ﻛﺎﻟﺲ ﻟﻨﺒﺎت اﻟﺮوﺟﺔ n=3 . ﻣﺪة اﻹﺿﺎءة اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ Contro l 0 12 16 24 LSD ≤0.05 Catchin 0.1657 0.1657 0.03225 0.03396 0.19482 * 0.02148 Hypersoid 0.09482 n.s 0.09482 n.s 0.14156 0.07915 0.0859 0.01452 Hypericin 0.6899 0.6899 1.10258* 0.39179 n.s 0.63666 n.s 0.06288 n.s Pseudohyper icin 0.1785 0.17856 0.3278 0.08531 0.17848 0.01673 Hyperforin 0.1796 0.1796 0.92429* 0.36769 0.57084 0.01842 Prenylated phlorglucin 0.2275 0.2275 0.21169 0.18438 0.16503 0.01396 ﺗ ﺄﺛﯿﺮ ﻣ ﺪة اﻟﺘﻌ ﺮﯾﺾ إﻟ ﻰ اﻷﺷ ﻌﺔ ﻓ ﻮق اﻟﺒﻨﻔﺴ ﺠﯿﺔ ﻓ ﻲ إﻧﺘ ﺎج اﻟﻤﺮﻛﺒ ﺎت اﻟﺜﺎﻧﻮﯾ ﺔ ﻣ ﻦ ﻛ ﺎﻟﺲ ﻧﺒ ﺎت اﻟﺮوﺟ ﺔ واﻟﺘﻘﺪﯾﺮ اﻟﻜﻤﻲ واﻟﻨ ﻮﻋﻲ ﻟﮭ ﺬه اﻟﻤﺮﻛﺒ ﺎت : ﺗﻮﺿ ﺢ اﻟﻨﺘ ﺎﺋﺞ ﻓ ﻲ اﻟﺸ ﻜﻞ 4 ﻣﻨﺤﻨ ﻲ اﻟﻤﺮﻛﺒ ﺎت اﻟﺜﺎﻧﻮﯾ ﺔ ﻟﻠﻜ ﺎﻟﺲ اﻟﻤﻌﺮض إﻟﻰ اﻷﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ وﻟﻠﻤﺪﺗﯿﻦ 10 و20 دﻗﯿﻘﺔ . اﻟﻤﻘﺪﻣﺔ ﻋﻨﺪ ﺣﺴﺎب ﺗﺮﻛﯿﺰ اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ اﻋﺘﻤ ﺎدا ﻋﻠﻰ ﻣﺪة اﻟﺘﻌﺮﯾﺾ وﻣﻼﺣﻈﺔ اﻟﺠﺪول 4 وﺟ ﺪ إن ھﻨ ﺎك ﻓﺮوﻗ ﺎ ﺑ ﯿﻦ ﺗﺮاﻛﯿ ﺰ اﻟﻤﺮﻛ ﺐ اﻟﻮاﺣ ﺪ ﻣﻘﺎرﻧ ﺔ ﺑﻤﻌﺎﻣﻠ ﺔ اﻟﺴﯿﻄﺮة . ﺗﺤﻔ ﺰ إﻧﺘ ﺎج اﻟﻤﺮﻛﺒ ﺎت ﻋﻨ ﺪ ﺗﻌ ﺮﯾﺾ اﻟﻜ ﺎﻟﺲ إﻟ ﻰ اﻷﺷ ﻌﺔ ﻓ ﻮق اﻟﺒﻨﻔﺴ ﺠﯿﺔ وﻟﻤ ﺪة 10 دﻗ ﺎﺋﻖ واﻟﺘ ﻲ ﺷ ﻤﻠﺖ Catchin, Hypericin, Hyperforin وﻛ ﺎن ﺗﺮﻛﯿﺰھ ﺎ0.14708, 0.84623, 0.85525 ﻣﻠﻐ ﻢ / ﻏ ﻢ ﻋﻠ ﻰ اﻟﺘ ﻮاﻟﻲ , أﻣ ﺎ ﻋﻨ ﺪ ﺗﻌ ﺮﯾﺾ اﻟﻜ ﺎﻟﺲ إﻟ ﻰ اﻷﺷ ﻌﺔ ﻓ ﻮق اﻟﺒﻨﻔﺴ ﺠﯿﺔ ﻟﻤ ﺪة 20 دﻗﯿﻘ ﺔ ﺗﺤﻔ ﺰ إﻧﺘ ﺎج اﻟﻤﺮﻛﺒﺎت Hypersoid, Pseudohypericin, Prenylated phlorglucin وﻛﺎن ﺗﺮﻛﯿﺰھﺎ0.21636, 0.16578, 0.14189 ﻣﻠﻐﻢ / ﻏ ﻢ ﻋﻠ ﻰ اﻟﺘ ﻮاﻟﻲ . وﻋﻨ ﺪ ﻣﻘﺎرﻧ ﺔ اﻟﻨﺘ ﺎﺋﺞ ﻣ ﻊ ﻣﻌﺎﻣﻠ ﺔ اﻟﺴ ﯿﻄﺮة وﺟ ﺪ ارﺗﻔ ﺎع ﻓ ﻲ ﺗﺮاﻛﯿﺰ اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ ﻋﻨﺪ اﻟﺘﻌﺮﯾﺾ ﻟﻸﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ . ﺟﺪول )4 ( ﺗﺄﺛﯿﺮ ﻣﺪة اﻟﺘﻌﺮض إﻟﻰ اﻷﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ ) دﻗﯿﻘﺔ ( ﻓﻲ إﻧﺘﺎج اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ ﻣﻦ ﻛﺎﻟﺲ ﻧﺒﺎت اﻟﺮوﺟﺔ اﻟﻤﺴﺘﺤﺚ ﻣﻦ اﻷﺟﺰاء اﻟﻮرﻗﯿﺔ ,n=3 . اﻟﺰﻣﻦ اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ Control 10 20 LSD ≤0.05 Catchin 0.1657 0.14708 0.01781 0.02218 Hypersoid 0.09482 0.09825 0.21636 0.02596 Hypericin 0.6899 0.84623 0.71438 0.01739 Pseudohypericin 0.1785 0.13707 0.16578 0.00866 ﺟﺪول )3 ( ﺗﺄﺛﯿﺮ ﻣﺪد اﻹﺿﺎءة ) ﺳﺎﻋﺔ ( ﻓﻲ إﻧﺘﺎج اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ ) ﻣﻠﻐﻢ ( ﻏﻢ / ﻣﻦ ﻛﺎﻟﺲ ﻟﻨﺒﺎت اﻟﺮوﺟﺔ n=3 . ﻣﺪة اﻹﺿﺎءة اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ Contro l 0 12 16 24 LSD ≤0.05 Catchin 0.1657 0.1657 0.03225 0.03396 0.19482 * 0.02148 Hypersoid 0.09482 n.s 0.09482 n.s 0.14156 0.07915 0.0859 0.01452 Hypericin 0.6899 0.6899 1.10258* 0.39179 n.s 0.63666 n.s 0.06288 n.s Pseudohyper icin 0.1785 0.17856 0.3278 0.08531 0.17848 0.01673 Hyperforin 0.1796 0.1796 0.92429* 0.36769 0.57084 0.01842 Prenylated phlorglucin 0.2275 0.2275 0.21169 0.18438 0.16503 0.01396 ﺗ ﺄﺛﯿﺮ ﻣ ﺪة اﻟﺘﻌ ﺮﯾﺾ إﻟ ﻰ اﻷﺷ ﻌﺔ ﻓ ﻮق اﻟﺒﻨﻔﺴ ﺠﯿﺔ ﻓ ﻲ إﻧﺘ ﺎج اﻟﻤﺮﻛﺒ ﺎت اﻟﺜﺎﻧﻮﯾ ﺔ ﻣ ﻦ ﻛ ﺎﻟﺲ ﻧﺒ ﺎت اﻟﺮوﺟ ﺔ واﻟﺘﻘﺪﯾﺮ اﻟﻜﻤﻲ واﻟﻨ ﻮﻋﻲ ﻟﮭ ﺬه اﻟﻤﺮﻛﺒ ﺎت : ﺗﻮﺿ ﺢ اﻟﻨﺘ ﺎﺋﺞ ﻓ ﻲ اﻟﺸ ﻜﻞ 4 ﻣﻨﺤﻨ ﻲ اﻟﻤﺮﻛﺒ ﺎت اﻟﺜﺎﻧﻮﯾ ﺔ ﻟﻠﻜ ﺎﻟﺲ اﻟﻤﻌﺮض إﻟﻰ اﻷﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ وﻟﻠﻤﺪﺗﯿﻦ 10 و20 دﻗﯿﻘﺔ . ﻋﻨﺪ ﺣﺴﺎب ﺗﺮﻛﯿﺰ اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ اﻋﺘﻤ ﺎدا ﻋﻠﻰ ﻣﺪة اﻟﺘﻌﺮﯾﺾ وﻣﻼﺣﻈﺔ اﻟﺠﺪول 4 وﺟ ﺪ إن ھﻨ ﺎك ﻓﺮوﻗ ﺎ ﺑ ﯿﻦ ﺗﺮاﻛﯿ ﺰ اﻟﻤﺮﻛ ﺐ اﻟﻮاﺣ ﺪ ﻣﻘﺎرﻧ ﺔ ﺑﻤﻌﺎﻣﻠ ﺔ اﻟﺴﯿﻄﺮة . اﻟﻤﻘﺪﻣﺔ ﻣﺘﻮﻟﻲ , رﻗﯿﺔ ﻣﺤﻤﺪ طﮫ )2011 .(ﻛﯿﻒ ﺗﺘﺤﻤﻞ اﻟﻨﺒﺎﺗﺎت وھﺞ اﻟﺸﻤﺲ اﻟﺤﺎرﻗﺔ , ﻗﺴﻢ اﻟﺒﺴﺎﺗﯿﻦ , ﻛﻠﯿﺔ اﻟﻌﻠﻮم اﻟﺰراﻋﯿﺔ , ﺟﺎﻣﻌﺔ أﺳﯿﻮط , ﺻﻔﺤﺔ 85 - 102 ,ﺟﻤﮭﻮرﯾﺔ ﻣﺼﺮ اﻟﻌﺮﺑﯿﺔ . ر [1]. ﺧﯿﻨﻜﺎﻧﻲ , ﻋﻠﻲ ﺣﺴﯿﻦ ﺟﺎﺳﻢ )2010 .(ﻣﻨﻈﻤﺎت اﻟﻨﻤﻮ ﻓﻲ اﻟﻨﺒﺎﺗﺎت , ﻗﺴﻢ أﻟﺒﺴﺘﻨﮫ واﻟﻨﺨﯿﻞ , ﻛﻠﯿﺔ اﻟﺰراﻋﺔ , ﺟﺎﻣﻌﺔ ﺑﺎﺑﻞ , ﺻﻔﺤﺔ 12 - 30 ,ﺟﻤﮭﻮرﯾﺔ اﻟﻌﺮاق . 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[14].Kirakosyan, A. اﻟﻤﻘﺪﻣﺔ ﺗﺤﻔ ﺰ إﻧﺘ ﺎج اﻟﻤﺮﻛﺒ ﺎت ﻋﻨ ﺪ ﺗﻌ ﺮﯾﺾ اﻟﻜ ﺎﻟﺲ إﻟ ﻰ اﻷﺷ ﻌﺔ ﻓ ﻮق اﻟﺒﻨﻔﺴ ﺠﯿﺔ وﻟﻤ ﺪة 10 دﻗ ﺎﺋﻖ واﻟﺘ ﻲ ﺷ ﻤﻠﺖ Catchin, Hypericin, Hyperforin وﻛ ﺎن ﺗﺮﻛﯿﺰھ ﺎ0.14708, 0.84623, 0.85525 ﻣﻠﻐ ﻢ / ﻏ ﻢ ﻋﻠ ﻰ اﻟﺘ ﻮاﻟﻲ , أﻣ ﺎ ﻋﻨ ﺪ ﺗﻌ ﺮﯾﺾ اﻟﻜ ﺎﻟﺲ إﻟ ﻰ اﻷﺷ ﻌﺔ ﻓ ﻮق اﻟﺒﻨﻔﺴ ﺠﯿﺔ ﻟﻤ ﺪة 20 دﻗﯿﻘ ﺔ ﺗﺤﻔ ﺰ إﻧﺘ ﺎج اﻟﻤﺮﻛﺒﺎت Hypersoid, Pseudohypericin, Prenylated phlorglucin وﻛﺎن ﺗﺮﻛﯿﺰھﺎ0.21636, 0.16578, 0.14189 ﻣﻠﻐﻢ / ﻏ ﻢ ﻋﻠ ﻰ اﻟﺘ ﻮاﻟﻲ . وﻋﻨ ﺪ ﻣﻘﺎرﻧ ﺔ اﻟﻨﺘ ﺎﺋﺞ ﻣ ﻊ ﻣﻌﺎﻣﻠ ﺔ اﻟﺴ ﯿﻄﺮة وﺟ ﺪ ارﺗﻔ ﺎع ﻓ ﻲ ﺗﺮاﻛﯿﺰ اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ ﻋﻨﺪ اﻟﺘﻌﺮﯾﺾ ﻟﻸﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ . ﺟﺪول )4 ( ﺗﺄﺛﯿﺮ ﻣﺪة اﻟﺘﻌﺮض إﻟﻰ اﻷﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ ) دﻗﯿﻘﺔ ( ﻓﻲ إﻧﺘﺎج اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ ﻣﻦ ﻛﺎﻟﺲ ﻧﺒﺎت اﻟﺮوﺟﺔ اﻟﻤﺴﺘﺤﺚ ﻣﻦ اﻷﺟﺰاء اﻟﻮرﻗﯿﺔ ,n=3 . اﻟﺰﻣﻦ اﻟﻤﺮﻛﺒﺎت اﻟﺜﺎﻧﻮﯾﺔ Control 10 20 LSD ≤0.05 Catchin 0.1657 0.14708 0.01781 0.02218 Hypersoid 0.09482 0.09825 0.21636 0.02596 Hypericin 0.6899 0.84623 0.71438 0.01739 Pseudohypericin 0.1785 0.13707 0.16578 0.00866 5 ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 ﻧ ﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypercom triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ Ғƶ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 Hyperforin 0.1796 0.85525 0.40633 0.04833 Prenylated phlorglucin 0.2275 0.11275 0.14189 0.02105 ﯾﺴﺘﻨﺘﺞ ﻣﻦ ﻧﺘﺎﺋﺞ ھﺬه اﻟﺪراﺳﺔ : ﺑﺎن اﻟﻤﻌﺎﻣﻠﺔ ﺑﺎﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻛﺎﻟﻀﻮء ادت إﻟﻰ زﯾﺎدة إﻧﺘ ﺎج اﻟﻤﺮﻛﺒ ﺎت اﻟﺜﺎﻧﻮﯾﺔ ﺧﺎﺻﺔ ﻋﻨﺪ ﺗﻌﺮﯾﺾ اﻟﻜﺎﻟﺲ ﻟﻤﺪة 12 ﺳﺎﻋﺔ ﺿﻮء . ﻛﻤﺎ اﺧﺘﻠﻔﺖ اﺳﺘﺠﺎﺑﺔ اﻷﻧﺴﺠﺔ ﻟﺘﺮاﻛﻢ ﻣﻮاد اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻣﻊ زﻣﻦ اﻟﺘﻌﺮﯾﺾ ﻟﻸﺷﻌﺔ ﻓﻮق اﻟﺒﻨﻔﺴﺠﯿﺔ , ﻓﻔﻲ ﻧﺒﺎت اﻟﺮوﺟﮫ اﺳﺘﺠﺎﺑﺖ ﺧﻼﯾﺎ اﻟﻜﺎﻟﺲ ﻟﻠﺘﺤﻔﯿﺰ وإﻧﺘ ﺎج اﻟﻤﺮﻛﺒ ﺎت اﻟﺜﺎﻧﻮﯾ ﺔ وﺣﺴ ﺐ ﻣ ﺪة اﻟﺘﻌ ﺮﯾﺾ ﻟﻸﺷ ﻌﺔ ﻓ ﻮق اﻟﺒﻨﻔﺴ ﺠﯿﺔ , إذ ازدادت اﻟﻤﺮﻛﺒ ﺎت Catchin, Hypericin, Hyperforin ﻋﻨ ﺪ ﺗﻌ ﺮﯾﺾ اﻟﻜ ﺎﻟﺲ ﻟﻤ ﺪة 10 دﻗ ﺎﺋﻖ ﻓ ﻲ اﻟﻮﻗ ﺖ اﻟ ﺬي زادت اﻟﻤﺮﻛﺒ ﺎت Hypersoid, Pseudohypericin, Prenylated phlorglucin ﻋﻨﺪ ﺗﻌﺮﯾﺾ اﻟﻜﺎﻟﺲ ﻟﻼﺷ ﻌﺔ ﻟﻤ ﺪة20 دﻗﯿﻘﺔ . Hyperforin 0.1796 0.85525 0.40633 0.04833 Prenylated phlorglucin 0.2275 0.11275 0.14189 0.02105 ر [1]. ﺧﯿﻨﻜﺎﻧﻲ , ﻋﻠﻲ ﺣﺴﯿﻦ ﺟﺎﺳﻢ )2010 .(ﻣﻨﻈﻤﺎت اﻟﻨﻤﻮ ﻓﻲ اﻟﻨﺒﺎﺗﺎت , ﻗﺴﻢ أﻟﺒﺴﺘﻨﮫ واﻟﻨﺨﯿﻞ , ﻛﻠﯿﺔ اﻟﺰراﻋﺔ , ﺟﺎﻣﻌﺔ ﺑﺎﺑﻞ , ﺻﻔﺤﺔ 12 - 30 ,ﺟﻤﮭﻮرﯾﺔ اﻟﻌﺮاق . [2]. اﻟﻤﻘﺪﻣﺔ B., Vardapetyan, H.R. and Charchoglyan, A. G. (2000). The content of hypericin and pseudohypecicin in cell culture of Hypericum 6 ﻧ ﺄﺛﯿﺮ ﺑﻌﺾ اﻟﻤﺤﻔﺰات اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻓﻲ إﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻻﯾﺾ اﻟﺜﺎﻧﻮي ﻟﻨﺒﺎت اﻟﺮوﺟﺔ Hypercom triquetrifolium ﺧﺎرج اﻟﺠﺴﻢ اﻟﺤﻲ ӨƶҰƺƵǚ ¤ǛǊ үǄ ƵǄ ƾƲҚƵǚǃ ҒƃӨƾǂ Ƶǚ ҒƶҰƹ 32 ơƀҰƵǚ¤ ) (B əӨƞƵǚ .2 ¤ 2014 perforatum L. (st. johns wort) and production of hypericin. Russ. J. Plant Physiology, 47:270-273. perforatum L. (st. johns wort) and production of hypericin. Russ. J. Plant Physiology, 47:270-273. [15].Meloan, C.E. (1999). Techniques in Analytical Chemistry, Chemical Separation, Principles, Techniques, and Experiments, USA. [16].Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15:473-497. [17].Namli, S., Akbas, F., Isikalan, G., Tilkat, E.A. and Davut B. (2010). The effect of different plant hormones on multiple shoots of Hypericum retusum L. (St. Johns Wort). Department of Biology, Faculty of Science and Art, The University of Batman, Batman, Turkey. [18].Silva, B.A., Ferreres, F., Malva, J.O., and Dias, A.C.P. (2005). Phytochemical and antioxidant characterization of Hypericum perforatum alcoholic extracts. Food Chem., 90:157-167. [19].Sirvent, T.M. and Gibson, D.M. (2000). Rapid Isocratic HPLC analysis of Hypericins. J. of Liquid Chromatography 23(2):251-259. 7 7
https://openalex.org/W4293173520	https://www.pure.ed.ac.uk/ws/files/292599666/fimmu_13_984799.pdf	English	null	Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida	Frontiers in immunology	2,022	cc-by	14,000	General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Edinburgh Research Explorer Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida Citation for published version: Taylor, R, Ruiz Daniels, R, Dobie, R, Naseer, S, Clark, TC, Henderson, NC, Boudinot, P, Martin, SAM & Macqueen, D 2022, 'Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida', Frontiers in Immunology, pp. 1-17. https://doi.org/10.3389/fimmu.2022.984799 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 TYPE Original Research PUBLISHED 24 August 2022 DOI 10.3389/fimmu.2022.984799 Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. OPEN ACCESS and Daniel J. Macqueen 1* 1The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom, 2Centre for Inﬂammation Research, The Queen’s Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, Edinburgh, United Kingdom, 3MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom, 4School of Biological Sciences, University of Aberdeen, Aberdeen, United Kingdom, 5 Universite´ Paris-Saclay, INRAE, UVSQ, VIM, Jouy-en-Josas, France CITATION Taylor RS, Ruiz Daniels R, Dobie R, Naseer S, Clark TC, Henderson NC, Boudinot P, Martin SAM and Macqueen DJ (2022) Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida. The liver is a multitasking organ with essential functions for vertebrate health spanning metabolism and immunity. In contrast to mammals, our understanding of liver cellular heterogeneity and its role in regulating immunological status remains poorly deﬁned in ﬁshes. Addressing this knowledge gap, we generated a transcriptomic atlas of 47,432 nuclei isolated from the liver of Atlantic salmon (Salmo salar L.) contrasting control ﬁsh with those challenged with a pathogenic strain of Aeromonas salmonicida, a problematic bacterial pathogen in global aquaculture. We identiﬁed the major liver cell types and their sub-populations, revealing poor conservation of many hepatic cell marker genes utilized in mammals, while identifying novel heterogeneity within the hepatocyte, lymphoid, and myeloid lineages. This included polyploid hepatocytes, multiple T cell populations including gd T cells, and candidate populations of monocytes/macrophages and dendritic cells. A dominant hepatocyte population radically remodeled its transcriptome following infection to activate the acute phase response and other defense functions, while repressing routine functions such as metabolism. These defense-specialized hepatocytes showed strong activation of genes controlling protein synthesis and secretion, presumably to support the release of acute phase proteins into circulation. The infection response further involved up-regulation of numerous genes in an immune-cell speciﬁc manner, reﬂecting functions in pathogen recognition and killing, antigen presentation, phagocytosis, regulation of inﬂammation, B cell differentiation by Aeromonas salmonicida. Front. Immunol. 13:984799. doi: 10.3389/fimmu.2022.984799 Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida OPEN ACCESS EDITED BY Uwe Fischer, Friedrich-Loefﬂer-Institute, Germany REVIEWED BY Alexander Rebl, Research Institute for Farm Animal Biology (FBN), Germany, Charlotte Scott, Flanders Institute for Biotechnology, Belgium *CORRESPONDENCE Daniel J. Macqueen daniel.macqueen@roslin.ed.ac.uk †These authors have contributed equally to this work and share ﬁrst authorship SPECIALTY SECTION This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology RECEIVED 02 July 2022 ACCEPTED 03 August 2022 PUBLISHED 24 August 2022 CITATION Introduction zebraﬁsh (Danio rerio), single cell transcriptomics has recently been used to reveal hepatic cellular heterogeneity, including within the hepatocyte, myeloid and lymphoid lineages (17, 18). However, zebraﬁsh are distantly related to salmonids, leaving a gap in knowledge on the role of liver cellular heterogeneity in this key group of ﬁshes, where such information can be applied to understand and manipulate health and immune traits relevant to sustainable aquaculture and food production. The vertebrate liver is a multitasking organ with diverse physiological functions, including nutrient metabolism, transport and storage, growth signaling, endocrine regulation, and immunity (1). In mammals, these roles are performed by the cooperative actions of several distinct cell types including hepatocytes, cholangiocytes (epithelial cells of the bile duct), stellate cells, Kupffer cells (resident liver macrophages) and lymphocytes (1). Recent advances in single cell transcriptomics have revealed functional heterogeneity within the major hepatic cell types of mammals (2, 3), providing insights into liver spatial organization (4, 5) while revealing cellular and molecular drivers of disease and malignancy states (6, 7). The aim of this study was to reveal the major cell lineages within the liver of Atlantic salmon and uncover the role of hepatic cellular heterogeneity in the host response to bacterial infection. Using single-nuclei RNA-Seq (snRNA-Seq), we report a comprehensive single cell transcriptomic atlas of the Atlantic salmon liver, identifying novel heterogeneity across multiple hepatic cell types. By comparing cell- speciﬁc responses in control animals to those challenged with Aeromonas salmonicida, we uncover a dramatic transcriptomic remodeling of hepatocytes that underpins the acute phase response, alongside major changes in gene expression speciﬁc to distinct immune cell populations. The adult liver of all vertebrates contains both immune and non-immune cells with important immunological functions (2, 7–9) that support immune homeostasis and tolerance (10) and the generation of inﬂammatory responses upon pathogen challenge, leading to secretion of acute phase proteins (APPs) into circulation by hepatocytes (7, 11). The liver is the major site of haematopoiesis in the mammalian fetus and hence an important organ for early immune cell development (12), though this feature is not conserved in ﬁshes (13). The multifaceted functions of the liver must demand tight coordination of different cell types to achieve appropriate responses to prevailing physiological and environmental conditions, inclusive of immune system status following pathogen challenge. Immunological functions may need to be prioritized at the cost of investment into metabolic functions in such scenarios (14). Introduction In this regard, the role of the liver in co- regulating metabolism and immunity makes it an interesting organ to understand the coordinated responses of different cell types following pathogenic challenge. COPYRIGHT COPYRIGHT © 2022 Taylor, Ruiz Daniels, Dobie, Naseer, Clark, Henderson, Boudinot, Martin and Macqueen. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. frontiersin.org Frontiers in Immunology 01 Taylor et al. 10.3389/ﬁmmu.2022.984799 10.3389/ﬁmmu.2022.984799 10.3389/ﬁmmu.2022.984799 and T cell activation. Overall, this study greatly enhances our understanding of the multifaceted role played by liver immune and non-immune cells in host defense and metabolic remodeling following infection and provides many novel cell-speciﬁc marker genes to empower future studies of this organ in ﬁshes. KEYWORDS liver, single cell transcriptomics, bacterial infection, salmonid ﬁsh, immune-metabolism cross talk liver, single cell transcriptomics, bacterial infection, salmonid ﬁsh, immune-metabolism cross talk snRNA-Seq library construction killed using a Schedule 1 method following anesthesia overdose using 2-phenoxyethanol (0.1% v/v) and destruction of the brain. Fish were immediately sampled, with liver samples (approx. 100mg) ﬂash frozen on dry ice before storage at -80°C prior to snRNA-Seq library construction. Separate liver samples were placed in 1.5ml of Tri Reagent (Sigma-Aldrich) and used for quantitative PCR (qPCR) to validate the expected response in infected ﬁsh. Based on the results of qPCR, n=4 samples were taken forward for snRNA-Seq library construction, representing n=2 control ﬁsh and n=2 infected ﬁsh (see Results; Supplementary Figure 1). A protocol adapted from ref (21) was used for nuclear extraction, employing a tween with salt and tris (TST) buffer. Approximately 45 mg of each frozen liver sample was placed in a 6-well tissue culture plate (Stem Cell Technologies) with 1 mL TST (2 mL of 2X ST buffer + 120 µL of 1% Tween-20 + 20 µL of 2% BSA brought up to 4 mL with nuclease-free water). The tissue was minced using Noyes spring scissors for 10 min on ice. The resulting homogenate was ﬁltered through a 40 µm Falcon cell strainer, and a further 1 mL of TST was added to wash the well and ﬁlter. The volume was brought up to 5 mL using 3 mL of 1X ST buffer (diluted from 2xST buffer [292 µl of 146 mM NaCl, 100 µl of 10 mM Tris-HCl pH 7.5, 10 µl of 1 mM CaCl2, 210 µl of 21 mM MgCl2, brought up to 10 mL with nuclease-free water]). The sample was centrifuged at 4°C for 5 min at 500g before the resulting pellet was re-suspended in 1 mL 1X ST buffer and the recovered nuclei were ﬁltered through a 40 µm Falcon cell strainer, Hoechst stained, visually inspected under a ﬂuorescent microscope, and counted using a Bio-Rad TC20. Liver nuclei were processed through the 10X Chromium™ Single Cell Platform using the Chromium™Single Cell 30 Library and Gel Bead Kit v3.1 and Chromium™Single Cell A Chip Kit (both 10X Genomics) as per the manufacturer’s protocol. For each sample, the nuclei were loaded into a channel of a Chromium 3’ Chip and partitioned into droplets using the Chromium controller before the captured RNA for each nucleus was barcoded and reverse transcribed. The resulting cDNA was PCR ampliﬁed for 14 cycles, fragmented, and size selected before Illumina sequencing adaptor and sample indexes were attached. snRNA-Seq library construction Libraries were sequenced on a NovaSeq 6000 by Novogene UK Ltd (2x150bp paired end reads). Validation of immune response by qPCR qPCR validation was performed on the twenty liver samples (comparing n=10 control vs. n=10 infected animals) to conﬁrm an inﬂammatory response in the infected ﬁsh. RNA was extracted from 100 mg liver tissue using 1 mL Trizol reagent. The tissue homogenization was performed using two Tungsten Carbide Beads (Qiagen) (3 mm) on a Tissuelyser II (Qiagen) at a frequency of 30.0 I/sec for 2.5 min. 200 µl of chloroform (Sigma- Aldrich) was added and the mixture centrifuged at 12,000 g for 20 minutes at 4°C to separate the aqueous phase, which was retained. RNA precipitation was performed using 700 µl of isopropanol (Sigma-Aldrich) and centrifuged at 12,000 g for 20 min at 4°C and washed 3 times with 80% ethanol. The concentration and purity of total RNA was estimated using a NanoDrop 1000 Spectrophotometer (Thermo Scientiﬁc). A QuantiTect Reverse Transcription kit (Qiagen) was used to synthesize ﬁrst-strand cDNA from 1 µg total RNA per sample, with a genomic DNA removal step included, in a total volume of 20 µl. The resulting cDNA was diluted 20-fold (working stock) with RNase/DNase free water. qPCR was performed using primers targeting the APP encoding genes saa and hamp (11). For normalization of gene expression, primers targeting rps13 and rps29 were used (19). qPCR was performed with 2x SYBR Green I (Invitrogen) master mix on a Mx3005P System (Agilent Technologies). Each assay was run in triplicate using 15 µl of reaction mix containing 7.5 µl Brilliant III Ultra-Fast SYBR Green (Agilent Technologies), 500 nM forward and reverse primers and 5 µl of cDNA (2.5 ng of total reverse-transcribed RNA). Assays were run with 1 cycle of 95°C for 3 min, followed by 40 cycles of 95°C for 20 s and 64°C for 20 s. A melting curve (thermal gradient 55°C - 95°C) was used to conﬁrm single qPCR products. Each qPCR plate included two no-template (i.e. water) controls. LinRegPCR (20) was used to establish the efﬁciency of each assay. Gene expression data were analyzed using GeneX 5.4.3 (MultiD Analysis), correcting for differences in efﬁciency across genes, before target gene expression was normalized to rps13 and rps29 and placed on a scale of relative expression. Separate Kruskal-Wallis tests were performed in Minitab® 17.1.0 to compare gene expression values for saa and hamp between the control (n=10) and infected (n=10) liver samples. Disease challenge and sampling Animal work was carried out in compliance with the Animals (Scientiﬁc Procedures) Act 1986 under Home Ofﬁce license PFF8CC5BE and was approved by the ethics committee of the University of Aberdeen. Atlantic salmon were kept in 250 L freshwater tanks at aquarium facilities of the University of Aberdeen. Water temperature was maintained at 14°C and ﬁsh were fed a commercial pellet diet at 2% body weight per day. After two weeks, twenty ﬁsh were anaesthetized using 2- phenoxyethanol (2.5 mL in 10L water/0.0025% v/v) and given an intraperitoneal injection of either PBS (0.5 mL) (n=10), or the pathogenic Hooke strain of A. salmonicida (2 × 105 colony- forming units/mL in PBS; 0.5 mL/ﬁsh) (n=10). Sampling was performed 24 h post-injection (after ref 11). All twenty ﬁsh were Bulk RNA-Seq and proteomics have been widely applied to understand liver functions in commercially important ﬁshes. For example, in Atlantic salmon (Salmo salar L.), which is among the most important aquaculture species globally (15), such work has uncovered the role of this organ in innate immune defense and the acute phase response (11, 16). In the biomedical model Frontiers in Immunology frontiersin.org 02 Taylor et al. 10.3389/ﬁmmu.2022.984799 Generation of snRNA-Seq count matrix Raw sequencing data were aligned to the unmasked ICSASG_v2 reference assembly (Ensembl release 104) of the Atlantic salmon genome (22). The analysis was restricted to protein coding genes. Mapping of reads to the genome, assignment of reads to cellular barcodes, and collapsing of unique molecular identiﬁers (UMIs) was performed with StarSolo v2.7.7a (23). The genome index was generated with standard settings and “sjdbOverhang” set to 149. The reads were then mapped with the “STAR” command and following settings: “soloType=CB_UMI_Simple”, “outFilterMultimapNmax = 20”, “outMultimapperOrder=random”, “soloUMIdedup=1MM_Directional”, “soloFeatures = GeneFull”, “soloBarcodeReadLength = 0”, “outFilterMatchNminOverLread = 0”, “soloCellFilter = TopCells 100000”. The top 100,000 cell Frontiers in Immunology frontiersin.org 03 Taylor et al. 10.3389/ﬁmmu.2022.984799 using the established Seurat pipeline. The appropriate number of PCs to use for clustering of each sample to minimise technical noise, was determined through visualisations generated through the “ElbowPlot” and “DimHeatmap” functions. The resolution parameter in the “FindClusters” function was tuned to return biologically meaningful heterogeneity with each cell type. At this stage, a further quality control step was performed to remove doublets from each lineage. Speciﬁcally, differential gene expression tests were performed and clusters that exhibited canonical markers (Supplementary Table 2) or lineage distinguishing markers (Supplementary Table 3) from two distinct cell lineages yet lacked any unique distinguishing marker genes of their own, were removed as likely doublets, before the data was re- clustered. This process was repeated until no doublet populations remained. For the immune cells, the Seurat object was further split into T cell, Myeloid, NK-like, Neutrophil and B cell objects (see Results) and the same process was repeated (using markers in Supplementary Table 3) and cell sub-clusters identiﬁed, before each object was merged back into a single “immune cell” Seurat object. Finally, all ﬁve major lineages were merged into a ﬁnal global liver cell atlas object, with cell identities retained from the cell lineage speciﬁc analyses. We used this strategy as opposed to global clustering, as markedly more biologically meaningful heterogeneity could be established in the cell lineage speciﬁc analyses. barcodes ranked by UMI number were retained to ensure the capture of transcriptionally quiet nuclei, lost when using the automated StarSolo ﬁltering algorithm. Mapping statistics for each snRNA-Seq sample are provided in Supplementary Table 1. Nuclei ﬁltering and quality control Nuclei ﬁltering was performed manually on each of the four samples using Seurat v3.1 (24). Ranked barcode plots of UMIs and gene counts were used to identify the lower ‘elbow point’ and cell barcodes with UMI count or gene counts below the elbow point removed as empty droplets. Further steps were used to identify additional empty droplets: the “SCTransform” function (25) was used to normalise the data, prior to centering, scaling, principal component analysis (PCA) and high-resolution clustering (using 30 PCs and resolution of 2). Wilcoxon rank sum differential gene expression tests were used to identify up-regulated genes in each cluster (see Methods Section ‘Differential gene expression tests’). Cell clusters that both lacked distinguishing markers and had a low median UMI or gene counts (typically 2 median absolute deviations lower than the median across all nuclei) were removed as likely empty droplets or poor-quality nuclei. This process was repeated iteratively on each sample until all such low-quality populations were removed. Likely doublets were identiﬁed and removed later in the analysis, after cell identity was established. Assignment of cellular identity Throughout this study, differentially expressed genes were deﬁned using the Seurat function “FindAllMarkers”, applying the Wilcoxon rank sum test with default cut-offs (multi-test adjusted p-value < 0.05, log2-fold change > 0.25, with expression of the gene in at least 20% of nuclei in the cluster tested). For the global analyses of major cell types, differential gene expression tests were performed for each deﬁned cluster in turn versus all other nuclei in the dataset as the background. For the hepatocyte-speciﬁc analysis we compared each hepatocyte cluster to all other hepatocyte nuclei. For the immune cell nuclei, the comparisons made were between each T cell sub- cluster and all other T cell nuclei, each myeloid sub-cluster and all other myeloid nuclei, and between each of the other immune cell clusters (i.e. NK-like, Neutrophil and B cell clusters) and all other immune cell nuclei. To assess which genes were up- regulated in infection in the deﬁned immune sub-clusters, a differential gene expression test was performed within each cluster to compare nuclei that originated from control vs. infected ﬁsh (Wilcoxon rank sum test, p-value < 0.05, log2- fold change > 0.25, with expression of the gene in at least 20% of nuclei from either the infected or control nuclei). Each cell cluster was assigned to one of the major liver cell lineages using a priori marker genes (Supplementary Table 2). Populations identiﬁed as erythrocytes and thrombocytes were not included in downstream analyses due to their likely origin from contaminating blood. Populations identiﬁed as hepatocytes, cholangiocytes, mesenchymal cells, endothelial cells, and immune cells were merged into ﬁve separate Seurat objects (using the Seurat “merge” function) for separate analyses of each cell lineage. Batch effects across samples were removed in each merged object with Harmony (26). Gene annotations were taken from the Ensembl annotation for Atlantic salmon, and in cases where the gene was not assigned a name, the name of the nearest annotated putative orthologue in rainbow trout (Oncorhynchus mykiss), zebraﬁsh or mouse (Mus musculus) was used, or in some cases informed by BLASTp searches against the NCBI non-redundant database. For each of the ﬁve major liver cell lineages retained, data was log normalised, then scaled and centered, before PCA was performed and used as the input to graph-based clustering, Frontiers in Immunology frontiersin.org 04 10.3389/ﬁmmu.2022.984799 Taylor et al. Single-nuclei RNA-Seq atlas of the Atlantic salmon liver Across all samples, we generated 47,432 nuclei transcriptomes with median UMI and gene counts per nucleus of 2,105 and 1,065, respectively. This was split across control ﬁsh as 11,679 and 11,433 nuclei, and Aeromonas-challenged ﬁsh as 19,148 and 5,172 nuclei, respectively (Supplementary Table 1). The major cell lineages were identiﬁed using a guided graph-based clustering strategy (24), with cell identity assigned using a priori deﬁned marker genes (Figures 1A, B; markers in Supplementary Table 2). Hepatocytes comprised most of the proﬁled nuclei (88.1%), followed by cholangiocytes (4.3%), immune cells (3.5%), mesenchymal cells (2.6%) and endothelial cells (1.5%) (Supplementary Figure 2; Supplementary Table 1). Clustering and identiﬁcation of these major cell lineages was repeatable across individual samples, with each sample contributing a large proportion of the nuclei (Supplementary Figure 2). Differential gene expression analysis Hepatocytes, the major epithelial cell type within the liver, have been underrepresented in mammalian scRNA-Seq analyses (2), which may be caused by damage occurring during the dissociation step. As Atlantic salmon are ectotherms, we were also concerned that enzymatic dissociation (typically done at >30°C) would activate cell stress and heat shock responses. We consequently decided to generate a snRNA-Seq atlas of salmon liver, using nuclei isolated from freshly ﬂash frozen samples, an approach expected to provide an accurate representation of cell diversity (e.g. 27, 28). The proﬁled liver nuclei were from control ﬁsh (n=2) and animals infected by a pathogenic strain of Aeromonas salmonicida (n=2), the bacterial agent of furunculosis, a long-standing problem disease in salmonid aquaculture (29). The infected group were sampled 24 hours post Aeromonas challenge, previously shown to capture B B A B D C FIGURE 1 Major cell types in the Atlantic salmon liver deﬁned by 47,982 nuclear transcriptomes. (A) UMAP highlighting ﬁve main liver cell-type clusters according to a priori deﬁned marker genes (see Supplementary Table 2). (B) Bubble plots showing the expression of a priori marker genes for the ﬁve main liver cell types, including mean expression (bubble intensity) and percentage of nuclei expressing gene (bubble size). (C) Heatmap of the top 20 differentially expressed genes per each liver cell type deﬁned against the background of all other cell types. (D) Violin plots demonstrating the expression of the most speciﬁc marker genes per each of the main liver cell types (colours are matched to the colours of the 5 cell lineages deﬁned in part A). Results the inﬂammatory and acute phase response (11). The ﬁsh used for sequencing were further selected based on gene expression data using marker genes for the acute phase response (11), which were robustly and signiﬁcantly up-regulated in the infected group (Supplementary Figure 1). Single-nuclei RNA-Seq atlas of the Atlantic salmon liver Adding to this challenge, genetic orthology is not a prerequisite for conservation of gene function or expression, which limits our ability to transfer knowledge about cell marker genes from mammalian studies to Atlantic salmon. While the reader must be aware of these caveats, they represent a general issue in functional genomics studies using non-model taxa like salmonids. expressed genes into the ambient RNA. Similar examples of this type of leakage can be observed in many liver scRNA-Seq datasets with, for example, hepatocyte expressed genes Alb and Hp being widely ‘expressed’ across non-hepatocyte cells in past mammalian studies (e.g. 3, 32). The immune compartment contained identiﬁable T and B cells, along with candidate populations of neutrophils, myeloid cells, and NK-like cells (Figure 2B). The transcriptome of cholangiocyte nuclei was homogeneous, while limited heterogeneity was identiﬁed in the endothelial and mesenchymal cells (Figure 1A; Supplementary Figure 5 and 6). The endothelia sub-clusters had a clear biological interpretation, with Atlantic salmon orthologues to marker genes from mammals distinguishing arterial and venous derived endothelial cells (33) (Supplementary Figure 5, marker genes in Supplementary Table 5). However, the mesenchymal sub- clusters were not readily biologically interpretable (Supplementary Figure 6; Supplementary Table 6). While it was possible to identify the ﬁve major liver cell lineages of Atlantic salmon using orthologues to marker genes deﬁned in mammalian liver scRNA-Seq datasets (e.g. 6), many markers were notably absent or expressed at very low levels in our snRNA-Seq dataset (Supplementary Figure 3). For example, Atlantic salmon orthologues of the widely used epithelial marker Epcam did not show expression in the cholangiocyte cluster (Supplementary Figure 3). Likewise, salmon orthologues of Pecam1 and Pdgfrb, which are excellent markers of mammalian endothelial and mesenchymal cells, were not detected at signiﬁcant levels in these cell types in our dataset (Supplementary Figure 3). These results may be explained by differences in transcriptome composition between snRNA-Seq and scRNA-Seq datasets from liver, as identiﬁed in humans (30). In support of this idea, epcam and pecam1 were respective markers of cholangiocytes and endothelial cells in a recently published zebraﬁsh scRNA-Seq dataset (31), making it less likely that the lack of expression of these markers in our Atlantic salmon dataset represents a true evolutionary difference between mammals and teleosts. Single-nuclei RNA-Seq atlas of the Atlantic salmon liver Differences in expression were also observed between predicted Atlantic salmon orthologues of mammalian marker genes for the major hepatic cell types, with, for example, only one of two cdh5 co-orthologues marking the endothelial population, and only one of two hnf4a co- orthologues marking hepatocytes (Supplementary Figure 3). Single-nuclei RNA-Seq atlas of the Atlantic salmon liver A A B D D C FIGURE 1 Major cell types in the Atlantic salmon liver deﬁned by 47,982 nuclear transcriptomes. (A) UMAP highlighting ﬁve main liver cell-type clusters according to a priori deﬁned marker genes (see Supplementary Table 2). (B) Bubble plots showing the expression of a priori marker genes for the ﬁve main liver cell types, including mean expression (bubble intensity) and percentage of nuclei expressing gene (bubble size). (C) Heatmap of the top 20 differentially expressed genes per each liver cell type deﬁned against the background of all other cell types. (D) Violin plots demonstrating the expression of the most speciﬁc marker genes per each of the main liver cell types (colours are matched to the colours of the 5 cell lineages deﬁned in part A). frontiersin.org Frontiers in Immunology 05 10.3389/ﬁmmu.2022.984799 Taylor et al. revealed markers for each major liver cell type (Figures 1C, D; Supplementary Table 3). Marker genes for candidate cell types and sub-populations are hereafter reported according primarily to annotations provided by Ensembl, or in cases where no Ensembl annotation was available, using supplementary BLAST homology searches against the NCBI database to support our inferences. While Ensembl annotation utilizes phylogenetic information to inform homology relationships, it may nonetheless fail to correctly capture orthology of Atlantic salmon genes to mammalian species, particularly for fast evolving and complex gene families. Adding to this challenge, genetic orthology is not a prerequisite for conservation of gene function or expression, which limits our ability to transfer knowledge about cell marker genes from mammalian studies to Atlantic salmon. While the reader must be aware of these caveats, they represent a general issue in functional genomics studies using non-model taxa like salmonids. revealed markers for each major liver cell type (Figures 1C, D; Supplementary Table 3). Marker genes for candidate cell types and sub-populations are hereafter reported according primarily to annotations provided by Ensembl, or in cases where no Ensembl annotation was available, using supplementary BLAST homology searches against the NCBI database to support our inferences. While Ensembl annotation utilizes phylogenetic information to inform homology relationships, it may nonetheless fail to correctly capture orthology of Atlantic salmon genes to mammalian species, particularly for fast evolving and complex gene families. Hepatocyte remodelling dominates the liver response to bacterial infection To explore how hepatocyte heterogeneity contributes to the response to Aeromonas infection, we analysed 41,792 available hepatocyte nuclei transcriptomes. Clustering using the most variable genes in this compartment identiﬁed nine sub- populations (H1-H9) (Figure 3A; marker genes in Supplementary Table 7), with several showing marked differences in abundance between control and infected ﬁsh (Figure 3B). H1-H4 comprised 90.2% of hepatocyte nuclei, with H1 and H2 deriving mainly from control ﬁsh and showing highly correlated transcriptomes (Figures 3B, C). H3 and H4 comprised 70.0% of nuclei from infected ﬁsh and showed closely related transcriptome proﬁles (Figures 3B, C). Hepatocyte nuclei derived from infected ﬁsh increased from H1 (16.9%), to H2 (28.2%), to H3 (64.3%) to H4 (80.8%), with 2,842 genes differentially expressed on this gradient (Figures 3D, E; Supplementary Tables 8 and 9). 379 genes were up-regulated in infection-dominated H4 vs. control-dominated H1, showing overrepresented functions linked to host defense and the acute phase response (‘complement activation’, ‘defense response to other organism’, and ‘cellular iron homeostasis’), in addition to translational processes (e.g. ‘translational elongation’) (Figure 3F, Supplementary Table 10). This response was dominated by genes encoding APPs including hepcidin, haptoglobins, ferritins, transferrin, ﬁbrinogens, ceruloplasmin, angiotensinogen, serum albumins, apolipoproteins, and c- reactive protein (Supplementary Table 8). One of the top up- regulated genes (3.7-fold up-regulated; ENSSSAG00000046715) encodes mechanistic target of rapamycin kinase (mTOR), a master regulator of translation (34). mTOR biasedly promotes translation of ribosomal protein genes (34), which is notable as Higher resolution clustering captured varying degrees of transcriptomic heterogeneity for each of the ﬁve major liver cell types (Figures 2A, B; Supplementary Table 4), which was consistent across the four samples (Supplementary Figure 4). We observed a split of hepatocytes into sub-populations explained largely by infection status, with 75.9% of ‘control-associated’ hepatocytes deriving from control ﬁsh and 73.3% of ‘infection- associated’ hepatocytes deriving from Aeromonas-challenged ﬁsh (expanded in next section). Signiﬁcant markers for the latter were dominated by genes encoding APPs, many of which were observed to be expressed across all cell types (Figure 2B). This is likely a consequence of the numerically dominant hepatocytes (Figure 1A) leaking mRNA from highly Frontiers in Immunology frontiersin.org 06 Taylor et al. 10.3389/ﬁmmu.2022.984799 A B FIGURE 2 Higher resolution atlas of Atlantic salmon liver cells deﬁned by snRNA-Seq. (A) Unbiased graph-based clustering reveals varying heterogeneity levels across the major liver cell types, presented on a UMAP. Hepatocyte remodelling dominates the liver response to bacterial infection Each cell population retains the gene signature of the parent lineage (Supplementary Figure 2), while also displaying its own distinct transcriptomic proﬁle, presented here as a heatmap (B), inclusive of the top 10 marker genes based on differential gene expression against all other cell clusters. The colour bars above columns on the heatmap illustrate the cell types to which the genes shown were identiﬁed as markers (matched to part A). A B A B B B B FIGURE 2 Higher resolution atlas of Atlantic salmon liver cells deﬁned by snRNA-Seq. (A) Unbiased graph-based clustering reveals varying heterogeneity levels across the major liver cell types, presented on a UMAP. Each cell population retains the gene signature of the parent lineage (Supplementary Figure 2), while also displaying its own distinct transcriptomic proﬁle, presented here as a heatmap (B), inclusive of the top 10 marker genes based on differential gene expression against all other cell clusters. The colour bars above columns on the heatmap illustrate the cell types to which the genes shown were identiﬁed as markers (matched to part A). 28 such genes, encoding many proteins comprising the large and small ribosomal subunits of salmonid ﬁshes (35), were up- regulated in H4 vs. H1, with only one downregulated (Supplementary Tables 8 and 9). We further observed up- regulation of copb1 (ENSSSAG00000068484), grp78 (ENSSSAG00000054661), sec61a1 (ENSSSAG00000039008), and srprb (ENSSSAG00000046456), encoding major components of the COPI and translocon complexes, representing key protein secretion pathways (36). activator of transcription 3, which acts downstream of NF-kB in mammalian hepatocytes to activate inﬂammation driving the acute phase response (37). 2,278 genes were downregulated in H4 vs. H1 (Supplementary Table 9), enriched in functions related to signaling (e.g. ‘intracellular receptor signaling pathway’), metabolism (e.g. ‘gluconeogenesis’), cell differentiation (e.g. ‘stem cell differentiation’) and transcription (e.g. ‘regulation of transcription, DNA-templated’) (Figure 3F) (Supplementary Table 11). The top downregulated gene was prkag2a (4.0-fold down-regulated; ENSSSAG00000079550), encoding a subunit of the AMPK complex - a master regulator of metabolism including gluconeogenesis (38). Also downregulated were master hepatic transcription factors for lipid and glucose metabolism pathways that interact with AMPK (38), including genes encoding hepatocyte nuclear factor 1 (HNF1) (ENSSSAG00000006158) and HNF4 (ENSSSAG00000047055) (39), carbohydrate-responsive element-binding protein (ENSSSAG00000039257) (40), and forkhead box proteins O1/O3 H5 nuclei were mainly from infected ﬁsh (Figure 3B) and showed highly correlated transcriptomes to H3/H4 (Figure 3C), sharing many of the same key markers up-regulated in H4 vs. Hepatocyte remodelling dominates the liver response to bacterial infection H1, but also speciﬁc markers (Supplementary Table 7) associated with NF-kB signaling, including relb (ENSSSAG00000052551), encoding a component of the NF-kB transcription factor complex. This is notable, as H5 also showed the highest expression among all hepatocyte sub-populations of stat3 (ENSSSAG00000003657), encoding signal transducer and Frontiers in Immunology frontiersin.org 07 Taylor et al. 10.3389/ﬁmmu.2022.984799 A B D E F C FIGURE 3 Striking remodeling of the hepatocyte transcriptome in response to bacterial infection. (A) UMAP visualisation of 41,792 hepatocyte nuclei, with sub-clustering performed using the most variable genes restricted to this cell lineage. (B) Shows the same UMAP with nuclei coloured by infection state (top) and the proportion of nuclei originating from each sample after normalising for different nuclei numbers across samples (bottom). (C) Pearson correlation of the expression values for the top 2,000 most variable genes across the nine hepatocytes populations H1- H9. (D) Violin plots of mean expression for the 20 most down-regulated (left) and up-regulated (right) genes based on log-fold change in H4 vs. H1. (E) Heatmap of the top 20 most upregulated and top 20 most downregulated genes in H4 relative to H1, illustrating a gradient of expression from H1 ! H2 ! H3 ! H4 (F) Example enriched GO terms in H4 based on all up-regulated and all down-regulated genes in H4 vs. H1 (full data provided in Supplementary Tables 10 and 11). B A B A B C D D F E F F E FIGURE 3 Striking remodeling of the hepatocyte transcriptome in response to bacterial infection. (A) UMAP visualisation of 41,792 hepatocyte nuclei, with sub-clustering performed using the most variable genes restricted to this cell lineage. (B) Shows the same UMAP with nuclei coloured by infection state (top) and the proportion of nuclei originating from each sample after normalising for different nuclei numbers across samples (bottom). (C) Pearson correlation of the expression values for the top 2,000 most variable genes across the nine hepatocytes populations H1- H9. (D) Violin plots of mean expression for the 20 most down-regulated (left) and up-regulated (right) genes based on log-fold change in H4 vs. H1. (E) Heatmap of the top 20 most upregulated and top 20 most downregulated genes in H4 relative to H1, illustrating a gradient of expression from H1 ! H2 ! H3 ! H4 (F) Example enriched GO terms in H4 based on all up-regulated and all down-regulated genes in H4 vs. Additional hepatocyte heterogeneity includes polyploid cells substantial contributions from all samples, except the candidate neutrophils, which derived mainly from one Aeromonas- challenged ﬁsh (Supplementary Figure 4). Sub-clustering revealed heterogeneity in the T and myeloid cells, but not the NK-like cells, B cells (with no evidence of plasma cells) or candidate neutrophils. The remaining hepatocyte subclusters were not linked to infection status (Figure 3B). H7 likely represents hepatocytes that have undergone polyploidization, which occurs progressively during aging in mammals, such that 4n-16n cells make up a large fraction of liver cells by adulthood (43). Polyploidy in H7 is indicated by a striking concordance of genes representing highly speciﬁc markers for H7 and those shown elsewhere to be up-regulated in 4n vs. 2n mammalian cells, encoding DNA primase subunit 2 (ENSSSAG00000001875), replication protein A 70 kDa DNA- binding subunit (ENSSSAG00000050927, ENSSSAG00000050209) and DNA polymerase (ENSSSAG00000078390), among others (44). The close relationship of H7 to H1 (Figures 3A, C) implies that polyploid hepatocytes derive from those supporting routine metabolism. Five T cell sub-clusters were compared using differential expression tests (Figure 4A; Supplementary Table 12). Only T1 and T2 expressed cd4 genes to low levels, speciﬁcally ENSSSAG00000076631, encoding CD4-1, which is also expressed by macrophages, and ENSSSAG00000076595, encoding CD4-2; shown elsewhere to be expressed by all CD4+ T cells (48). We did not identify any CD8 expressing T cells, likely due to low expression levels. T1 was the largest sub-cluster but showed few speciﬁc markers. T2 expressed genes encoding receptors involved in T cell activation. This included cd28 (ENSSSAG00000060163), encoding the main co-stimulatory T cell receptor (49) and cd44 (ENSSSAG00000076128), a widely used T cell adhesion, co-stimulation and activation marker (50). However, T2 cells did not speciﬁcally express any genes annotated as ctla-4, an IgSF member induced during T cell activation that regulates CD28 activity. Furthermore, T1 and T2 both expressed tcf7 (ENSSSAG00000006857) at a much higher level than T3-T5, encoding the master Wnt pathway transcription factor, which is most highly expressed on naïve mammalian T cells (51) and was a speciﬁc marker for resting CD4+ T cells in humans (52). T1 and T2 also expressed foxp1b (ENSSSAG00000077820) at a higher level than T3-5; a gene essential for quiescent naive T cells in mammals (53). T1 and T2 therefore appear to be constituted mainly of resting T cells. T3 appears to be an activated T cell population based on speciﬁc expression of slamf1 (ENSSSAG00000043093) encoding CD150 (54). Additional hepatocyte heterogeneity includes polyploid cells T3 expressed many highly speciﬁc markers, including the integrin coding gene itgal (ENSSSAG00000046537; encoding CD11a) and itgb2 (ENSSSAG00000022772; encoding CD18), whose products form lymphocyte function-associated antigen 1 (LFA-1), a molecule with key roles in T cell activation and migration, in addition to cytotoxic and memory responses (55). In mammals, LFA-1 is an established marker for the migration of liver T resident memory (TRM) cells into liver, which is also the case for CD103 (also known as ITGAE) (56), which was expressed more highly in T3 (ENSSSAG00000076346) than other T sub-clusters. The human orthologue of a highly e x p r e s s e d T 3 s p e c i ﬁc m a r k e r , c a c n a 2 d 2 a (ENSSSAG00000071299), encoding a calcium voltage-gated channel, was not detected in any immune cell in the human protein atlas, implying a teleost-speciﬁc T cell marker. T4 expressed several activation markers including pou2f2a (aka oct2) (ENSSSAG00000071136) (57), CD226 (58), along with two distinct ctram genes (encoding cytotoxic and regulatory T-cell molecule), previously shown in mammals to be required for differentiation of cytotoxic CD4+ T cells (59). T5 speciﬁcally expressed two paralogues of sox13 (ENSSSAG00000077869, H9 showed many speciﬁc markers encoding mitosis proteins, e.g. cytoskeleton-associated protein 5 (ENSSSAG00000066206), centromere protein E (ENSSSAG00000073454), abnormal spindle- like micr oc ephaly-asso ciated protein homolog (ENSSSAG00000053226) and kinesin family member 23 (ENSSSAG00000044381), indicating these are cycling hepatocytes. H6 expressed a small number of highly speciﬁc markers, including genes encoding ligand of numb-protein X1 (lnx1) (ENSSSAG00000072728 and ENSSSAG00000070275), a E3 ubiquitin ligase that targets a wide range of proteins, including CD8 expressed on T-cells in mammals (45). H8 was biasedly represented by control ﬁsh and most correlated with H1/H2 in transcriptome proﬁle (Figure 3C), expressing marker genes described as ‘novel’ in the Ensembl annotation (Supplementary Table 7). Hepatocyte remodelling dominates the liver response to bacterial infection H1 (full data provided in Supplementary Tables 10 and 11). downstream of GHR to activate growth and cell proliferation genes (42). In addition, a gene was downregulated encoding glucocorticoid receptor (GR) (ENSSSAG00000062169), which promotes expression of gluconeogenesis genes, while its interaction with Stat5 is required for transcriptional activation of growth genes via GHR signalling (42). (ENSSSAG00000054669/ENSSSAG00000055241) (41) (Supplementary Table 9). Further evidence for repression of anabolism included downregulation of genes encoding growth hormone receptor (GHR) (ENSSSAG00000065355 and ENSSSAG00000081526) and Stat5a/b (ENSSSAG00000010616, ENSSSAG00000003584 and ENSSSAG00000048873), which act frontiersin.org Frontiers in Immunology 08 Taylor et al. 10.3389/ﬁmmu.2022.984799 Immune cell heterogeneity in the Atlantic salmon liver 3.4% (n=1,620) of the liver nuclei were derived from immune cells (Figures 4A, B). Combinations of canonical marker expression was used to classify T cells (cd3e, tox2 and tcf7), B cells (ighm, cd37, cd79a), NK-like cells (prf1.3 and runx3 with absence of cd3e) and myeloid cells (mpeg1, cd63, csf1r, lyz2) (Figure 4B; marker genes provided in Supplementary Table 12). We also identiﬁed a candidate population of neutrophils based on marker genes that showed highest speciﬁcity or expression for neutrophils among different immune cells in the human protein atlas, namely itgax (ENSSSAG00000049715), ncf1 (ENSSSAG00000079828), and mmp9 (ENSSSAG00000069874) (Figure 4B). Mmp9 was an effective marker for neutrophils in other teleost species, and has a role in driving neutrophil migration in mammals (46, 47). The NK-like cells represent a tentative annotation owing to a lack of certain markers for NK cells, i.e. genes encoding granzymes. Each immune cluster had 3.4% (n=1,620) of the liver nuclei were derived from immune cells (Figures 4A, B). Combinations of canonical marker expression was used to classify T cells (cd3e, tox2 and tcf7), B cells (ighm, cd37, cd79a), NK-like cells (prf1.3 and runx3 with absence of cd3e) and myeloid cells (mpeg1, cd63, csf1r, lyz2) (Figure 4B; marker genes provided in Supplementary Table 12). We also identiﬁed a candidate population of neutrophils based on marker genes that showed highest speciﬁcity or expression for neutrophils among different immune cells in the human protein atlas, namely itgax (ENSSSAG00000049715), ncf1 (ENSSSAG00000079828), and mmp9 (ENSSSAG00000069874) (Figure 4B). Mmp9 was an effective marker for neutrophils in other teleost species, and has a role in driving neutrophil migration in mammals (46, 47). The NK-like cells represent a tentative annotation owing to a lack of certain markers for NK cells, i.e. genes encoding granzymes. Each immune cluster had Frontiers in Immunology frontiersin.org 09 Taylor et al. 10.3389/ﬁmmu.2022.984799 A B D C FIGURE 4 Heterogeneity in Atlantic salmon immune cells. (A) UMAP visualization of 1,620 immune nuclei. (B) Heatmap of top 5 markers genes for each immune sub-cluster, sorted by log2 fold change. (C) Number of cell-speciﬁc genes up-regulated by infection in immune sub-clusters (D) Examples of genes showing cell-speciﬁc up-regulation in response to Aeromonas infection across the breath of immune cell heterogeneity identiﬁed. A C B B D B D FIGURE 4 D FIGURE 4 Heterogeneity in Atlantic salmon immune cells. (A) UMAP visualization of 1,620 immune nuclei. u e sub c uste sp Supplementary Table 13). B cells speciﬁcally up-regulated two genes, namely stat3 (2.4- fold up-regulated; ENSSSAG00000003657), which is essential for B cell differentiation (67) and a gene annotated zgc:174904 (2.1-fold up-regulated; ENSSSAG00000070511), encoding a 304 amino acid protein with a CD209/DC-Sign-like, C-type lectin- like domain (InterPro domain: IPR033989). C-type lectin/DC- SIGN is a broad-speciﬁcity PRR that detects bacteria by binding mannose or carbohydrate structures (68). In the candidate neutrophils, the top up-regulated gene was ladderlectin (38.9- fold up-regulated; ENSSSAG00000039613), encoding a soluble lectin that bound Aeromonas in salmonids, leading to bacterial killing actions (69). The NK-like cells up-regulated few genes speciﬁcally, one of which was pglyrp2 (2.3-fold up-regulated; ENSSSAG00000054105), a peptidoglycan recognition protein with enzymatic activity targeting and limiting the inﬂammatory effects of bacterial peptidoglycan (70). My3 speciﬁcally up-regulated 16 genes, including il12p40b2 (5.7-fold up-regulated; ENSSSAG00000069633), best known as a component of IL-12 and/or IL-23 heterodimers, but that also has deﬁned cytokine functions as a monomer protein, including promotion of DC migration in response to bacterial infection in mammals (82, 83). Another induced gene was afp4 (3.7-fold up- regulated; ENSSSAG00000072959), encoding type IV ice- structuring protein LS-12, an apolipoprotein-like molecule that dramatically increases in abundance in salmon plasma following Aeromonas infection (84). My4 speciﬁcally up-regulated 64 genes, including gas7 (8.56-fold up-regulated; ENSSSAG00000076110), which has a crucial role in phagocytosis (85), dnase1l3 (8.4-fold up-regulated; ENSSSAG00000066441), which controls inﬂammasome- induced cytokine secretion (86), and cd82 (6.9-fold up- regulated; ENSSSAG00000052206), previously shown to be up- regulated during DC activation, where is promotes stable interactions between DCs and T cells, and MHC-II maturation (87). My4 further upregulated irf4 (6.28-fold up-regulated; ENSSSAG00000039730), a gene essential to the ability of DCs to promote Th2 differentiation and inﬂammation (88). Among the T cell populations, T3, T4, and the gd T cells (T5), showed the strongest responses to Aeromonas infection. The top up-regulated genes in T3 included csl1 (4.3-fold up- regulated; ENSSSAG00000004327), encoding a L-rhamnose- binding lectin that binds bacteria and enhances phagocytosis in salmonids (71), and c8b (4.1-fold up-regulated; ENSSSAG00000073702), encoding a core component of the complement membrane attack complex. In T4, up-regulated genes included tln1, encoding Talin-1 (4.8-fold up-regulated; ENSSSAG00000063331), which is known to regulate the integrin LFA-1 complex (deﬁning T3; see last section), and is required for sustained interactions between APCs and T cells, as well as T cell proliferation (72). Immune cell heterogeneity in the Atlantic salmon liver (B) Heatmap of top 5 markers genes for each immune sub-cluster, sorted by log2 fold change. (C) Number of cell-speciﬁc genes up-regulated by infection in immune sub-clusters (D) Examples of genes showing cell-speciﬁc up-regulation in response to Aeromonas infection across the breath of immune cell heterogeneity identiﬁed. expression in My4 supports an annotation as dendritic cells (DCs) (61), with speciﬁc up-regulation of cd9 (ENSSSAG00000059637) and lamp2 (encoding CD107b) (ENSSSAG00000074801) genes, consistent with monocyte- derived DCs in mammals (62, 63). My3 expressed the second highest level of ﬂt3, while the top My3 marker gene, ptprsa (ENSSSAG00000051752), is a speciﬁc marker for plasmacytoid DCs (pDCs) in mice and human (64). The relationship of My3 to pDCs is also supported by speciﬁc expression of sbf1 (ENSSSAG00000071635) (65). However, another My4- speciﬁc marker gene, tcf4 (ENSSSAG00000071044), encodes a transcription factor required for pDC development (66). ENSSSAG00000058488), the deﬁning vertebrate transcription factor for the gd T lineage (60). My1 and My2 markers are associated with monocytes and macrophages (Supplementary Table 12). My1 speciﬁcally expressed cd4-1 (ENSSSAG00000076631), at a level higher than any T sub-cluster, likely representing a phagocytic CD4+ macrophage characterized in rainbow trout (48). My2 expressed speciﬁc monocyte marker genes including csf3r (ENSSSAG00000041566) and timp2 (ENSSSAG00000042353 and ENSSSAG00000064056). Two csf1r copies were identiﬁed with reciprocal higher expression in My1 (ENSSSAG00000004088) and My2 (ENSSSAG00000061479). High ﬂt3 (ENSSSAG00000009390) Frontiers in Immunology frontiersin.org 10 Taylor et al. 10.3389/ﬁmmu.2022.984799 u e sub c uste sp Supplementary Table 13). T4 also up-regulated itgb1 (encoding CD29, also called b1-integrin) (4.8-fold up-regulated; ENSSSAG00000007621), a signature marker for cytotoxic T cells in humans (73). In T5, among the top-up-regulated genes was prex1 (4.8-fold up-regulated; ENSSSAG00000044871), a signaling molecule that promotes expression of key interleukin cytokines in activated human T cells, including IL-2 (74). T5 also up-regulated catl1, encoding cathepsin L (8.0-fold up-regulated; ENSSSAG00000077309), which regulates T cell cytotoxicity (75) and an unannotated gene encoding a protein with saposin-like domains, which is annotated as prosaposin-like in NCBI (7.4- fold up-regulated; ENSSSAG00000009411). In mammals, Immune cell-speciﬁc responses to Aeromonas insult prosaposin encodes the precursor to all saposin lysosomal proteins, which are known to have antibacterial activity and play a key role in presenting lipid antigens to CD1-restricted T cells (76). To uncover the role of hepatic immune cell heterogeneity in the response to Aeromonas challenge, we performed differential expression tests comparing nuclei from control and infected ﬁsh within each immune sub-cluster (Figures 4C, D). 819 genes showed signiﬁcant up-regulation in nuclei from infected ﬁsh (criteria: P < 0.05; Log2FC > 1), among which 274 (33%) and 72 (8.8%) were up-regulated by most (≥7 of the 12 sub-clusters) or all immune cell types, respectively, and 271 (33%) showed immune sub-cluster speciﬁc up-regulation (Figure 4C; Supplementary Table 13). My1 speciﬁcally up-regulated 17 genes, including a different itgal paralogue (encoding CD11A) to that noted as a marker for T3 (4.2-fold up-regulated; ENSSSAG00000046996), encoding a component of LFA-1 essential to the immune response of mice to Mycobacterium tuberculosis, supporting T cell-mediated activation and recognition of infected macrophages (77). My1 also speciﬁcally up-regulated olfm4 (4.1-fold upregulated; ENSSSAG00000046003), encoding a glycoprotein induced in mice macrophages by Helicobacter pylori infection, which regulates inﬂammatory responses (78). My1 further speciﬁcally up-regulated cats (ENSSSAG00000070942) and ctsd (ENSSSAG00000027269), encoding cathepsin S and D, proteolytic enzymes with established macrophage roles in bacterial killing and antigen processing (79). My2 speciﬁcally up-regulated 32 genes, including cath2 (6.6-fold upregulated ENSSSAG00000049319), encoding an antimicrobial peptide that increased in abundance in response to Aeromonas infection in salmon plasma (80) and abr (6.6-fold upregulated; ENSSSAG00000080204), encoding a GTPase-activating protein that down-regulates the inﬂammatory actions of macrophages (81). Discussion This study greatly enhances our knowledge of liver function in a salmonid ﬁsh with global commercial and scientiﬁc importance. The major advancement compared to previous Frontiers in Immunology frontiersin.org 11 Taylor et al. 10.3389/ﬁmmu.2022.984799 work comes from our application of snRNA-Seq, which, in contrast to previously past bulk transcriptomic or proteomic studies, allowed us to identify multiple hepatic cell populations, before dissecting the role of this heterogeneity in host defense following bacterial infection. Furthermore, a plethora of novel marker genes are reported for developmentally and functionally diverse hepatic cell types, which will be useful for future studies investigating traits relevant to salmonid health and immunological status. is ﬁrst initiated. A previous scRNA-Seq study of zebraﬁsh liver failed to identify hepatocytes showing any equivalent specialization towards host defense (18), while another identiﬁed a minor hepatocyte population enriched for immune functions (17), which may be analogous to H3-H5. Differences with these past zebraﬁsh studies may reﬂect the fact that both studies utilized control zebraﬁsh lacking immune stimulation. However, the fact that H3-5 comprised a signiﬁcant fraction of hepatocytes in our control ﬁsh could also be explained by differences in liver function, potentially indicative of a higher baseline inﬂammatory state in the liver of the Atlantic salmon population we studied. Our results demonstrate the essential contribution of hepatocytes to the antibacterial and acute phase response in Atlantic salmon. Transcriptomic heterogeneity in the dominant hepatocyte population (i.e., H1-4; Figure 3) was inconsistent with distinct hepatocyte populations. Instead, our data supports a single hepatocyte population that can exist in radically distinct transcriptional states dependent on infection status. At one extreme are the hepatocytes that dominate the liver of healthy ﬁsh (H1/2), which appear to be performing routine functions controlled by master hepatic transcription factors and signaling pathways. Conversely, the liver of bacterially infected Atlantic salmon was dominated by hepatocytes (H3/4) that downregulated master hepatic pathways (e.g. controlling metabolic functions), and up-regulated a suite of genes encoding APPs and innate immune molecules. This includes many APPs routinely detected in Atlantic salmon plasma by proteomics (89) implying extremely high abundance. As most plasma proteins derive from liver, this aligns with our ﬁnding that these ‘defense-specialized’ hepatocytes strongly up- regulated mTOR, its target ribosomal protein-coding genes, and genes from protein secretion pathways, presumably to boost APP translation and secretion rates during the acute phase response. Discussion This striking repurposing of hepatocyte function upon infection illustrates the vital role these cells play as a hub for cross-talk between metabolism and immunity, presumably allowing energetic resources to be allocated towards clearing a pathogen at the short-term cost of limiting investment into routine hepatic functions e.g. supporting growth (14). While representing a small population of liver nuclei, we also offer evidence for polyploid hepatocytes in Atlantic salmon. The functional role of polyploidy in mammalian liver remains ill- deﬁned, despite extensive study over decades (43). Work done over 40 years ago showed that the liver of several teleosts contained polyploid hepatocytes (91), so our result is perhaps not unexpected. Polyploidy increases with aging in mammals (43), which may explain why this hepatocyte population was so limited in the ﬁsh used in our study, which were juveniles. However, polyploid hepatocytes were not reported in past scRNA-Seq studies of zebraﬁsh liver, which included an 18- month-old adult population (17), representing half the adult lifespan for this species. More work is required to understand the role of hepatocyte polyploidy in teleost health and disease. It is important to acknowledge that comparing our results with liver scRNA-Seq studies in other species has limitations due to fundamental differences in experimental execution, which, for example, is linked to striking differences in the composition of the cells captured. For example, a preprint by ref (18) surprisingly identiﬁed T cells as the dominant liver population in zebraﬁsh, with hepatocytes reﬂecting a smaller proportion than expected. While underrepresentation of hepatocytes has been observed in several mammalian liver scRNA-Seq studies, a separate zebraﬁsh scRNA-Seq study identiﬁed hepatocytes as the most abundant liver cell type (17), albeit at a smaller fraction than for our snRNA-Seq atlas in Atlantic salmon. This perhaps illustrates a recognized beneﬁt of snRNA-Seq compared to scRNA-Seq; a more accurate representation of the true tissue cell diversity (e.g. 27, 28, 94). Considering our limited knowledge of cellular diversity in most ﬁsh species, including salmonids, careful comparisons of results from scRNA-Seq and snRNA-Seq will be required to establish baseline expectations for future studies. Interestingly, a sub-population of defense-specialized hepatocytes (i.e. H5; Figure 3), almost exclusively derived from infected ﬁsh, speciﬁcally expressed genes associated with NF-kB signaling (e.g. relb was ~10-fold more highly expressed in this population compared to the average across the eight other hepatocyte subpopulations). Discussion While KCs were not expected among our macrophage populations, it is worth noting the expression of the predicted Atlantic salmon orthologues of mammalian Clec4f (ENSSSAG00000040735 and ENSSSAG00000076658). Clec4f encodes a C-Type Lectin (also known as Kupffer cell receptor) expressed speciﬁcally in KCs to the exclusion of non-liver macrophages (102, 103). In our Atlantic salmon dataset, one predicted orthologue of Clec4f is expressed across all four myeloid populations (ENSSSAG00000076658), showing h i g h e s t e x p r e s s i o n i n M y 2 , w h i l s t t h e o t h e r (ENSSSAG00000040735) was expressed across all immune cell types (Supplementary Table 11). Furthermore, both genes were among the top up-regulated genes in hepatocytes following Aeromonas infection (Supplementary Table 8). These ﬁndings highlight the potential for major differences in marker gene cell- speciﬁc expression and presumably function for homologous genes shared by mammals and teleosts. Having robust marker genes for different cell-types is clearly essential for accurate studies of cell biology in any species. However, single cell transcriptomics studies performed to date, including this study, demonstrate that marker genes can vary markedly across species, especially for immune cells (96, 104). This demands a broader uptake of single cell transcriptomics in more species to deﬁne conserved from non-conserved marker genes, and to separate true biological or evolutionary differences in cell heterogeneity and associated marker genes from differences introduced by technical reasons discussed earlier, including the use of snRNA-Seq vs. scRNA-Seq. In salmonid ﬁshes, the presence of an ancestral whole genome duplication, and the associated retention of numerous duplicated genes that have diverged extensively in tissue expression (22, 105–107), further challenges the transfer of knowledge on marker genes from model species - more work is required in this area. In summary, our comprehensive dissection of the Atlantic salmon liver using single cell transcriptomics has generated many species-speciﬁc marker genes for a range of immune and non- immune cell types that contribute to health and immunological traits of relevance to sustainable aquaculture. Our results can further be used to extract cell-speciﬁc information from existing and future bulk gene expression studies. Discussion In addition, H5, among all hepatocyte sub-populations, showed the highest expression of stat3, which is indispensable for activation of APP and protein secretion pathway gene expression during bacterial infection in mice, acting downstream of NF-kB (37, 90). As APP and secretory protein pathway genes were strongly up-regulated in the dominant sub-populations of defense-specialized hepatocytes (H3/4), which lacked signiﬁcant relb expression, H5 may represent an intermediate hepatocyte state, where the activation of the APP response and associated secretory pathway This is the ﬁrst single cell transcriptomic study to characterize immune cell heterogeneity in the liver of a salmonid ﬁsh, and the ﬁrst single cell study in any teleost to characterize transcriptomic responses of speciﬁc hepatic immune cell subtypes to infection. Past scRNA-Seq studies of zebraﬁsh liver paint very distinct pictures of lymphocyte diversity, with one reporting no B cells, and little T cell heterogeneity (17). Conversely, a recent preprint reported a Frontiers in Immunology frontiersin.org 12 Taylor et al. 10.3389/ﬁmmu.2022.984799 10.3389/ﬁmmu.2022.984799 small B cell population, and six T cell sub-clusters capturing distinct CD8 and CD4 subsets (18). While we also identiﬁed a single B cell population and multiple T cell sub-clusters, the identity of T cells was markedly less clear in our data, due partly to a general lack of cd4 and cd8 expression, which may reﬂect a limitation of snRNA-Seq, or the lower sequencing depth in our study compared to (18). However, unlike these previous studies, we identiﬁed a small population of sox13+ gd T cells, an ancient vertebrate T cell subtype with roles bridging adaptive and innate immunity. In zebraﬁsh, gd T cells possess phagocytic ability and act as APCs that activate CD4+ T cells, inducing B cell proliferation (93). In mammals, gd T cells increase dramatically in liver during inﬂammatory conditions (94) and produce IL-17 essential for the innate response to bacterial infection (95). While gd T cells have not been reported among the plethora of immune cells reported to date in scRNA-Seq studies spanning different teleost species (96), we ﬁnd them readily identiﬁable in Atlantic salmon by sox13 expression in multiple tissues (not shown). Consistent with their known functions, salmon liver gd T cells up-regulated genes with roles spanning innate and adaptive immunity during the early response to Aeromonas infection. Figure 9). Discussion Our study also identiﬁed evidence for myeloid heterogeneity within the Atlantic salmon liver, including two candidate DC populations that showed a strong response to bacterial infection, up-regulating genes required for interactions with T cells, phagocytosis and inﬂammasome activation, suggesting conserved roles between mammals and salmonids, as shown elsewhere (97). DCs were reported in a recent scRNA-Seq study of zebraﬁsh liver (31), but not in two other liver scRNA-Seq analyses from the same species (17, 18). DCs have also been reported in Atlantic cod spleen (98). We observed two distinct candidate macrophage populations, matching the level of heterogeneity recently reported in two previous zebraﬁsh studies (17, 18). A natural question relates to the relationship of teleost macrophages and mammalian Kupffer cells (KCs). Past work largely agrees that KCs are amniote-speciﬁc and hence not present in teleost liver (99), including for Atlantic salmon (100). Consistent with this notion, a recent study in zebraﬁsh identiﬁed that resident macrophages were not located in the sinusoidal space, and lacked phagocytic ability (101). However, a recent cross-vertebrate scRNA-Seq analysis of liver cell repertoires reported putative KCs in the liver of zebraﬁsh based on a conserved transcriptomic signature with mammals (31). This study did not report any heterogeneity within the macrophages beyond the putative KCs, notably lacking any monocyte-derived macrophages known to make up a large proportion of macrophages in mammalian livers (2, 3), and which share marker genes with our Atlantic salmon macrophage clusters. We failed to identify conserved expression of the signature marker genes for KCs (in addition to transcription factors associated with KCs) deﬁned in this past study (31) in any of our myeloid sub-clusters from Atlantic salmon (Supplementary Data availability statement All data generated in this study has been made available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/). Accession: GSE207655. Acknowledgments Procedures) Act 1986 under Home Ofﬁce license PFF8CC5BE and was approved by the ethics committee of the University of Aberdeen. For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) license to any Author Accepted Manuscript version arising from this submission. Ethics statement The animal study was reviewed and approved by Animal work was carried out in compliance with the Animals (Scientiﬁc Frontiers in Immunology frontiersin.org 13 Taylor et al. 10.3389/ﬁmmu.2022.984799 Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ ﬁmmu.2022.984799/full#supplementary-material Conﬂict of interest Designed study: DM, RRD, RT; coordinated sampling and disease challenge experiment: SM; performed ﬁsh sampling: SN, SM, DM; performed quantitative PCR: SN; optimized nuclear isolation: RRD; generated snRNA-Seq libraries: RD, RRD; provided infrastructure for snRNA-Seq: NH; performed bioinformatics: RT; interpreted immunological data: TC, PB; drafted manuscript: DM, RT, RRD; made ﬁgures and tables: RT, DM; contributed to data interpretation and ﬁnalization of manuscript: all authors. The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Funding This study was funded by grants from the Scottish Universities Life Sciences Alliance (Technology Seed Funding Call), the University of Edinburgh’s Data Driven Innovation Initiative (Scottish Funding Council Beacon ‘Building Back Better’ Call), and the Biotechnology and Biological Sciences Research Council, including the institutional strategic programme grants BBS/E/D/10002071 and BBS/E/D/20002174 and the responsive mode grant BB/W005859/1. NH is supported by a Wellcome Trust Senior Research Fellowship in Clinical Science (ref. 219542/Z/19/Z). Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. 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https://openalex.org/W3208441916	https://www.researchsquare.com/article/rs-3217050/latest.pdf	English	null	A Japanese Multi-Institutional Phase II Study of Moderate Hypofractionated Intensity-Modulated Radiotherapy With Image-Guided Technique for Prostate Cancer	International journal of radiation oncology, biology, physics	2,021	cc-by	4,212	A Japanese Multi-Institutional Phase II Study of Moderate Hypofractionated Intensity-Modulated Radiotherapy with Image-Guided Technique for P t t C Page 1/12 Radiotherapy with Image-Guided Technique for Prostate Cancer Katsumasa Nakamura Hamamatsu University School of Medicine: Hamamatsu Ika Daigaku https://orcid.org/0000-0003- 2090-881X Keiji Nihei Osaka Medical and Pharmaceutical University: Osaka Ika Yakka Daigaku Yoshihiro Saito Saitama cancer center Naoto Shikama Juntendo University: Juntendo Daigaku Shin-ei Noda Saitama Medical University International Medical Center: Saitama Ika Daigaku Kokusai Iryo Center Ryusuke Hara Chiba-ken Gan Center Toshiyuki Imagumbai Kobe City Medical Center General Hospital: Kobe Shiritsu Iryo Center Chuo Shimin Byoin Takashi Mizowaki Kyoto University: Kyoto Daigaku Takeshi Akiba Tokai University: Tokai Daigaku Etsuo Kunieda Tokai University: Tokai Daigaku Masanori Someya Sapporo Medical University: Sapporo Ika Daigaku Saiji Ohga Kyushu medical center Jiro Kawamori St Luke's Hospital Takuyo Kozuka Katsumasa Nakamura Takashi Mizowaki Kyoto University: Kyoto Daigaku Takeshi Akiba Tokai University: Tokai Daigaku Etsuo Kunieda Tokai University: Tokai Daigaku Page 1/12 Page 1/12 Toranomon Hospital: Toranomon Byoin Yosuke Ota Hyogo Cancer Center: Hyogo Kenritsu Gan Center Koji Inaba National Cancer Center Takeshi Kodaira Aichi-ken Gan Center Yoshiyuki Itoh Anjo Kosei Hospital: Anjo Kosei Byoin Kouta Funakoshi Kyushu University Faculty of Medicine Graduate School of Medical Science: Kyushu Daigaku Igakubu Daigakuin Igakukei Gakufu Daigakuin Igaku Kenkyuin Yoshikazu Kagami Showa University: Showa Daigaku Research Article Keywords: prostate cancer, external beam radiotherapy, moderate hypofractionation Posted Date: August 10th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3217050/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at International Journal of Clinical Oncology on April 17th, 2024. See the published version at https://doi.org/10.1007/s10147-024-02517-z. Toranomon Hospital: Toranomon Byoin Yosuke Ota Hyogo Cancer Center: Hyogo Kenritsu Gan Cente Koji Inaba National Cancer Center Takeshi Kodaira Aichi-ken Gan Center Yoshiyuki Itoh Anjo Kosei Hospital: Anjo Kosei Byoin Kouta Funakoshi Kyushu University Faculty of Medicine Graduate S Daigakuin Igakukei Gakufu Daigakuin Igaku Kenky Yoshikazu Kagami Showa University: Showa Daigaku Research Article Keywords: prostate cancer, external beam radiotherapy, moderate hypofractionation Posted Date: August 10th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3217050/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at International Journal of Clinical Oncology on April 17th, 2024. See the published version at https://doi.org/10.1007/s10147-024-02517-z. Page 2/12 Abstract Background Although moderate hypofractionated radiotherapy were one of standard regimens for localized prostate cancer, the efficacy of moderated hypofractionated regimens has not been well validated in Asian countries. The aim of this multi-institutional phase II study was to confirm the safety and the potential efficacy of moderately hypofractionated intensity-modulated radiotherapy (IMRT) with prostate-based image-guidance for Japanese patients. Methods Patients with low- or intermediate-risk localized prostate cancer were eligible. Patients with a part of high risk (having only one of the following factors, cT3a, 20 < PSA ≤ 30, or GS = 8 or 9) were also included in this trial. Hypofractionated IMRT using daily image-guided technique with prostate matching was performed with a total dose of 70 Gy in 28 fractions. Neoadjuvant hormonal therapy for 4-8 months was mandatory for patients with intermediate or high-risk prostate cancer. Results The number of patients with low, intermediate, and high-risk prostate cancer was 20, 80, and 34, respectively. The 5-year overall, biochemical failure-free, and clinical failure-free survival was 94.5%, 96.0%, and 99.2%, respectively. The 5-year biochemical failure-free survival for patients with low-, intermediate-, and high-risk disease was 94.1%, 97.4%, and 93.9%, respectively. The incidences of grade 2 gastrointestinal and genitourinary late toxicities at 5 years were 5.3% and 5.3%, respectively. There are no acute or late toxicities ≥ grade 3. vely. The 5-year biochemical failure-free survival for patients with low-, intermediate-, and high-risk disease was 94.1%, 97.4%, and 93.9%, respectively. The incidences of grade 2 gastrointestinal and genitourinary late toxicities at 5 years were 5.3% and 5.3%, respectively. There are no acute or late toxicities ≥ grade 3. Conclusion The safety and efficacy of moderately hypofractionated IMRT was confirmed in this trial among Japanese patients with prostate cancer. Conclusion The safety and efficacy of moderately hypofractionated IMRT was confirmed in this trial among Japanese patients with prostate cancer. Introduction Recently, high-precision external beam radiotherapy has been introduced widely in Japan. Intensity- modulated radiotherapy (IMRT) and image-guided radiotherapy (IGRT) are effective techniques for external beam radiotherapy for localized prostate cancer, and these techniques have been routinely used in most institutions in Japan [1–3]. Several large, randomized trials for localized prostate cancer in the United States or Europe have been published and demonstrated that moderate hypofractionation regimens were not inferior to the conventional fractionation regimens with respect to both prostate cancer control outcomes and rates of late toxicity [4–7]. Based on the high-quality evidence by these trials, several guidelines have recommended that moderately hypofractionated regimens should be offered to patients who were treated with external beam radiotherapy for prostate cancer, because moderate hypofractionation has potential advantages for patient convenience and resource utilization. In Japan, IMRT and IGRT were covered by Japanese national health insurance in 2008 and 2010, respectively. In addition, hypofractionation regimens have been supported by health insurance in 2018. Page 3/12 Page 3/12 However, the radiation dose administered to the prostate was 2 Gy per fraction at most facilities in Japan [1, 3] as well as Asian countries [8]. Under these circumstances, we started a multi-institutional phase II study to confirm the safety and the efficacy of moderately hypofractionated intensity-modulated radiotherapy (IMRT) with image-guided technique for Japanese patients. Here, we present the results of the trial after the follow-up of 5 years. Study design This was a single-arm multi-institutional prospective phase II study with the University Hospital Medical Information Network Clinical Trials Registry (http://www.umin.ac.jp/ctr/index-j.htm), identification number UMIN000007810. Patients with pathologically confirmed localized adenocarcinoma of the prostate with low(T1-T2a and PSA < 10, and Gleason score (GS) ≤ 7)or intermediate risk (cT1-T2c, PSA < 20 and GS < 7, but not low risk) or high risk (having one of the following factors, cT3a, 20 < PSA ≤ 30, or GS = 8 or 9) were eligible for participation. Other eligibility criteria were the age range of 50–80 years and Eastern Cooperative Oncology Group performance status of 0–1. Patients with lymph nodes involvement or metastatic disease, history of pelvic or prostate surgery, prior radiotherapy to the prostate or pelvis, severe medical disorders, a history of inflammatory bowel disease, active collagen vascular disease, and previous malignancy within 5 years of prostate carcinoma diagnosis (except completely treated carcinoma in situ or T1N0M0 glottic cancer) were excluded from this study. The study was conducted under an institutional review board-approved prospective clinical protocol at 20 institutions in Japan, and all the patients enrolled signed an informed consensus. Treatment Radiotherapy alone was recommended for patients with low-risk prostate cancer. However, hormonal therapy within 8 months before radiotherapy was permitted. For patients with intermediate- or high-risk prostate cancer, neoadjuvant hormonal therapy consisting of a luteinizing hormone-releasing hormone (LH-RH) analogue alone or a combination of LH-RH analog and antiandrogen for 4–8 months was mandatory. After radiotherapy, the use of hormonal therapy is not allowed until tumor relapse occurs. Hypofractionated radiotherapy was performed with a total dose of 70 Gy in 28 fractions (2.5 Gy per fraction). Patients were treated 5 days per week for 7 consecutive weeks. In this study, an α/β ratio was assumed as 1.5 Gy for prostate cancer. Under this assumption, the equivalent dose to 70 Gy delivered in 28 fractions for prostate cancer was estimated to be the same biologically equivalent dose of 80 Gy if delivered at 2 Gy per fraction. Computed tomography (CT) planning was performed with 0.3 cm slice thickness or less. All patients underwent CT scanning with an empty rectum and a moderately full bladder. The gross tumor volume (GTV) was defined as the prostate. However, extracapsular invasion was included in GTV in patient with Page 4/12 T3a tumor. The clinical target volume (CTV) was defined as the GTV only in low-risk patients, or the GTV and the base of the seminal vesicle (1 cm) in intermediate- or high-risk patients. The margin of the planning target volume (PTV) from the CTV should be 4–8 mm (at least 5 mm except on the rectal side) for organ motions and set-up errors. The bladder, rectum, small intestine, large intestine, and femoral head were defined as organs at risk. All organs should be contoured as the solid organ. The bladder is enclosed from the neck to the base of the bladder. The rectum is enclosed from the anus (level of the ischial tuberosity) to the rectosigmoid transition. The level is usually below the caudal end of the sacroiliac joint. The dose was prescribed to the D50 of the PTV (dose that covers 50% of the PTV). However, if it is judged to be medically dangerous to follow the protocol, compliance with dose constraints for organs at risk should be given priority over PTV and/or CTV coverage, and changes should be made as appropriate according to the medical judgment of the physician in charge. Treatment A maximum dose of 105% of the prescribed dose was allowed to < 2% of PTV, and D95 (dose that covers 95% of the PTV) was allowed to ≥ 95% of the prescribed dose. The dose constraints for organs at risk were as follows: bladder V70/V65/V60/V55, 15%/25%/35%/50%; rectum V65/V55/V50/V45, 15%/25%/35%/50%; small intestine V55 < 0.5 cc; large intestine V60 < 0.5 cc; femoral heads Vmax < 50 Gy. However, recommended dose constraints were; bladder V70/V65/V60/V55, 10%/20%/25%/40%; rectum V65/V55/V50/V45, 10%/20%/25%/40%. Radiotherapy was delivered by IMRT using daily IGRT with prostate matching. Ultrasound, CT, and intraprostatic gold fiducial markers was permitted as image guidance. Prophylactic irradiation to the pelvic lymphatic region was not performed. Hydrogel spacers were not inserted in all patients. After completion of radiotherapy, patients were observed without any treatment until recurrence was observed. Treatment after the event of recurrence is not prescribed. However, it was strongly recommended that hormone therapy in the patients with PSA recurrence should not be considered until the PSA level increased to 4 ng/mL or higher. Acute and late adverse events were prospectively scored by the radiation oncologist using an adapted CTCAE ver.4.0-based criteria. Follow-up appointments were scheduled at least every 3 months in the first 2 years after the completion of treatment, then every 6 months to collect data on acute/late bowel and bladder toxicities and serum PSA values. The primary endpoint is the cumulative incidence of late toxicities at 5 years, and the secondary endpoints are the cumulative incidence of the acute adverse events, 5-year biochemical failure-free survival rate according to Phoenix definitions (nadir + 2 ng/mL), clinical recurrence-free survival rate, and overall survival rate. Patients without biochemical relapse or clinical recurrence were censored at death or last follow-up. Statistics The sample size was calculated to 130 patients, considering as an acceptable and unacceptable grade ≥ 2 late toxicities of ≤ 7% and ≥ 15%, respectively, with 80% power and a one-sided significance level of Page 5/12 Page 5/12 5%. Results Between July 2012 to October 2014, 134 patients from 20 institutions were enrolled. The planned radiotherapy was completed in 131 patients, while three patients were not irradiated because of non- eligibility (n=1) or withdrawal of a consent (n=2). Patient characteristics are summarized in Table 1. Median age was 71.1 years. Of 121 patients (90.3%) who received neoadjuvant hormonal therapy, 93 patients (76.3%) were treated with a combination of LH-RH analog and antiandrogen. Of 131 patients who were treated with radiotherapy, grade 2 gastrointestinal (GI) and genitourinary (GU) acute toxicities occurred in 5 patients (3.8%) and 19 patients (14.5%), respectively, whereas grade 2 GI and GU late toxicities at 5 years were observed in 7 patients (5.3%) and 7 patients (5.3%), respectively. There are no acute or late toxicities ≥ grade 3. Overall survival rate at 5 years was 94.5%. There were no patients who died of prostate cancer. Three patients had died of lung cancer, two of gastric cancer, one pancreatic cancer, and one of heart failure. The 5-year biochemical failure-free survival and clinical failure-free survival rates were 96.0% (Fig. 1) and 99.2%, respectively. The 5-year biochemical failure-free survival for patients with low-, intermediate-, and high-risk disease was 94.1%, 97.4%, and 93.9%, respectively (Fig. 2). Discussion The grade 2 ≥ late GI and GU toxicity was 14.0% and 22.8% in the 73.8 Gy/41 fraction group, 22.4% and 29.7% in the 70 Gy/28 fraction group, respectively. There are few multi-institutional trials of moderately hypofractionated radiotherapy for prostate cancer using both IMRT and IGRT techniques. Lieng et al published the results of a prospective single-institution phase II study of moderately hypofractionated IMRT with IGRT using implanted intraprostatic fiducial markers [11]. They showed that the grade 2 ≥ late GI and GU toxicity at 8 years was 4% and 12% respectively in patients treated with 60 Gy in 20 fractions. Similarly, in our multi-institutional prospective trial of radiotherapy using both IMRT and IGRT techniques, the incidences of grade 2 ≥ late GI and GU toxicities at 5 years were 5.3% and 5.3%, respectively. Hypofractionated IMRT with daily prostate- targeted image-guided techniques may decrease the late toxicities, although it should be noted that one of reasons why the low late toxicities were achieved in our study may be that the dose was prescribed to the D50 of the PTV. In CHHiP trial, median dose was prescribed to PTV with a minimum 95% isodose coverage [4]. On the other hand, the PTV was planned to receive at least 95% of the intended dose in PROFIT trial [7]. In RTOG 0415 trial, the minimum dose to ≥ 98% of the PTV was prescribed [6]. There are few prospective studies of moderately hypofractionated radiotherapy for localized prostate cancer using modern radiation techniques in Asian countries, although these fractionated regimens have been established as a standard of care [12]. Liu et performed a prospective phase I-II study using helical tomotherapy with daily image guidance in 67 patients on the feasibility of two fractionated regimens (76 Gy in 34 fractions and 71.6 Gy in 28 fractions) and showed acute toxicities were acceptable [13]. Nakamura et al reported a pilot study of hypofractionated IMRT of 54 Gy in 15 fractions in combination with neoadjuvant hormonal therapy for 24 patients with low- or intermediate-risk prostate cancer [14]. At the follow-up period of 77 months, neither grade 2 ≥ late GI toxicity nor grade 3 ≥ late GU toxicity was observed. As for retrospective studies, Kubo et al treated 75 patients with intermediate- and high-risk prostate cancer using tomotherapy with 63 Gy in alternate-day 21 fractions [15]. Discussion This multi-institutional phase II study started in 2012. In Europe and the United States, several large- scale phase III trials comparing between conventional and moderately hypofractionated regimens for localized prostate cancer had already started. Therefore, this small single-arm phase II study was focused to confirm whether the late toxicities of moderately hypofractionated RT can be controlled within an acceptable level in Asian people. Although large-scale phase III studies have now proved non- inferiority of hypofractionated regimens particularly regarding PSA relapse-free survival [4-7], it is of great importance to clarify whether moderately hypofractionated radiotherapy achieved excellent low- levels of late toxicities in Asian populations including Japanese. Moderately hypofractionated radiotherapy for localized prostate cancer has been established as a standard treatment strategy by several large, multi-institutional phase III trials such as CHHiP [4], PROFIT [7], HYPRO [5], and RTOG 0415 [6]. However, as to adverse events, it has not been well known whether moderately hypofractionated radiotherapy is equivalent or not. In addition, although patients were treated with IMRT or IGRT, these techniques were not mandatory in these trials. In CHHiP trial, forward or inverse IMRT was used, but the use of IGRT was not required [4]. The 5-year grade 2 ≥ late GI and GU toxicity was 13.7% and 9.1% in the 74 Gy/37 fraction group, 11.9% and 11.7% in the 60 Gy/20 fraction group, 11.3% and 6.6% in the 57 Gy/19 fraction group, respectively. In PROFIT trial, although daily IGRT was mandatory, 3DCRT was permitted in addition to IMRT [7]. The grade 2 ≥ late GI and GU toxicity was 13.9% and 22.0% in the 78 Gy/39 fraction group, 8.9% and 22.2% in the 60 Gy/20 fraction group, Page 6/12 respectively. In HYPRO trial, image-guided IMRT with daily online positioning of the prostate was performed in 99% of the patients [9]. As for IGRT technique, cone beam CT was used in 23% and portal imaging devices was used in 77% of the patients. The grade 2 ≥ late GI and GU toxicity was 17.7% and 39.0% in the the 78 Gy/39 fraction group, 21.9% and 41.3% in the 64.6 Gy/19 fraction group, respectively [10]. In RTOG 0415 trial, 3D-CRT or IMRT were used with daily IGRT with intraprostatic fiducial markers or other means to the prostate [6]. Discussion The incidence of late grade 2 GI and GU toxicity at 5 years was 1.3% and 17.1%, respectively, with excellent PSA relapse-free rate of 92.1%. Takasugi et al showed that hypofractionated IMRT of 66 Gy in alternate-day 22 fractions resulted in PSA relapse-free rate at 5 years of 98.9% with late grade 2 or more GU or GI toxicity of 21.8% or 14.1%, respectively [16]. Tamaki et al. showed that the long-term efficacy and safety of hypofractionated IMRT in 412 patients with prostate cancer [17]. At the median follow-up of 71.5 months, the 5-year PSA relapse-free rate was 93.2%, and the late GU and GI toxicity rates were 4.5% and 3.3%, respectively. There are few prospective studies of moderately hypofractionated radiotherapy for localized prostate cancer using modern radiation techniques in Asian countries, although these fractionated regimens have been established as a standard of care [12]. Liu et performed a prospective phase I-II study using helical tomotherapy with daily image guidance in 67 patients on the feasibility of two fractionated regimens (76 Gy in 34 fractions and 71.6 Gy in 28 fractions) and showed acute toxicities were acceptable [13]. In 2018, radiation oncologists’ attitudes and practice patterns of radiotherapy for hormone-naïve prostate cancer with bone metastases were explored in Japan [3]. Most Japanese respondents chose Page 7/12 Page 7/12 the conventional fractionation of 2 Gy to the prostate despite the existence of bone metastases. Although hypofractionation regimens have been supported by health insurance in 2018, conventional fractionation regimens to the prostate have been still used at many facilities in Japan. Our multi- institutional prospective study revealed the excellent PSA relapse-free survival rate with low late GU and GI toxicities. We believe that our trial will be a milestone for the widespread use of moderately hypofractionated radiotherapy for prostate cancer in Asian countries. the conventional fractionation of 2 Gy to the prostate despite the existence of bone metastases. Although hypofractionation regimens have been supported by health insurance in 2018, conventional fractionation regimens to the prostate have been still used at many facilities in Japan. Our multi- institutional prospective study revealed the excellent PSA relapse-free survival rate with low late GU and GI toxicities. We believe that our trial will be a milestone for the widespread use of moderately hypofractionated radiotherapy for prostate cancer in Asian countries. Acknowledgment This work was supported by a Health Labor Sciences Research Grant (H24-ganrinsho-ippan-007) from the Ministry of Health, Labor and Welfare of Japan. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Katsumasa Nakamura and Kouta Funakoshi. The first draft of the manuscript was written by Katsumasa Nakamura and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Declarations Acknowledgment References Anticancer Res 38 :4207-4212 17. Tamari K, Oh R-J, Masai N, et al (2018) Long-term Outcomes of Radiotherapy Regimen of 72 Gy in 30 Fractions for Prostate Cancer. Anticancer Res 38 :4207-4212 References 1. Nakamura K, Akimoto T, Mizowaki T, et al (2012) Patterns of practice in intensity-modulated radiation therapy and image-guided radiation therapy for prostate cancer in Japan. Jpn J Clin Oncol 42:53-57 1. Nakamura K, Akimoto T, Mizowaki T, et al (2012) Patterns of practice in intensity-modulated radiation therapy and image-guided radiation therapy for prostate cancer in Japan. Jpn J Clin Oncol 42:53-57 2. Numasaki H, Teshima T, Ando Y, et al (2020) Japanese structure survey of radiation oncology in 2012. J Radiat Res 61:146-160 3. Nakamura K, Ishikawa H, Akimoto T, et al (2020) National survey of radiation oncologists’ practice patterns regarding hormone-naïve prostate cancer with bone metastases. Jpn J Clin Oncol 50 :1188-1194 4. Dearnaley D, Syndikus I, Mossop H, et al (2016) Conventional versus hypofractionated high-dose intensity-modulated radiotherapy for prostate cancer: 5-year outcomes of the randomised, non- inferiority, phase 3 CHHiP trial. Lancet Oncol 17 :1047-1060 5. Incrocci L, Wortel RC, Alemayehu WG, et al (2016) Hypofractionated versus conventionally fractionated radiotherapy for patients with localised prostate cancer (HYPRO): final efficacy results from a randomised, multicentre, open-label, phase 3 trial. Lancet Oncol 17 :1061-1069 6. Lee WR, Dignam JJ, Amin MB, et al (2016) Randomized phase III noninferiority study comparing two radiotherapy fractionation schedules in patients with low-risk prostate cancer. J Clin Oncol 34:2325- 2332 6. Lee WR, Dignam JJ, Amin MB, et al (2016) Randomized phase III noninferiority study comparing two radiotherapy fractionation schedules in patients with low-risk prostate cancer. J Clin Oncol 34:2325- 2332 Page 8/12 7. Catton CN, Lukka H, Gu C-S, et al (2017) Randomized trial of a hypofractionated radiation regimen for the treatment of localized prostate cancer. J Clin Oncol 35 :1884-1890 8. Zhong Q-Z, Xia X, Gao H et al (2021) Hypofractionated versus conventionally fractionated image- guided volumetric-modulated arc radiotherapy for localized prostate cancer: a phase II randomized trial from China. Aging 13:6936 9. Heemsbergen WD, Incrocci L, Pos FJ, et al (2020) Local dose effects for late gastrointestinal toxicity after hypofractionated and conventionally fractionated modern radiotherapy for prostate cancer in the HYPRO trial. Front Oncol:469 10. Aluwini S, Pos F, Schimmel E, et al (2016) Hypofractionated versus conventionally fractionated radiotherapy for patients with prostate cancer (HYPRO): late toxicity results from a randomised, non- inferiority, phase 3 trial. Lancet Oncol 17:464-474 10. References Aluwini S, Pos F, Schimmel E, et al (2016) Hypofractionated versus conventionally fractionated radiotherapy for patients with prostate cancer (HYPRO): late toxicity results from a randomised, non- inferiority, phase 3 trial. Lancet Oncol 17:464-474 11. Lieng H, Pintilie M, Bayley A, et al (2017) Long-term outcomes of a phase II trial of moderate hypofractionated image-guided intensity modulated radiotherapy (IG-IMRT) for localized prostate cancer. Radiother Oncol 122 :93-98 11. Lieng H, Pintilie M, Bayley A, et al (2017) Long-term outcomes of a phase II trial of moderate hypofractionated image-guided intensity modulated radiotherapy (IG-IMRT) for localized prostate cancer. Radiother Oncol 122 :93-98 12. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: Prostate Cancer V1, 2023. Available at: http://www.nccn.org/ 12. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: Prostate Cancer V1, 2023. Available at: http://www.nccn.org/ 13. Liu H-X, Du L, Yu W, et al (2016) Hypofractionated helical tomotherapy for older aged patients with prostate cancer: preliminary results of a phase I-II trial. Technol Cancer Res Treat 15:546-554 13. Liu H-X, Du L, Yu W, et al (2016) Hypofractionated helical tomotherapy for older aged patients with prostate cancer: preliminary results of a phase I-II trial. Technol Cancer Res Treat 15:546-554 14. Nakamura K, Ikeda I, Inokuchi H, et al (2023) Long-Term Outcomes of a Prospective Study on Highly Hypofractionated Intensity Modulated Radiation Therapy for Localized Prostate Cancer for 3 Weeks. Pract Radiat Oncol S1879-8500(23)00175-3. 14. Nakamura K, Ikeda I, Inokuchi H, et al (2023) Long-Term Outcomes of a Prospective Study on Highly Hypofractionated Intensity Modulated Radiation Therapy for Localized Prostate Cancer for 3 Weeks. Pract Radiat Oncol S1879-8500(23)00175-3. 15. Kubo N, Kawamura H, Oike T, et al (2019) Hypofractionated intensity-modulated radiotherapy for intermediate-And high-risk prostate cancer: A retrospective study. In Vivo 33 :1235-1241 15. Kubo N, Kawamura H, Oike T, et al (2019) Hypofractionated intensity-modulated radiotherapy for intermediate-And high-risk prostate cancer: A retrospective study. In Vivo 33 :1235-1241 16. Takakusagi Y, Kawamura H, Okamoto M, et al (2019) Long-term outcome of hypofractionated intensity-modulated radiotherapy using TomoTherapy for localized prostate cancer: A retrospective study. PLoS One 14 :e0211370 16. Takakusagi Y, Kawamura H, Okamoto M, et al (2019) Long-term outcome of hypofractionated intensity-modulated radiotherapy using TomoTherapy for localized prostate cancer: A retrospective study. PLoS One 14 :e0211370 17. Tamari K, Oh R-J, Masai N, et al (2018) Long-term Outcomes of Radiotherapy Regimen of 72 Gy in 30 Fractions for Prostate Cancer. 17. Tamari K, Oh R-J, Masai N, et al (2018) Long-term Outcomes of Radiotherapy Regimen of 72 Gy in 30 Fractions for Prostate Cancer. Anticancer Res 38 :4207-4212 Table Page 9/12 Figures Page 10/12 Figure 1 Proportion Free of Biochemical Failure in all patients Page 11/12 Figure 1 Proportion Free of Biochemical Failure in all patients Proportion Free of Biochemical Failure in all patients Page 11/12 Figure 2 Proportion Free of Biochemical Failure according to the risk groups Page 12/12 Figure 2 Proportion Free of Biochemical Failure according to the risk groups Figure 2 Proportion Free of Biochemical Failure according to the risk groups Page 12/12
W3161621225.txt	https://www.frontiersin.org/articles/10.3389/fonc.2021.657595/pdf	en		Prognosis and Immune Infiltration of Chromobox Family Genes in Sarcoma	Frontiers in oncology	2,021	cc-by	7,790	ORIGINAL RESEARCH published: 11 May 2021 doi: 10.3389/fonc.2021.657595 Prognosis and Immune Inﬁltration of Chromobox Family Genes in Sarcoma Jian Zhou 1,2†, Ziyuan Chen 1†, Ming Zou 3, Rongjun Wan 4,5, Tong Wu 1, Yingquan Luo 6, Gen Wu 1,7, Wanchun Wang 1,2 and Tang Liu 1,2* Edited by: Lori Rink, Fox Chase Cancer Center, United States Reviewed by: Xiaojun Ren, University of Colorado Denver, United States Shuai Ye, Fox Chase Cancer Center, United States Correspondence: Gen Wu wu.gen@csu.edu.cn Wanchun Wang wanchun.wang@csu.edu.cn Tang Liu liutang1204@csu.edu.cn † These authors have contributed equally to this work Specialty section: This article was submitted to Cancer Molecular Targets and Therapeutics, a section of the journal Frontiers in Oncology Received: 25 January 2021 Accepted: 13 April 2021 Published: 11 May 2021 Citation: Zhou J, Chen Z, Zou M, Wan R, Wu T, Luo Y, Wu G, Wang W and Liu T (2021) Prognosis and Immune Inﬁltration of Chromobox Family Genes in Sarcoma. Front. Oncol. 11:657595. doi: 10.3389/fonc.2021.657595 Frontiers in Oncology \| www.frontiersin.org 1 Department of Orthopedics, The Second Xiangya Hospital, Central South University, Changsha, China, 2 Hunan Key Laboratory of Tumor Models and Individualized Medicine, The Second Xiangya Hospital, Central South University, Changsha, China, 3 Department of Orthopedics, Brain Hospital of Hunan Province (The Second People’s Hospital of Hunan Province), Changsha, China, 4 Department of Respiratory and Critical Care Medicine, Xiangya Hospital, Central South University, Changsha, China, 5 National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha, China, 6 Department of General Medicine, The Second Xiangya Hospital, Central South University, Changsha, China, 7 Clinical Medicine Eight-Year Program, Central South University, Changsha, China Background: Chromobox family genes (CBXs) are known to play roles in numerous modiﬁcations of the chromatin in order to inhibit the transcription of target genes. CBXs have been shown to be expressed at high levels in many types of cancer and can also serve as a target gene for therapeutic purposes. However, little is known about the expression and prognostic value of CBXs in human sarcomas. Methods: The transcription level of CBXs was analyzed using the Oncomine dataset, and the differential expression of CBXs in sarcoma was reported by the Gene Expression Proﬁling Interactive Analysis (GEPIA) dataset. We also used the CCLE dataset to evaluate the expression of CBXs in a sarcoma cell line. The prognostic value of CBXs was analyzed using GEPIA and Kaplan–Meier analysis. In addition, the corrections between CBXs and their co-expressed genes were reported using Oncomine and GEPIA datasets. DAVID was used to perform GO function enrichment analysis for the CBXs and their coexpression genes. Finally, TIMER was used to analyze the immune cell inﬁltration of CBXs in patients with sarcoma. Results: HP1-a/b/g (CBX1/3/5) and CBX4/6/8 were found to be overexpressed in human sarcoma, and CBXs were upregulated in almost all the sarcoma cell line. The expression levels of HP1-a/b/g (CBX1/3/5) and CBX7 were associated with overall survival (OS) in patients with sarcoma, while high expression levels of CBX7 were related to disease-free survival (DFS). In addition, the expression levels of CBX2/6/7 were related to recurrencefree survival (RFS). We also found that the CBX family was positively correlated with the inﬁltration of immune cells, including CD8+ T cells, CD4+ T cells, B cells, macrophages, neutrophils, and dendritic cells, in sarcoma. Conclusions: The results from the present study indicated that CBXs were signiﬁcantly associated with prognosis and immunological status in sarcoma. These data suggest that 1 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs CBXs could serve as potential biomarkers for prognosis and immune inﬁltration in human sarcoma. Keywords: CBX family genes, prognosis, immune inﬁltration, sarcoma, survival (DFS) in patients with non-small-cell lung cancer. Furthermore, the elevated expression of HP1-g (CBX3) has been demonstrated to exert impact on tumor diameter and lymph node metastasis (16). A previous study reported that the increased mRNA expression of HP1-b/g (CBX1/3) and CBX2/6/8 was correlated with a worse OS, while the overexpression of CBX7 was related to a greater OS in patients with hepatocellular carcinoma (17). However, the role of CBXs in sarcoma remains unclear. Here, we aimed to investigate the expression of CBX in sarcomas and the relationship between CBXs and prognosis/immune cell inﬁltration in patients with sarcoma. INTRODUCTION Sarcomas are rare but aggressive bone and soft tissue malignancies that afﬂict patients of all ages. Sarcomas are usually incurable because chemotherapy and surgery are not effective (1); these malignant tumors also exhibit broad differentiation (2). Although some causative factors have been established for sarcomas, including environmental factors, stimulation by foreign matter, and endocrine dyscrasia, the precise underlying causes of sarcoma have yet to be elucidated. Although generally rare, sarcomas can be found in patients of any age and tend to occur more commonly in adolescents and teenagers than in the elderly (3). Osteosarcoma is the most common form of primary bone sarcoma and accounts for 1% of all tumors; the incidence of osteosarcoma in children was previously reported to be ﬁve per million (4). Chromobox family genes (CBXs) are associated with a variety of modiﬁcations to the chromatin that inhibit the transcription of target genes as key elements of polycomb repressive complex 1 (PRC1) (5). Currently, eight members of the CBX family have been identiﬁed, based on their single N-terminal chromosomal domains, consisting of three b folds and an a helix. There are two broad categories of CBX genes: heterochromatin protein 1 and polycomb complexes. HP1-a/b/g (CBX1/3/5) are heterochromatic proteins while CBX2/4/6/7/8 are polycomb complexes. The heterochromatin protein 1 group consists of an N-terminal chromodomain and a C-terminal chromodomain, while the polycomb group contains only a conservative Nterminal chromodomain (6). Different CBX proteins have been associated with different parts of the chromatin, leading to the speciﬁc transcription of target genes (7, 8). According to previous studies, CBXs are known to be involved in the occurrence and development of a diverse range of tumors through various pathways (9, 10). For example, Han et al. reported that CBX2 could act as a tumor promoter in osteosarcoma by targeting miRNA let-7a (11). In addition, Ma et al. reported that CBX3 was related to an unfavorable prognosis and tumorigenesis in patients with osteosarcoma (12). In another study, Wang et al. demonstrated that targeting the CK1a/CBX4 axis may provide beneﬁt to patients with metastasis of osteosarcoma (13). Liang et al. found that HP1-a/b/g (CBX1/3/5) and CBX2/4/6/7 exerted an effect on breast cancer, thus indicating that CBX2 is expressed at high levels in basal-like and HER-2 subtypes (14). In addition, CBX4/7 is highly expressed in Luminal A and Luminal B subtypes of breast cancer (14, 15). The increased mRNA expression of HP1b/g (CBX1/3) and CBX2 has been linked to a poorer Relapse free survival (RFS) via survival analysis; better outcomes were found to be associated with higher expression levels of CBX4/5/6/7 (14). Moreover, the high expression levels of HP1-b/g (CBX1/3) were associated with overall survival (OS) and disease-free survival Frontiers in Oncology \| www.frontiersin.org METHODS Ethics Statement This study was approved by the Second Xiangya Hospital of Central South University Committee for Clinical Research and all methods were carried out in accordance with the Declaration of Helsinki. ONCOMINE Analysis The Oncomine database (https://www.oncomine.org/resource/ login.html) is usually adopted to analyze DNA or RNA sequences from a comprehensive cancer microarray database and genome-wide expression data for malignant tumors. In the present study, we used the Oncomine database to identify the differential transcriptional expression of CBXs in multiple cancer tissues and corresponding normal tissues. Moreover, three databases including Detwiller sarcoma database (18), Barretina sarcoma database (19), and Quade uterus database (20) were used to analyze the differential transcriptional expression of CBXs in subtypes of sarcoma and corresponding normal tissues using p < 0.05 as the signiﬁcance threshold. GEPIA Analysis The GEPIA dataset (http://gepia.cancer-pku.cn/) is an online service that includes a spectrum of cancer expression data. The GEPIA dataset contains 9,736 tumor samples and 8,587 normal samples from the TCGA. The GEPIA dataset is usually used to analyze data arising from the TCGA project. In this study, we used the GEPIA database to analyze the expression levels of CBX genes in sarcoma tissues and normal tissues. CCLE Dataset Evaluation The CCLE dataset (https://www.broadinstitute.org/ccle) is usually used to accurately depict the genetic characteristics of cancer cells. The CCLE dataset can provide information relating to DNA mutation and gene expression. We used the CCLE database to investigate the expression of CBXs in sarcoma cell lines. 2 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs genes and proteins, was used to perform GO enrichment analyses of CBXs and their co-expression genes (21). Kaplan–Meier Plotter Database Analysis The Kaplan–Meier Plotter Database (https://kmplot.com/ analysis/index.php?p=service&cancer=pancancer_rnaseq) is used to evaluate the inﬂuence of genes on the survival of patients afﬂicted with various forms of cancers. In this study, we used the Kaplan–Meier Plotter Database to analyze the relationship between the expression of CBXs in sarcoma and associated survival rates. RESULTS Transcriptional Levels of the CBX Family in Patients With Sarcoma According to data arising from the Oncomine database, we found that HP1-a/b/g (CBX1/3/5) and CBX4/6/8 were all highly expressed in sarcoma (Figure 1). Additionally, three databases including Detwiller sarcoma database, Barretina sarcoma database, and Quade uterus database were used to analyze the differential transcriptional expression of CBXs in subtypes of sarcoma and corresponding normal tissues. The Detwiller sarcoma database, reported by Detwiller et al. (18) identiﬁed distinctly different patterns of expression between sarcomas and normal tissues as assessed by hierarchical clustering analysis. The Barretina sarcoma database, established by Barretina et al. (19), described an integrative analysis of DNA sequence, copy number, and mRNA expression in 207 sarcoma samples encompassing seven major subtypes. The Quade uterus database, reported by Quade et al. (20), depicted RNAs proﬁled TIMER Dataset Analysis The TIMER database (https://cistrome.shinyapps.io/timer/) is an online service that can be used to investigate the inﬁltration of different immune cells and their clinical signiﬁcance. In the present study, CBXs were input into the ‘Gene module’ tool of TIMER in order to generate scatterplots to investigate the association between CBX expression and immune inﬁltration in sarcoma. GO Enrichment Analyses of DEGs The Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.ncifcrf.gov) (version 6.7), an online biological information database that provides a comprehensive set of functional annotation information for FIGURE 1 \| The expression levels of CBX genes in different types of human cancers and normal samples. The red cells represent evidence of gene overexpression. The blue cells represent evidence of reduced gene expression. The numbers in each cell represent the evidential frequencies. The deeper the color, the higher the signiﬁcance. Frontiers in Oncology \| www.frontiersin.org 3 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs sarcoma database) were raised by 2.068- and 5.159fold, respectively. from four normal uterine myometria, seven uterine leiomyomas, and nine uterine leiomyosarcomas. As shown in Figure 2, the expression levels of HP1-b (CBX1) were upregulated in the Detwiller sarcoma database; the fold-changes for the expression of these genes in synovial sarcoma, pleomorphic liposarcoma, leiomyosarcoma, ﬁbrosarcoma, and malignant ﬁbrous histiocytoma were 4.180, 2.460, 3.155, 4.010, and 3.294, respectively. With regard to the Barretina sarcoma database, the expression levels of HP1-b (CBX1) in pleomorphic liposarcoma showed a fold-change of 2.826 when compared with normal samples and a fold-change of 3.315 when compared with normal samples in leiomyosarcoma. With regard to the Detwiller sarcoma database, the foldchanges of HP1-g (CBX3) expression in ﬁbrosarcoma, malignant ﬁbrous histiocytoma, pleomorphic liposarcoma, and round cell liposarcoma were 3.515, 3.249, 2.242, and 2.645, respectively. The expression levels of HP1-g (CBX3) in dedifferentiated liposarcoma, myxoid/round cell liposarcoma, myxoﬁbrosarcoma, and pleomorphic liposarcoma were upregulated by 2.270, 2.803, 2.439, and 2.785, fold for the Barretina sarcoma database. The fold-change for CBX4 expression was 6.516 in synovial sarcoma when compared with normal tissues for the Detwiller sarcoma database. High expression levels of HP1-a (CBX5) were found in uterine corpus leiomyosarcoma, synovial sarcoma, pleomorphic liposarcoma, dedifferentiated liposarcoma, and ﬁbrosarcoma, with fold-changes of 2.005, 3.713, 3.401, 2.018, and 2.673, respectively (Quade uterus database and Detwiller sarcoma database). The expression levels of CBX6/8 in leiomyosarcoma (Barretina sarcoma database) and synovial sarcoma (Detwiller The mRNA Levels of CBX Genes in Sarcoma Next, we used the GEPIA dataset to compare the expression of CBX family mRNAs between sarcoma and normal tissues. The mRNA levels of HP1-a/b/g (CBX1/3/5) and CBX2/4/8 were signiﬁcantly higher than those in normal tissues. In contrast, the mRNA expression levels of CBX6/7 in sarcoma were lower than those in normal tissues (Figure 3). The Expression Levels of CBX Genes in Sarcoma Cell Lines We used the CCLE dataset to investigate the expression levels of CBXs in human cancer. Data showed that all eight members of the CBX family were expressed at high levels in sarcoma cell lines (Figure 4). The Prognostic Value of CBXs in Sarcoma The prognostic value of CBX gene expression in sarcoma was evaluated by GEPIA and the Kaplan–Meier Plotter Database. As shown in Figure 5 (GEPIA), increased expression levels of HP1-a/g (CBX3/5) were associated with a poorer OS in patients with sarcoma, while elevated expression levels of CBX7 were associated with a better OS. HP1-b (CBX1) and CBX2/4/6/8 also tended to exert impact on the OS, but without statistical signiﬁcance. High expression levels of CBX6 appear to be associated with a better DFS. Similar ﬁndings were evident in our Kaplan–Meier analysis (Figure 6). The expression levels of FIGURE 2 \| Signiﬁcant changes of CBX gene expression at the transcription level between different types of sarcoma and normal tissues. The horizontal axis represents the type of tissue, and the vertical axis represents the expression level of CBX genes. The number next to the name of the sarcoma subtype represents the speciﬁc subtype vs ‘normal’ cases. Frontiers in Oncology \| www.frontiersin.org 4 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs A B C D E F G H J I FIGURE 3 \| The expression levels of CBX genes in sarcoma. (A–H) The expression levels of CBX1-8 in pan-cancer, (I–J) The expression levels of CBX genes in sarcoma. The expression levels of CBX genes in sarcomas. Each dot represents an individual sample, P < 0.05. SPTLC2, and ZDHHHC2. CBX6 was co-expressed with SYP11, ANKRD35, PRICKLE1, MXD4, ZBTB20, CD99L2, RNF150, JAM3, CLIP3, and MFAP4. CBX7 was co-expressed with PDCD6IP, CNOT6L, UNC80, SRI, KGFLP2, MRPS6, PCM1, S1PR1, SEPSECS, and LPP. CBX8 was co-expressed with CYP46A1, ALOX12B, C1orf109, TET2, MGC16384, MYO1A, HTR7, PNKD, C7orf28B, and ASPHD2 (Figure 7A). Similarly, we analyzed the co-expression relationships between different CBX genes. HP1-b (CBX1) was positively correlated with CBX2 (R = 0.63, P < 0.05), HP1-g (CBX3) (R = 0.47, P < 0.05), CBX4 (R = 0.49, P < 0.05), HP1-a (CBX5) (R = 0.61, P < 0.05), CBX6 (R = 0.32, P < 0.05), and CBX8 (R = 0.49, P < 0.05), but there was no signiﬁcant correlation between HP1-b (CBX1), and CBX7. CBX2 was signiﬁcantly correlated with HP1g (CBX3) (R = 0.38, P < 0.05), CBX4 (R = 0.57, P < 0.05), HP1-a (CBX5) (R = 0.46, P < 0.05), CBX6 (R = 0.19, P < 0.05), and CBX8 (R = 0.66, P < 0.05). HP1-g (CBX3) was signiﬁcantly correlated with CBX4 (R = 0.25, P < 0.05), HP1-a (CBX5) (R = 0.39, P < 0.05), CBX7 (R = −0.15, P < 0.05) and CBX8 (R = 0.2, P < 0.05). CBX4 signiﬁcantly correlated with HP1-a (CBX5) (R = 0.35, P < 0.05), CBX6 (R = 0.41, P < 0.05), and CBX8 (R = 0.79, P < 0.05). HP1-a (CBX5) was signiﬁcantly correlated with CBX6 HP1-a/b/g (CBX1/3/5) and CBX2 were associated with a poorer OS, while the levels of CBX7 were associated with a better OS. Furthermore, high expression levels of CBX6/7 were associated with a better RFS, while high expression levels of CBX2 were associated with a worse RFS. The Identiﬁcation of Genes That Were Co-Expressed With CBX Genes in Sarcoma We used the Oncomine database to identify a series of genes that were co-expressed with CBX genes in sarcoma. HP1-b (CBX1) was co-expressed with S100A6, STAG1, MBD4, CHMP2B, PMM1, PCNP, C5orf54, TTC33, CEP70, and KIAA1586. CBX2 was coexpressed with USPS, TTLL4, ASRBK1, SMYDS, MDFI, BSG, MTA1, YBX1, MCM4, and GGA2. HP1-g (CBX3) was coexpressed with HNRNPAZ2B1, CCT6A, HNRNPR, SFRS13A, DDX46, SFRS2, SFRS3, TRA2B, NONO, MSH2, and HP1-g (CBX3). CBX4 was co-expressed with SF1, RABSB, MBOAT7, PRKACA, AKT2, WASL, CCNG1, UBL4A, PRKAR2A, and TRIB1. HP1-a (CBX5) was co-expressed with WDHD1, CDK2, ARHGAP28, PCDH19, RORB, CYP39A1, ST85IA1, PAR1GD52, Frontiers in Oncology \| www.frontiersin.org 5 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs A B D C E F G H FIGURE 4 \| The expression of CBXs in sarcoma cell lines. (A–H) The expression of CBX1-8 in sarcoma cell lines. The number next to the lineage name represents number of cell lines in the lineage. The dashed line within a box is the mean. Frontiers in Oncology \| www.frontiersin.org 6 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs A B FIGURE 5 \| The prognostic signiﬁcance of CBXs in sarcoma patients. (A) The association between the elevated expression of CBX genes and overall survival in patients with sarcoma. (B) The association between high expression levels of CBX genes and disease-free survival in patients with sarcoma. HR, hazard ratio; TPM, Transaction per million. (R = 0.35, P < 0.05) and CBX8 (R = 0.42, P < 0.05). In addition, CBX6 was signiﬁcantly correlated with CBX7 (R = 0.49, P < 0.05) and CBX8 (R = 0.32, P <0.05). However, there was no signiﬁcant correlation between CBX7 and CBX8 (Figure 7B). Next, we used the DAVID online tool to perform GO function enrichment analysis from three aspects: biological processes (BPs), cellular components (CCs), and molecular functions (MFs). We found that CBXs and their co-expressed genes were mainly involved in GO:0000398 (mRNA splicing, via spliceosome) for BP, GO:0005634 (nucleus) for CC, and GO:0003682 (chromatin binding) for MF. More detailed information is given in Figure 8 and Table 1. immune inﬁltration of sarcoma. We found that HP1-b (CBX1) expression was signiﬁcantly associated with the inﬁltration of CD8+ T cells (Correlation coefﬁcient (cor) = −0.131, p < 0.05), CD4+ T cells (cor = −0.362, p < 0.05), macrophages (cor = −0.27, p < 0.05), and dendritic cells (cor = −0.4, p < 0.05). CBX2 was signiﬁcantly associated with the inﬁltration of CD4+ T cells (cor = −0.293, p < 0.05), macrophages (cor = −0.402, p < 0.05), neutrophils (cor = −0.173, p < 0.05), and dendritic cells (cor = −0.384, p < 0.05). HP1-g (CBX3) was signiﬁcantly associated with inﬁltration by CD4+ T cells (cor = −0.247, p < 0.05). CBX4 was signiﬁcantly associated with inﬁltration by B cells (cor = 0.137, p < 0.05), CD4+ T cells (cor = −0.187, p < 0.05), macrophages (cor = −0.28, p < 0.05), and dendritic cells (cor = −0.197, p < 0.05). HP1-a (CBX5) was signiﬁcantly associated with inﬁltration by CD4+ T cells (cor = −0.279, p < 0.05), macrophages (cor = −0.293, p < 0.05) and dendritic cells Immune Inﬁltration Levels in Sarcoma In the present study, the TIMER dataset was used to analyze whether the expression of CBX genes were correlated with the Frontiers in Oncology \| www.frontiersin.org 7 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs A B FIGURE 6 \| The prognostic value of CBX genes in sarcoma patients. (A) The association between high expression levels of CBX genes and the overall survival of patients with sarcoma. (B) The association between elevated expression levels of CBX genes and the recurrence-free survival of patients with sarcoma. HR, hazard ratio. (cor = −0.371, p < 0.05). CBX6 was signiﬁcantly associated with inﬁltration by CD8+ T cells (cor = 0.157 p < 0.05), CD4+ T cells (cor = −0.341 p < 0.05), macrophages (cor = −0.217 p < 0.05), and dendritic cells (cor = −0.178 p < 0.05). CBX7 was signiﬁcantly associated with inﬁltration by CD8+ T cells (cor = 0.142 p < 0.05). Finally, CBX8 was signiﬁcantly associated with inﬁltration by CD8+ T cells (cor = −0.146 p < 0.05), CD4+ T cells (cor = −0.182 p < 0.05), macrophages (cor = −0.357 p < 0.05), neutrophils (cor = −0.264 p < 0.05), and dendritic cells (cor = −0.284 p < 0.05) (Figure 9). CBXs and sarcoma has not been extensively reported. Currently, there is a renewed interest in immunotherapy for cancer. Most immunotherapy studies have focused on melanoma. These have revealed that the overexpression of GM2, GD2, and GD3 immunogenicity gangliosides can trigger an antibody response and improve the survival rate of patients with melanoma. Other studies have investigated MHC-restricted cancer-testicular antigens; these have also raised the prospect of immunotherapy (22). Subsequently, the concept of tumor vaccines was proposed for speciﬁc tumor epitopes by stimulating an immune response. Therefore, immune-related studies on sarcomas, combined with radiotherapy, chemotherapy, and several targeted therapies, have become increasingly popular (23). Therefore, we conducted this study to reveal the relationships between CBX genes and human sarcoma, particularly with respect to their prognostic value and the immune inﬁltration of CBXs. DISCUSSION The CBX family of genes is thought to represent vital elements in a variety of tumors (14–16). However, the association between Frontiers in Oncology \| www.frontiersin.org 8 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs A B FIGURE 7 \| Co-expression analysis. (A) Genes that were co-expressed with CBX genes in sarcoma. (B) Correlation between CBX genes in sarcoma. results which indicated that the up-regulated expression of HP1-a/ g (CBX3/5) is related to a poorer OS, while the up-regulated expression of CBX7 was associated with a better OS. Similar results were obtained from our Kaplan–Meier Plotter analyses. According to a previous study, CBX2 is a key member of the polycomb group (PcG) family and CBX2 mRNA was predominately localized in spermatogonia and spermatocytes, as demonstrated by in situ hybridization (ISH) (25). Tatavosian et al. found that CBX2 undergoes phase separation to form condensates and that these CBX2 condensates concentrate DNA and nucleosomes (26). Plys et al. further reported that the domain of CBX2 was the same as the domain related to chromatin compaction and development, thus improving the possibility of an evolutionary or mechanistic link between these activities (27). In another study, Alexandra et al. reported that the stabilization of the testis required CBX2-mediated repression The up-regulated expression of HP1-b (CBX1) has been reported for many forms of cancer. For example, Liang et al. reported high expression levels of HP1-b (CBX1) mRNA in breast cancer and found that this was associated with a poorer RFS. HP1b (CBX1) has also been associated with chemoresistance in patients with breast cancer. Therefore, these authors deﬁned HP1-b (CBX1) as a potential target for the treatment of breast cancer (14). In addition, Yang et al. found that the overexpression of HP1-b (CBX1) in hepatocellular carcinoma could activate the Wnt/bCatenin signaling pathway by interacting with the transcription factor HMGA2, thus suggesting that HP1-b (CBX1) was an independent factor for hepatocellular carcinoma (24). Similar results were reported by Gang et al. (17). Higher expression levels of HP1-b/g (CBX1/3) and CBX2/6/8 were reported to be related to a worse OS while higher expression levels of CBX7 were associated with a better OS. These results are similar to our present Frontiers in Oncology \| www.frontiersin.org 9 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs FIGURE 8 \| GO analysis of CBX genes and co-expressed genes. GO analysis was based on three aspects: (BP) biological processes, (CC) cellular components, and (MF) molecular function. regulate genes associated with the cell cycle, mismatch repair, and immune-related pathways. The expression level of HP1-g (CBX3) was also signiﬁcantly and inversely related to the expression levels of TILs, PDCD1, and PDCD2, and immunotherapy responses, thus implying that HP1-g (CBX3) could inﬂuence the efﬁcacy of immunotherapy and chemotherapy (33). Ma et al. further conﬁrmed the function and role of HP1-g (CBX3) in osteosarcoma, by showing that the expression of HP1-g (CBX3) was associated with a poorer DFS and OS, as well as a larger tumor size, a higher distant metastasis rate, and a higher clinical stage. These authors used HP1-g (CBX3) siRNA to knockdown HP1-g (CBX3) and thus block proliferation ability, thus resulting in increased levels of apoptosis and cell cycle arrest at the G0 and G1 phase (12); these ﬁndings were consistent with those reported in the present study. Another study reported that the elevated expression of HP1-g (CBX3) was related to unfavorable OS in patients with human sarcoma. CBX4 is known to recruit GCN5 to the Runx2 promoter to transcriptionally upregulate Runx2; in this manner, of bivalent ovary-determining genes (28). In hepatocellular carcinoma, the knockdown of CBX2 restrained the proliferation of HCC cells and increased the phosphorylation of YAP. These data suggest that CBX2 could be a potential target for hepatocellular carcinoma (29). In addition, Zheng et al. showed that higher expression levels of CBX2 were signiﬁcantly and independently associated with a worse OS by affecting the PI3K/AKT signaling pathway in breast cancer (30). In our present study, we observed elevated expression levels of CBX2 and found that CBX2 was associated with a poorer OS, thus indicating that this gene might be an independent prognostic factor for human sarcoma. High expression levels of HP1-g (CBX3) [encoded by HP1-g (CBX3)] are known to accelerate HCC cell proliferation, thus suggesting that HP1-g (CBX3) is a crucial oncogene in hepatocellular carcinoma (31). Similar ﬁndings have been reported for glioma; high expression levels of HP1-g (CBX3) are known to predict a worse prognosis (32). In gastric cancer, Lin et al. found that HP1-g (CBX3) was overexpressed and could Frontiers in Oncology \| www.frontiersin.org 10 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs TABLE 1 \| GO analysis of CBXs and the co-expression genes. The enriched BP BP BP BP BP BP BP BP BP BP BP BP BP BP BP CC CC CC CC CC CC CC CC CC GO-ID Description 0000398 mRNA splicing, via spliceosome 0016569 covalent chromatin modiﬁcation 0016925 protein sumoylation 0000122 negative regulation of transcription from RNA polymerase II promoter 0045892 negative regulation of transcription, DNA-templated 0048662 negative regulation of smooth muscle cell proliferation 0030522 intracellular receptor signaling pathway 0007257 activation of JUN kinase activity 0006397 mRNA processing 0006351 transcription, DNA-templated 0046929 negative regulation of neurotransmitter secretion 0061157 mRNA destabilization 0002318 myeloid progenitor cell differentiation 0043401 steroid hormone mediated signaling pathway 0000086 G2/M transition of mitotic cell cycle 0005654 nucleoplasm 0031519 PcG protein complex 0035102 spliceosomal complex 0000792 heterochromatin :0005634 nucleus 0000784 nuclear chromosome, telomeric region 0044853 plasma membrane raft 0005720 nuclear heterochromatin 0000785 chromatin Count P-Value Genes 8 3.86E-04 DDX46, TRA2B, NONO, SRRT, HNRNPA2B1, HNRNPR, YBX1, SF1 6 5.98E-04 CBX7, CBX6, SMARCD3, CBX4, CBX3, CBX2 6 7.01E-04 CBX8, STAG1, MTA1, CBX4, KIAA1586, CBX2 13 0.001273 CBX8, CBX7, CBX6, KDM1A, CBX4, CBX2, NR2F1, ZBTB20, YBX1, NR2F2, HNRNPA2B1, MDFI, MXD4 10 0.003125 CBX5, KDM1A, CBX4, CBX3, NONO, CBX1, RORB, NR2F2, PRICKLE1, MDFI 3 0.013084 VIPR2, TRIB1, SF1 3 0.021878 NR2F1, RORB, NR2F2 3 0.021878 PTPN1, ZAK, MDF 5 20 2 0.022920 CNOT6L, NONO, HNRNPA2B1, HNRNPR, PRKACA 0.023107 CBX8, CBX7, CBX6, SMARCD3, CNOT6L, KDM1A, CBX4, CBX3, NONO, CBX2, RFX2, NR2F1, ZBTB20, WASL, RORB, NR2F2, MTA1, BRWD1, MXD4, SF1 0.029718 PNKD, SYT11 2 0.035555 CNOT6L, HNRNPR 2 0.041358 TET2, JAM3 3 0.046159 NR2F1, RORB, NR2F2 4 0.049691 PCM1, CEP70, CDK2, PRKACA 34 5 9.53E-06 SMARCD3, KDM1A, SRRT, ZBTB20, HNRNPR, RORB, YBX1, SRI, DCAF7, MTA1, EXOSC4, AKT2, TRA2B, CEP70, PRKACA, CBX8, CBX7, WDHD1, MBD4, CBX6, CBX5, CBX4, NONO, CBX2, CBX1, NR2F1, ARHGAP28, STAG1, MSH2, CCNG1, HNRNPA2B1, CDK2, MCM4, SF1 1.41E-05 CBX8, CBX7, CBX6, CBX4, CBX2 4 4.54E-05 CBX8, CBX7, CBX4, CBX2 4 47 6 2.04E-04 CBX8, CBX7, CBX6, CBX2 5.39E-04 SMARCD3, DDX46, KDM1A, ZAK, ZBTB20, WASL, RORB, PRICKLE1, YBX1, LPP, PCNP, MTA1, EXOSC4, AKT2, TRA2B, PRKACA, UBL4A, CBX8, CBX7, MBD4, CBX6, CNOT6L, CBX5, CBX4, CBX3, NONO, CBX2, CBX1, TET2, RFX2, NR2F1, NR2F2, GDAP1, SEPSECS, PNKD, STAG1, HNRNPA2B1, CDK2, CHMP2B, S100A6, MCM4, TRIB1, BRWD1, MDFI, TSPYL5, MXD4, SF1 7.84E-04 CBX5, MSH2, KDM1A, CBX3, CBX1, MCM4 3 0.001067 MYO1A, PRKAR2A, PRKACA 3 0.006535 CBX5, CBX3, CBX1 4 0.013301 MBD4, STAG1, CBX3, CBX1 (Continued) Frontiers in Oncology \| www.frontiersin.org 11 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs TABLE 1 \| Continued The enriched GO-ID Description Count CC CC 0005925 focal adhesion 0016020 membrane 7 20 CC 0031618 nuclear pericentric heterochromatin 0005829 cytosol 2 CC CC CC CC MF MF MF MF MF MF MF MF MF 0005952 cAMP-dependent protein kinase complex 0031965 nuclear membrane 0000775 chromosome, centromeric region 0005515 protein binding 0003682 chromatin binding 0035064 methylated histone binding 0003727 single-stranded RNA binding 0008270 zinc ion binding 0004879 RNA polymerase II transcription factor activity, ligand-activated 0003714 transcription corepressor activity 1990226 histone methyltransferase binding 0003707 steroid hormone receptor activity 27 P-Value Genes 0.021639 PDCD6IP, STX16, PRKAR2A, BSG, NHS, CD99L2, LPP 0.032225 UBL4A, RAB5B, VIPR2, PDCD6IP, DDX46, STX16, MBOAT7, NONO, SRI, GDAP1, PNKD, PCM1, MSH2, PRKAR2A, BSG, HNRNPA2B1, MCM4, ASPHD2, PRKACA, CD320 0.032800 CBX5, CBX3 2 0.036723 STX16, RAP1GDS1, ALOX12B, WASL, PRICKLE1, SRI, PCM1, EXOSC4, PRKAR2A, AKT2, CEP70, PRKACA, NADK, ATP6V1C1, UBL4A, PTPN1, CNOT6L, PDCD6IP, PMM1, ARHGAP28, CCT6A, CLIP3, SEPSECS, STAG1, CDK2, CHMP2B, S100A6 0.038162 PRKAR2A, PRKACA 5 0.038575 PCM1, CEP70, PRICKLE2, PRICKLE1, YBX1 3 0.039848 STAG1, CBX3, CBX1 77 11 5 2.58E-06 KDM1A, ZAK, HNRNPR, WASL, YBX1, RORB, DCAF7, ROBO1, PCM1, AKT2, BSG, PRKACA, WDHD1, CNOT6L, PDCD6IP, USP5, TTC33, TET2, RFX2, MRPS6, CPT1B, CLIP3, MSH2, S100A6, MCM4, TRIB1, MDFI, TSPYL5, SF1, RAB5B, STX16, SRRT, RAP1GDS1, CD99L2, ALOX12B, PRICKLE1, SRI, LPP, PCNP, GGA2, NEURL1B, MTA1, EXOSC4, TRA2B, C1ORF109, PRKAR2A, CEP70, ATP6V1C1, NADK, JAM3, UBL4A, CBX8, PTPN1, CBX7, MBD4, CBX6, CBX5, CBX4, PMM1, CBX3, MBOAT7, NONO, CBX2, CBX1, NR2F1, NR2F2, CCT6A, MFAP4, SEPSECS, STAG1, PNKD, SYT11, CCNG1, HNRNPA2B1, CDK2, CHMP2B, MXD4 1.27E-04 CBX7, STAG1, MTA1, CBX5, SMARCD3, KDM1A, CBX4, NONO, CBX2, CBX1, YBX1 2.93E-04 CBX8, CBX7, CBX5, CBX4, CBX2 4 0.002218 CBX8, CBX7, CBX6, CBX4 15 3 0.010524 PTPN1, USP5, PRICKLE2, TET2, NR2F1, PRICKLE1, RORB, NR2F2, ZDHHC2, LPP, NEURL1B, MTA1, S100A6, RNF150, SF1 0.019929 NR2F1, RORB, NR2F2 5 0.034675 MTA1, CBX4, NR2F2, MXD4, SF1 2 0.035716 CBX3, CBX1 3 0.045081 NR2F1, RORB, NR2F2 In the present study, we found that CBX4 was highly expressed in human sarcoma tissues. Guo et al. were the ﬁrst to conﬁrm the high expression levels of HP1-a (CBX5) in gastric cancer tissues, and then revealed that HP1-a (CBX5) could promote the proliferation, migration, and invasion, of gastric cancer cells in vitro (37). In lung cancer, Yu et al. used a panel of tumor stem-like cells (hESCs) to verify the materiality of HP1-a (CBX5) (38). In our study, increased levels of HP1-a (CBX5) expression were related to a poorer OS, both in the GEPIA dataset and the Kaplan–Meier Plotter dataset. Wang et al. reported that the level of anti-HP1-a (CBX5) antibody was associated with age, cigarette-smoking habits, and blood pressure, in patients who had suffered from transient ischemic attack, thus indicating that serum levels of antibodies against HP1-a (CBX5) could potentially serve as tools for diagnosing transient ischemic attack (39). The elevated expression of CBX6 CBX4 can promote the metastasis of osteosarcoma (13). In another paper, Hu et al. reported that the cell growth and migration of human lung cancer cell was suppressed by the knockdown of CBX4, both in vitro and in vivo. Furthermore, CBX4 has been shown to promote proliferation and metastasis by regulating the BMI-1 pathway, thus suggesting that CBX4 might be a potential therapeutic target in lung cancer (34). In the digestive system, the suppression of Runx2 by CBX4 resulted in the inhibition of cell migration, invasion, and metastasis (35). Interestingly, Ren et al. reported that CBX4 counteracts senescence in human mesenchymal stem cells (HMSC) by maintaining nucleolar homeostasis, and that CBX4 maintained nucleolar homeostasis by recruiting nucleolar protein-ﬁbrillin and heterochromatin KRAB associated protein 1 (KAP1) within nucleolar rDNA, thereby limiting rRNA overexpression and attenuating the development of osteoarthritis in mice (36). Frontiers in Oncology \| www.frontiersin.org 12 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs FIGURE 9 \| Relationship between differentially expressed CBX genes and immune cell inﬁltration. The immune cells we analyzed included B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. Frontiers in Oncology \| www.frontiersin.org 13 May 2021 \| Volume 11 \| Article 657595 Zhou et al. Prognosis and Immune of CBXs In addition, the performance of immune inﬁltration should be validated by co-localization with different members of the CBX family and immune inﬁltration markers. has been reported in HCC tissues and cell lines; CBX6 was also correlated with a larger tumor size (≥5 cm; p = 0.011). Moreover, HCC patients with higher expression levels of CBX6 showed a worse OS and RFS than patients with lower expression levels of CBX6 (40). Deng et al. identiﬁed CBX6 as a downregulated gene in breast cancer via a comprehensive analysis of The Cancer Genome Atlas (TCGA); their ﬁnal ﬁndings supported CBX6 as a cancer suppressor in breast cancer (41). Similarly, our present study suggested that higher expression levels of CBX6 were related to DFS in patients with sarcoma. Reduced expression levels of CBX7 have been found to be associated with a poorer OS and the aggressiveness of thyroid cancers, colorectal cancers and breast cancers (42–44), In HCC, the downregulation of CBX7 may be related to a short OS (17). However, high expression levels of CBX7 were associated with a reduced OS and DFS in patient with prostate cancers and ovarian cancers (45, 46). In sarcoma, we found that the expression level of CBX7 was downregulated in sarcoma, and that the elevated expression of CBX7 was associated with a better OS and DFS. Therefore, further studies are still needed to fully evaluate the role of CBX7 in cancers. CBX8 was found to have the ability to promote invasion and migration in breast cancer, lung cancer, and glioblastoma (47), and in HCC. A previous study showed that CBX8 could act as an oncogene and play an important role in upregulating the Akt/b-catenin pathway stimulated by EGR1 and miR-365-3p. The upregulated expression of CBX8 was also correlated with a poorer OS (48). In our study, high expression levels of CBX8 tended to exert impact on the survival rate but without statistical signiﬁcance. The tumor microenvironment can affect the progression and recurrence of multiple cancers. Immune cells within the tumor microenvironment have been shown to promote or suppress cancer activities and are considered as an important determining factor in clinical outcome and immune therapy. In the present study, we found that the expression levels of CBX genes were signiﬁcantly related to immune cell inﬁltration, thus indicating that CBXs could reﬂect the immune status of sarcoma. Our study can provide more detailed immune information to sustain immune therapy for patients with sarcoma. Our study has several limitations that need to be considered. First, the data used for analysis were obtained from online services. We need to carry out more cell-based studies and clinical experiments to conﬁrm our ﬁndings and to further explore interactions between relevant molecules, the precise mechanisms involved, and the potential clinical applications of CBX genes in sarcoma. CONCLUSION In conclusion, our study showed that HP1-a/b/g (CBX1/3/5) and CBX4/6/8 were highly overexpressed in human sarcoma tissues. The high expression levels of HP1-a/g (CBX3/5) were closely associated with a poorer OS while the high expression levels of CBX7 were associated with a greater OS. CBX genes were positively correlated with the inﬁltration of immune cells, including CD8+ T cells, CD4+ T cells, B cells, macrophages, neutrophils, and dendritic cells, in sarcoma. These results indicated the crucial value of CBX genes in the prognosis and immune therapy of human sarcoma. Our ﬁndings may provide new insight and comprehensive analysis to select novel prognostic and immune biomarkers for sarcoma. DATA AVAILABILITY STATEMENT The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. AUTHOR CONTRIBUTIONS JZ and WW conceived, designed, and conducted the experiments. JZ and ZC wrote the paper. JZ and YL collected the data. JZ and WW edited the paper. JZ and GW provided the research guide. RW, JZ, MZ, and TW revised the manuscript. YL, WW, and TL supervised this project. All authors contributed to the article and approved the submitted version. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 48. Zhang CZ, Chen SL, Wang CH, He YF, Yang X, Xie D, et al. Cbx8 Exhibits Oncogenic Activity Via AKT/Beta-Catenin Activation in Hepatocellular Carcinoma. Cancer Res (2018) 78:51–63. doi: 10.1158/0008-5472.CAN-17-0700 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Frontiers in Oncology \| www.frontiersin.org 16 May 2021 \| Volume 11 \| Article 657595
https://openalex.org/W2623308034	http://journal.iain-manado.ac.id/index.php/jiep/article/download/429/353	Indonesian	null	Merancang Pengembangan Madrasah Unggul	Journal of islamic education policy	2,017	public-domain	8,358	95 \| Agustini Buchari, Erni Moh. Saleh ISSN 2528-0295 (print)/ISSN 2528-0309 (online) 95 \| Agustini Buchari, Erni Moh. Saleh ISSN 2528-0295 (print)/ISSN 2528-0309 (online) Journal of Islamic Education Policy 2016, Vol.1, No.2, 95—112 Journal of Islamic Education Policy 2016, Vol.1, No.2, 95—112 Diterbitkan Online Desember 2016 (http://journal.iain-manado.ac.id/index.php/jiep) Agustini Buchari Agustini Buchari Madrasah Aliyah Negeri Model Manado, Sulawesi Utara, Indonesia Email: agustinibuchari@yahoo.co.id Erni Moh. Saleh Madrasah Aliyah Negeri Model Manado, Sulawesi Utara, Indonesia Email: ernysaleh28@gmail.com Erni Moh. Saleh Madrasah Aliyah Negeri Model Manado, Sulawesi Utara, Indonesia Email: ernysaleh28@gmail.com ABSTRACT Preparation of human resources that are well-qualified and excellent is both an option and orientation for all types and levels of education. Madrasah as com- munity-based educational institutions (community based education) have responsibility in preparing human resources and superior quality. To be able to continue actively pre- paring qualified human resources and excellent insight, innovative development of superior madrasah is required; which is developed to achieve excellence in output. To achieve this excellence, the input, the process of education, teachers and education per- sonnels, management, education, and supporting facilities should be directed to support the achievement of this goal. Excellenct madrasas should be designed in accordance with the vision, mission and institutional objectives, and the need analysis of academic and institutional system. This should be done with an adequate understanding of the geographic and cultural context. Keywords: Excellent madrasah, educational development, quality education ABSTRAK Penyiapan sumber daya manusia yang berkualitas dan unggul merupakan pilihan sekaligus orientasi pendidikan pada semua jenis dan strata pendidikan. Madra- sah sebagai lembaga pendidikan berbasis masyarak memiliki peran tanggung jawab yang sama dalam menyiapkan sumber daya manusia yang berkualitas dan unggul. Un- tuk dapat terus berperan aktif menyiapkan sumber daya manusia yang berkualitas dan memiliki wawasan keunggulan, diperlukan inovasi pengembangan madrasah unggul, yang dikembangkan untuk mencapai keunggulan dalam keluaran (output) pendidika- nya. Untuk mencapai keunggulan tersebut, masukan (input), proses pendidikan, guru dan tenaga kependidikan, manajemen, layanan pendidikan, serta sarana penunjangnya harus diarahkan untuk menunjang tercapainya tujuan tersebut. Madrasah unggulan harus dirancang sesuai dengan visi-misi dan tujuan kelembagaan, analisis kebutuhan sistem akademik dan kelembagaan, dan memahami konteks geografis dan budaya. Kata Kunci: Madrasah unggulan, pengembangan pendidikan, pendidikan berkualitas 1. Pendahuluan Sebagai lembaga pendidikan yang sudah lama berkembang di Indonesia, pendidik- an madrasah telah berhasil membina dan mengembangkan kehidupan beragama di Indonesia dan ikut berperan dalam menanamkan rasa kebangsaan ke dalam jiwa rakyat Agustini Buchari, Erni Moh. Saleh \| 96 Indonesia. Madrasah juga telah berperan penting dalam mencerdaskan kehidupan bangsa. Oleh karena itu, peningkatan mutu, relevansi, dan daya saing madrasah di masa depan diharapkan dapat memberi dampak bagi perwujudan eksistensi sumber daya ma- nusia Islam, sehingga dapat hidup bersama dalam keragaman sosial dan budaya. Upaya peningkatan mutu dan relevansi pendidikan madrasah pada gilirannya diharapkan dapat meningkatkan taraf hidup masyarakat serta daya saing bangsa, bagi warga masyarakat, khususnya masyarakat muslim. y y Salah satu indikator mutu pendidikan madrasah adalah kualitas lulusannya. Sedang- kan, aspek yang sangat dominan dalam peningkatan mutu penyelenggaraan pendidikan adalah kualifikasi guru, mutu manajemen, fasilitas (sarana dan prasarana), kurikulum, sistem yang diterapkan dan dana yang tersedia. Oleh karena itu, peningkatan kualitas SDM di lembaga-lembaga pendidikan Islam, merupakan suatu keharusan. Selain itu, penguatan tata kelola organisasi dan akuntabilitas pengelolaan madrasah juga dinilai signifikan dalam memengaruhi citra pendidikan madrasah. Sudah barang tentu, sebagai lembaga pendidikan Islam, madrasah tidak hanya di- arahkan kepada aktivitas penggalian ilmu pengetahuan semata, tetapi juga menjadi wahana pelatihan untuk mengaplikasikan ilmu pengetahuan pada tataran realitas. Selain itu, pendidikan di madrasah tidak hanya mengarah kepada keunggulan akademis (aca- demic excellence), tetapi justru menegaskan orientasi pembentukan karakter (character building) yang berasaskan prinsip akhlakul karimah. Madrasah sebagai lembaga pengembangan dakwah, dengan sendirinya menjadi salah satu tanda syiar agama sekaligus tampil sebagai komponen penting dari gerakan amar ma’ruf nahi munkar. Sebagai lembaga pendidikan masyarakat, madrasah berperan dalam pengembangan masyarakat sekitar (stakeholders) terutama terkait dengan ma- salah keagamaan maupun pemberdayaan sektor nonkeagamaan. Dengan demikian, madrasah merupakan pendidikan berbasis masyarakat (community based education). Peran aktif madrasah dalam pemberdayaan masyarakat sekitar dan sebaliknya peran aktif masyarakat dalam pengembangan madrasah akan menjadi amat penting, sehingga satu sama lain saling mendukung dalam meraih kemanfaatan dan kemaslahatan bersama (mutual support for the sake of mutual benefit). 2. Hakikat Madrasah Unggul Secara terminologis sekolah unggul adalah sekolah yang dikembangkan untuk men- capai keunggulan dalam keluaran (output) pendidikannya. Untuk mencapai keunggulan tersebut maka masukan (input), proses pendidikan, guru dan tenaga kependidikan, manajemen, layanan pendidikan, serta sarana penunjangnya harus diarahkan untuk me- nunjang tercapainya tujuan tersebut. Di Indonesia, istilah sekolah unggul pertama kali digagas oleh Wardiman Djojo- negoro (Mantan Menteri Pendidikan dan Kebudayaan) pada tahun 1994. Gagasan ter- sebut lahir dari adanya suatu keinginan kuat untuk menghadirkan sekolah sebagai lembaga pendidikan yang mempunyai visi yang jauh menjangkau ke depan dan ber- wawasan keunggulan. Bahkan Djojonegoro mengekspektasikan bahwa kehadiran se- kolah unggul di setiap propinsi di Indonesia akan membuat terjadinya distribusi ilmu pengetahuan dan dengan demikian sasaran berikutnya ialah terjadinya akselerasi peningkatan sumber daya manusia (SDM). Oleh karena itu, kehadiran sekolah unggul bukan untuk diskriminasi, tetapi untuk menyiapkan SDM yang berkualitas dan memiliki wawasan keunggulan (Djojonegoro, 1998, p. 15). Gagasan tentang sekolah unggul tersebut telah mendorong dikembangkannya konsep madrasah unggul. Madrasah unggulan adalah madrasah program unggulan yang lahir dari keinginan untuk memiliki madrasah yang mampu berprestasi di tingkat nasional dan dunia dalam penguasaan ilmu pengetahuan dan teknologi ditunjang oleh akhlakul karimah (Depag RI, 2004, p. 41). Dengan kata lain, pengembangan madrasah unggulan sejajar dengan pengembangan sekolah unggulan yang masing-masing di- kembangkan untuk mencapai keunggulan dalam keluaran (output) pendidikan- nya. Untuk mencapai keunggulan tersebut, masukan (input), proses pendidikan, guru dan tenaga kependidikan, manajemen, layanan pendidikan, serta sarana penunjangnya harus diarahkan untuk menunjang tercapainya tujuan tersebut. Dalam praktik di lapangan terdapat tiga tipe madrasah atau sekolah Islam unggulan (Moedjiarto, 2002, p. 34). Ketiga madrasah atau sekolah Islam unggulan tersebut, yaitu pertama, tipe madrasah atau sekolah Islam berbasis pada anak cerdas. Jenis tipe ini yaitu sekolah atau madrasah hanya menerima dan menyeleksi secara ketat calon siswa yang masuk dengan kriteria memiliki prestasi akademik yang tinggi. Meskipun proses belajar-mengajar di lingkungan madrasah atau sekolah Islam tersebut tidak terlalu istimewa bahkan biasa-biasa saja, namun karena input siswa yang unggul, maka memengaruhi outputnya tetap berkualitas. Kedua, tipe madrasah atau sekolah Islam berbasis pada fasilitas. Sekolah Islam atau madrasah semacam ini cenderung menawarkan fasilitas yang serba lengkap dan me- madai untuk menunjang kegiatan pembelajarannya. Tipe ini cenderung memasang tarif lebih tinggi ketimbang rata-rata sekolah atau madrasah pada umumnya. Ketiga, tipe madrasah atau sekolah Islam berbasis pada iklim belajar. Tipe ini cen- derung menekankan pada iklim belajar yang positif di lingkungan madrasah. 1. Pendahuluan Dalam konteks itulah, pengembangan madrasah tidak dapat ditangani secara parsial atau setengah-setengah, tetapi memerlukan pemikiran pengembangan yang utuh dan komprehensif serta langkah dan upaya yang visibel, fleksibel, dan kredibel, terutama ketika dihadapkan pada kebijakan pembangunan nasional bidang pendidikan yang mempunyai visi terwujudnya sistem pendidikan sebagai pranata sosial yang kuat dan berwibawa untuk memberdayakan semua warga negara Indonesia berkembang menjadi manusia yang berkualitas, sehingga mampu dan proaktif menjawab tantangan zaman yang selalu berubah (baca penjelasan UU No. 20/2003 tentang Sisdiknas). Ditambah lagi era globalisasi telah memberi dampak yang cukup luas dalam berbagai aspek kehidupan, termasuk tuntutan mutu dalam penyelengaraan pendidikan. Pada era ini setiap bidang menuntut SDM bermutu yang memiliki kemampuan tinggi, handal, dan kompetitif. Untuk memenuhi tuntutan ini, perbaikan dan pengembangan sistem penyelenggaraan pendidikan di madrasah secara berkesinambungan perlu dilakukan sejalan dengan dinamika perkembangan ilmu pengetahuan dan teknologi serta dinamika perubahan masyarakat itu sendiri. Penguatan keunggulan madrasah tersebut dengan cara membangun cita-cita dan kultur akademik yang kokoh hingga memunculkan output yang unggul dan baik sesuai harapan masyarakat. 97 \| Agustini Buchari, Erni Moh. Saleh 3. Rasional dan Dasar Pemikiran Madrasah Unggul Undang-undang Dasar 1945 yang secara historis disebut sebagai Indonesian Dec- laration of Independence, dalam pembukaannya secara jelas mengungkapkan alasan di- dirikannya negara untuk (1) mempertahankan bangsa dan tanah air, (2) meningkatkan kesejahteraan rakyat, (3) mencerdaskan kehidupan bangsa, dan (4) ikut serta dalam me- wujudkan perdamaian dunia yang abadi dan berkeadilan. Konsep pencerdasan kehidup- an bangsa berlaku untuk semua komponen bangsa. Oleh karena itu, Undang-undang Dasar 1945 pada pasal 31 ayat (1) menyebutkan bahwa setiap warga negara berhak mendapatkan pendidikan, dan ayat (3) menegaskan bahwa pemerintah mengusahakan dan menyelenggarakan satu sistem pendidikan nasional yang meningkatkan keimanan dan ketaqwaan serta akhlak mulia. Sebagai lembaga pendidikan yang sudah lama ber- kembang di Indonesia, madrasah selain telah berhasil membina dan mengembangkan kehidupan beragama di Indonesia, juga ikut berperan dalam menanamkan rasa ke- bangsaan ke dalam jiwa rakyat Indonesia. Di samping itu, madrasah juga sangat ber- peran dalam mencerdaskan kehidupan bangsa. Namun demikian, performa madrasah sampai saat ini masih sangat rendah. Beberapa permasalahan telah berhasil diidentifikasi menjadi penyebabnya, baik pada tingkat pengelolaan maupun kebijakan. Masalah kurikulum madrasah yang masih belum “fokus” dan proses pendidikan yang belum mendukung visi dan misi madrasah, merupakan contoh kasus di tingkat pengelolaan, sedangkan kebijakan pengembangan madrasah yang masih bersifat “tambal sulam” serta belum adanya blue print (cetak biru) pengembangan madrasah merupakan contoh kasus di bidang kebijakan. Secara rinci dapat dikemukakan beberapa pokok permasalahan, baik pada tingkat pengelolaan maupun kebijakan sebagai berikut: 1. Pengembangan madrasah masih bersifat tambal sulam, hal ini misalnya terlihat dengan diadakannya program “keterampilan” yang ditempelkan pada program reguler, sebagai respon terhadap tingginya lulusan Madrasah Aliyah yang tidak bisa melanjutkan pada jenjang Pendidikan Tinggi. Demikian juga dengan prog- ram “keagamaan” sebagai respon terhadap lemahnya penguasaan ilmu keagama- an siswa, juga munculnya Madrasah Aliyah Unggulan (Insan Cendekia), yang merupakan langkah penyelamatan. Program-program tersebut meskipun banyak manfaat yang dapat diambil untuk proses pengembangan madrasah, tetapi langkah-langkah tersebut tampaknya tidak didasari oleh konsep yang terencana yang matang. y g g 2. Kurikulum madrasah yang belum “fokus”, hal ini terlihat misalnya, banyak- nya materi yang diajarkan sementara waktu tidak memadai. Pada tingkat Aliyah, misalnya siswa yang ingin mendalami ilmu-ilmu keagamaan masih juga di- bebani mata pelajaran lain yang tidak relevan dalam jumlah yang cukup banyak. Sebaliknya siswa yang mengambil jurusan IPA harus pula dibebani dengan banyaknya mata pelajaran lain yang tidak berhubungan secara langsung. 2. Hakikat Madrasah Unggul Lembaga pendidikan dapat menerima dan mampu memproses siswa yang masuk (input) dengan prestasi rendah menjadi lulusan (output) yang bermutu tinggi. Tipe ketiga ini termasuk agak langka, karena harus bekerja ekstra keras untuk menghasilkan kualitas yang bagus. Dari uraian di atas dapat didefinisikan bahwa madrasah unggulan adalah lembaga pendidikan Islam yang memiliki komponen unggul, yang tercermin pada sumber daya manusia (pendidik, tenaga kependidikan, dan siswa) sarana prasarana, serta fasilitas pendukung lainnya untuk menghasilkan lulusan yang mampu menguasai ilmu penge- Agustini Buchari, Erni Moh. Saleh \| 98 tahuan dan teknologi secara terampil, memiliki kekokohan spiritual (iman dan/atau Islam), dan memiliki kepribadian akhlak mulia. 3. Rasional dan Dasar Pemikiran Madrasah Unggul Hal lainnya dalam kurikulum madrasah adalah masih adanya duplikasi materi yang diajarkan berulang-ulang pada mata pelajaran yang berbeda dan juga pada tingkat yang berbeda. g y g 3. Akibat dari kurikulum yang belum “fokus” (bahan terlalu berat dan tumpang tin- dih), maka proses pendidikan yang terjadi di madrasah tidak sesuai dengan visi 3. Akibat dari kurikulum yang belum “fokus” (bahan terlalu berat dan tumpang tin- dih), maka proses pendidikan yang terjadi di madrasah tidak sesuai dengan visi 99 \| Agustini Buchari, Erni Moh. Saleh dan misi pendidikan madrasah. Program-program pengembangan yang se- potong-potong (parsial), dan tidak berangkat dari suatu desain yang terencana, juga diidentifikasi sebagai penyebab tidak bertemunya visi-misi madrasah dengan pendidikan yang diberikan. 4. Ketidakadaan cetak biru (blue print) pengembangan madrasah, ini barangkali permasalah yang paling mendasar, sehingga pengembangan madrasah menjadi tidak memiliki arah (Depag RI, 2004, pp. 1—5). 4. Ketidakadaan cetak biru (blue print) pengembangan madrasah, ini barangkali permasalah yang paling mendasar, sehingga pengembangan madrasah menjadi tidak memiliki arah (Depag RI, 2004, pp. 1—5). Dari uraian di atas, dapat ditarik benang merah bahwa munculnya sekolah unggulan berangkat dari keinginan untuk menciptakan madrasah yang menjadi central for exel- lence untuk mempersiapkan SDM yang siap pakai untuk masa depan. Selama ini data menunjukkan bahwa mutu pendidikan nasional belum merata. Adanya sekolah unggul- an dapat membekali mereka dengan pengalaman belajar yang berkualitas, dengan sendirinya mereka mempunyai peluang yang lebih besar untuk memasuki jenjang pendidikan yang lebih tinggi sesuai dengan pilihannya. Pada sisi lain, harus diakui bahwa masyarakat Indonesia tidak sedikit yang lebih memercayai lembaga pendidikan madrasah daripada sekolah umum. Lembaga pendidik- an Islam ini diminati oleh masyarakat yang menghendaki para putra-putrinya memper- oleh pendidikan agama yang cukup sekaligus pendidikan umum yang memadai. Namun, ada empat masalah utama yang sedang dihadapi oleh madrasah pada umumnya, yaitu: masalah identitas diri madrasah, masalah jenis pendidikan yang dipilih sesuai titik tekan keagamaan, masalah kemunduran kualitas ajaran Islam yang berimplikasi pada kedangkalan pemahaman Islam dan masalah sumber daya internal yang ada dan pe- manfaatannya bagi pembangunan madrasah sendiri di masa depan (Muhaimin, 2005, p. 186). Untuk itu, pemikiran pengembangan madrasah unggul dinilai relevan dengan upaya pemberdayaan sumber daya manusia di Indonesia. Dalam hal lain, juga perlu dikemukakan bahwa gagasan pengembangan madrasah dapat dilihat dari dua dasar utama, yaitu dasar religius dan dasar yuridis. Berikut adalah penjabaran kedua dasar yang dimaksud. Dasar Yuridis Penyelenggaraan Madrasah secara yuridis diatur dalam tata perundangan kita. Sila pertama yang menyebutkan Ketuhanan Yang Maha Esa memiliki makna bahwa agama dijadikan sebagai pembimbing sekaligus keseimbangan hidup bangsa Indonesia. Ini ber- arti bahwa lembaga keagamaan seperti madrasah diakui sebagai tempat pembinaan mental spiritual bangsa indonesia. Secara konstitusional pasal 29 ayat 2 negara menjamin kebebasan rakyatnya dalam melaksanakan ajaran agamanya. Termasuk ke- bebasan belajar di madrasah. Pasal 31 ayat 3 menyebutkan bahwa pemerintah meng- usahakan satu sistem pendidikan nasional yang meningkatkan keimanan dan ketakwaan serta akhlak mulia dalam mencerdaskan kehidupan bangsa, salah satunya adalah pe- nyelenggaraan Madrasah. Secara operasional ketentuan Madrasah terakhir diatur dalam keputusan menteri agama No. 1 tahun 2001 setelah lahirnya Direktorat Pendidikan Keagamaan dan Pondok Pesantren khususnya melayani pondok pesantren dan mad- rasah. Keberadaan madrasah sebagai bagian dari sistem pendidikan nasional diperkuat dengan lahirnya Undang-undang No. 20 tahun 2003 terutama pasal 30 ayat 1 hingga 4 yang menyatakan bahwa pendidikan keagamaan dapat diselenggarakan oleh pemerintah dan atau kelompok masyarakat dari pemeluk agama sesuai dengan peraturan perundangan. Berdasarkan pada Undang-undang No. 20 tahun 2003 itu, maka dipahami bahwa (1) pendidikan keagamaan dapat diselenggarakan oleh pemerintah (pendidikan ke- agamaan negeri) dan dapat diselenggarakan oleh masyarakat (pendidikan keagamaan swasta); (2) Pendidikan keagamaan berfungsi mempersiapkan peserta didik menjadi anggota masyarakat yang memahami dan mengamalkan nilai-nilai agamanya dan atau menjadi ahli ilmu agama; (3) Pendidikan keagamaan dapat diselenggarakan pada jalur formal, nonformal dan informal. Ketentuan ini memberikan ruang yang sangat luas pada lembaga pendidikan keagamaan untuk menyelenggarakan pendidikan pada jalur formal persekolahan, nonformal seperti kursus, pelatihan, kelompok belajar keagamaan (majelis taklim), atau jalur informal seperti pendidikan dalam keluarga; dan (4) Pen- didikan keagamaan berbentuk pendidikan madrasah, pesantren, dan bentuk lain yang sejenis. Berdasarkan alasan-alasan tersebut, maka perlu dikembangkan madrasah-madrasah unggul dengan manajemen yang profesional dalam rangka meningkatkan mutu atau kualitas pendidikan, khususnya pendidikan yang berbasis agama. Dasar Religius Islam memerintahkan belajar pada ayat pertama yang diturunkan pada Rasulullah Saw. Oleh karena itulah belajar merupakan kewajiban utama dan sarana terbaik mencerdaskan umat (Muhaimin, 2005, p. 186). Perintah belajar tersebut tidak terbatas pada urusan duniawi saja, tetapi juga dalam urusan ukhrawi. Firman Allah Swt., dalam Al Qur’an surat At-Taubah ayat 122, yang terjemahannya: “Tidak sepatutnya bagi orang-orang yang mukmin itu pergi semuanya (ke medan perang). Mengapa tidak pergi dari tiap-tiap golongan di antara mereka beberapa orang untuk memperdalam pengetahuan mereka tentang agama dan untuk memberi peringatan kepada kaumnya apabila mereka telah kembali kepadanya, supaya mereka itu dapat menjaga dirinya.” (Q.S At-Taubah: 122) “Tidak sepatutnya bagi orang-orang yang mukmin itu pergi semuanya (ke medan perang). Mengapa tidak pergi dari tiap-tiap golongan di antara mereka beberapa orang untuk memperdalam pengetahuan mereka tentang agama dan untuk memberi peringatan kepada kaumnya apabila mereka telah kembali kepadanya, supaya mereka itu dapat menjaga dirinya.” (Q.S At-Taubah: 122) Lafadz “liyatafaqqahuu fidiin” dalam surat At-Taubah ayat 122 itu memberi isya- rat tentang kewajiban memperdalam ilmu agama (Nata, 2002, p. 159). Artinya, seorang muslim perlu mendalami ilmu agama dan mengajarkan kepada orang lain berdasarkan kadar yang diperkirakan dapat memberikan kemaslahatan bagi mereka, sehingga memberikan pengetahuan hukum-hukum agama yang pada umumnya harus diketahui oleh orang-orang beriman. Hal ini disebabkan banyaknya orang yang pintar dalam urusan duniawi namun mereka lalai dalam urusan akherat. Firman Allah Swt., dalam Alquran surat Ar Rum ayat 7, yang terjemahannya: Agustini Buchari, Erni Moh. Saleh \| 100 “Mereka hanya mengetahui yang lahir (saja) dari kehidupan dunia; sedang mereka tentang (kehidupan) akhirat adalah lalai.” (Q.S. Ar Rum: 7). “Mereka hanya mengetahui yang lahir (saja) dari kehidupan dunia; sedang mereka tentang (kehidupan) akhirat adalah lalai.” (Q.S. Ar Rum: 7). Jadi belajar agama merupakan suatu hal yang sangat penting bagi seorang muslim sebagai benteng yang dapat menjaga diri dan tetap dalam koridor yang disyariatkan. Begitu pentingnya belajar agama sehingga Allah Swt., memberikan kedudukan tinggi pada orang yang memusatkan perhatian mendalami ilmu agama sebagaimana derajatnya orang-orang berjihad dengan harta dan dirinya dalam rangka meninggikan kalimah Allah Swt. Salah satu cara yang bisa dilakukan dengan belajar di sebuah lembaga yang khusus mengajarkan ilmu-ilmu agama yaitu madrasah. 4. Karakteristik Madrasah Unggul Secara teoretis, karakteristik madrasah unggul bisa diklasifikasi ke dalam beberapa kriteria, antara lain: keunggulan proses pembelajaran, keunggulan sarana-prasarana atau fasilitas belajar, keunggulan lingkungan pendidikan, keunggulan kognitif, keunggulan 101 \| Agustini Buchari, Erni Moh. Saleh ekstrakuriler, keunggulan mulok, keunggulan life skill, keunggulan administrasi, dan lain-lain. Tentu yang dikehendaki adalah keunggulan multifacet. Kemendikbud umpamanya, menetapkan kriteria sekolah unggul sebagai berikut: (1) input siswa melalui seleksi yang ketat berdasarkan kriteria dan standar; (2) sarana prasarana belajar yang memadai, baik intra maupun ekstra; (3) lingkungan sekolah yang kondusif; (4) guru dan tenaga kependidikan yang memiliki kualifikasi sesuai dengan persyaratan dan ketetapan Undang-undang Guru dan Dosen tahun 2005; (5) kurikulum yang diperkaya, yang mampu mengembangkan dan memfasilitasi potensi, bakat, kreasi siswa; (6) Rentang waktu belajar di sekolah lebih panjang; (7) proses belajar mengajar yang berkualitas; (8) perlakuan tambahan di luar kurikulum nasional; dan (9) menjadi pusat keunggulan bagi sekolah-sekolah di sekitarnya (Subhan, 2006, p. 19). Berbeda dengan madrasah model yang secara umum persyaratan yang dikriteriakan ialah (1) memiliki manajemen madrasah yang baik; (2) SDM yang berkualitas; (3) kelengkapan sarana dan prasarana pendidikan; (4) bantuan pendidikan yang memadai; dan (5) keunggulan kualitas lulusan (Fachruddin, 1998, p. 80). Harris and Bennett (dalam Jalal, 2012) mengemukakan bahwa karakrektistik sekolah unggul, yaitu: (1) Kepemimpinan yang profesional (Profesional Leadership); (2) Visi dan tujuan bersama (Shared Vision and Goals); (3) Lingkungan belajar (a Learning Environment); (4) Konsentrasi pada belajar-mengajar (Concentration on Learning and Teaching); (5) Harapan yang tinggi (High Expectation); (6) Penguat- an/pengayaan/ pemantapan yang positif (Positive Reinforcement); (7) Pemantauan kemajuan (Monitoring Progress); (8) Hak dan tanggung jawab peserta didik (Pupil Rights and Responsibility); (9) Pengajaran yang penuh makna (Purposeful Teaching); (10) Organisasi pembelajar (a Learning Organization); dan (11) Kemitraan keluarga- sekolah (Home-School Partnership). Dari semua kriteria tentang madrasah atau sekolah unggul atau madrasah model pasti dirujuk pada Peraturan Pemerintah No. 19/2005 tentang Standar Nasional Pen- didikan (SNP) yang menjadi kriteria minimal sistem pendidikan di seluruh Indonesia. Standarisasi pendidikan ini berfungsi sebagai dasar dalam perencanaan, pelaksanaan, dan pengawasan pendidikan dalam rangka mewujudkan pendidikan nasional yang ber- mutu. Artinya, jika madrasah telah memenuhi semua atau sebagian besar kriteria SNP, maka madrasah tersebut disebut sebagai Madrasah Standar Nasional (MSN) atau Mad- rasah Kategori Mandiri (MKM). Dalam posisi ini madrasah berada pada level Unggulan atau Model, selanjutnya meningkat statusnya menjadi Madrasah Standar Internasional (MSI), akan berada pada level Model yang menjadi idealisasi sebuah madrasah. Dalam konteks madrasah, Zayadi (2005, p. 4. Karakteristik Madrasah Unggul 57) mengemukakan beberapa unsur karakteristik madrasah unggul ialah: pertama, aspek administrasi atau manajemen, yaitu (1) maksimal 6 kelas untuk tiap tingkatan, (2) tiap kelas terdiri atas 30 siswa, (3) rasio guru kelas adalah 1:25, (4) mendokumentasi perkembangan tiap siswa, dan (5) transpar- an dan akuntabel. Kedua, aspek ketenagaan, yaitu (1) kepala madrasah yaitu (a) minimal S2 untuk MA, S1 untuk Madrasah Tsanawiyah (MTs) dan Madrasah Ibtidaiyah (MI), (b) pengalaman minimal 5 tahun menjadi kepala madrasah, (c) mampu berbahasa Arab atau berbahasa Inggris, (d) lulus tes (fit and proper test), (e) sistem kontrak satu tahunan, dan (f) siap tinggal di kompleks madrasah, (2) guru, yaitu (a) minimal S1, (b) spesialisasi sesuai mata pelajaran, (c) pengalaman mengajar minimal 5 tahun, (d) mampu berbahasa Arab atau bahasa Inggris, (e) lulus test (fit and proper test), dan (f) sistem kontrak 1 tahun, (3) tenaga kependidikan, yaitu (a) minimal S1, (b) spesialisasi sesuai dengan bidang tugas, dan (c) pengalaman mengelola minimal 3 tahun. Ketiga, Agustini Buchari, Erni Moh. Saleh \| 102 aspek kesiswaan terdiri dari (1) input, yaitu (a) sepuluh besar MTs (untuk MA), (b) sepuluh besar MI (untuk MTs), dan (c) lulus tes akademik (bahasa Arab dan Inggris) dan (2) output, yaitu (a) menguasai berbagai disiplin ilmu, (b) mampu berbahasa Arab maupun bahasa Inggris, (c) terampil menulis dan berbicara (Indonesia) dengan baik, dan (d) siap bersaing untuk memasuki jenjang lebih tinggi yakni universitas atau institut bermutu di dalam negeri. Keempat, aspek kultur belajar, yaitu (1) full day school, (2) student centered learning, (3) kurikulum dikembangkan dengan melibatkan seluruh elemen madrasah termasuk siswa, (4) bahasa pengantar Arab dan Inggris, (5) sis- tem droup out, dan (6) pendekatan belajar dengan fleksibelitas tinggi dengan mengikuti perkembangan metode-metode pembelajaran terbaru. Dan, kelima, aspek sarana dan prasarana, yaitu (1) perpustakaan yang memadai, (2) laboratorium (IPA, Bahasa dan Matematika), (3) laboratorium alam yang memadai, (4) mushalla, dan (5) lapangan dan fasilitas olahraga lainnya yang memadai. Terkait dengan madrasah unggul dan model, Mastuhu (1994, p. 58) menglasifikasi kriteria madrasah unggul ke dalam dua hal, yaitu: (1) sumber daya manusia (SDM) dan (2) perangkat pendidikan. Sumber daya manusia terdiri atas pimpinan madrasah, guru, siswa, dan tenaga kependidikan. Perangkat keras (hardware) berupa bangunan madrasah, masjid, lapangan olahraga, dan fasilitas pendidikan lainnya. Perangkat lunak (software) berupa visi, misi, tujuan, kurikulum, metode pembelajaran sistem penilaian, dan lain-lain (Mastuhu, 1994, p. 58). 4. Karakteristik Madrasah Unggul Dengan demikian, kriteria, karakteristik dan standar madrasah dapat dikelompokkan ke dalam dua substansi utama, yaitu sistem kelembagaan dan sistem pembelajaran. Dalam konteks pendidikan di Indonesia, pemerintah telah memberikan rambu- rambu, bagaimana menciptakan sekolah/madrasah yang memiliki kualitas baik. Salah satunya adalah dengan menerbitkan Peraturan Pemerintah Nomor 19 Tahun 2005 tentang Standar Nasional Pendidikan (SNP) yang menjadi acuan minimal kualitas pendidikan, yaitu standar isi, standar proses, standar sarana prasarana, standar kompe- tensi lulusan, standar pengelolaan, standar pembiayaan, standar pendidik dan tenaga pendidik, dan standar penilaian. Madrasah yang bermutu karenanya adalah madrasah yang secara konsisten berorientasi pada implementasi SNP sebagaimana ditetapkan oleh pemerintah. Dengan kata lain, pemenuhan SNP tersebut akan menjadi men- jadi tool untuk mempercepat meningkatkanya kualitas pendidikan di Indonesia. Se- kolah/madrasah berkualitas akan menghasilkan lulusan (output) yang memiliki daya saing tinggi, mampu berkompetensi di dunia global, yang pada akhirnya akan menjadi salah satu satu tolok ukur kemajuan suatu bangsa. 5. Analisis SWOT Pengembangan Madrasah Unggul Analisis terhadap lingkungan strategis baik internal maupun eksternal diperlukan guna mengetahui sasaran prioritas apa yang direncanakan di dalam menanggulangi masalah pendidikan dan mengembangkan mutu pendidikan madrasah. Untuk itu di- perlukan analisis SWOT (Strength, Weakness, Opportunity and Threat) tentang kondisi pendidikan madrasah, yang sebenarnya tidak bisa lepas dari kondisi umum pendidikan di Indonesia. Asumsi penyusunan analisis SWOT ini adalah bahwa pendidikan mad- rasah merupakan bagian yang inheren dan tidak terpisahkan dari pendidikan nasional. Berdasarkan asumsi tersebut, faktor pendukung keberhasilan pencapaian rencana strategis yang pada hakikatnya merupakan kekuatan (Strength) dalam pendidikan madrasah dapat diidentifikasi antara lain: 103 \| Agustini Buchari, Erni Moh. Saleh 1. Pengakuan atas hak dasar untuk memperoleh pendidikan telah dijamin di dalam Pembukaan Undang-Undang Dasar 1945 yang bertujuan untuk men- cerdaskan kehidupan bangsa dan memajukan kesejahteraan umum; 2. Adanya aspek legal yang menjamin pengelolaan pendidikan menuju pen- didikan yang bermutu dan memberdayakan seperti adanya Undang-undang Nomor 20 Tahun 2003 tentang Sistem Pendidikan Nasional dan Undang- undang Nomor 14 Tahun 2005 tentang Guru dan Dosen; 3. Komitmen pemerintah untuk merealisasikan keputusan Mahkamah Konsti- tusi Nomor 13/PUU-VI/2008 tentang kewajiban pemerintah untuk meng- alokasikan 20% dari APBN untuk pendanaan pendidikan, merupakan pe- luang untuk secara lebih leluasa merencanakan dan mengelola pendidikan madrasah yang berorientasi mutu; 4. Adanya reformasi dan regulasi yang pro pembangunan pendidikan seperti ketentuan Standar Pelayanan Minimal, Standar Nasional Pendidikan bahkan acuan internasional dalam meningkatkan mutu pendidikan; 5. SDM pengelola pendidikan madrasah yang sinergi membangun kinerja yang berkualitas; y g ; 6. Kebanggaan seluruh warga masyarakat dan rasa memiliki madrasah; 7. Koordinasi dan pemanfaatan terpadu sumber daya; 8. Kerja sama yang saling mendukung; 9. Sarana dan prasarana yang memadai untuk mendukung program aksi; 10. Kekayaan dan keragaman tradisi, seni dan budaya merupakan dasar untuk membentuk dan membangun watak, karakter dan budi pekerti luhur. g p Selain kekuatan, kita juga melihat kelemahan yang harus diantisipasi dan dicarikan sinya. Kelemahan tersebut antara lain adalah: 1. Sebagai imbas kualitas pendidikan nasional yang mutunya masih di bawah pencapaian mutu sejumlah negara pendiri ASEAN, yakni Singapura, Malay- sia, Thailand dan Filipina maka masih diperlukan upaya yang besar demi meningkatkan mutu pendidikan madrasah; 2. Masih adanya anggapan masyarakat di sebagian wilayah tentang kurang pentingnya pendidikan formal, sehingga mereka cukup mendidik anaknya di pendidikan informal, yang berakibat menyulitkan pemberantasan buta aksara; 3. Lembaga pendidikan dinilai belum sepenuhnya mampu melahirkan lulusan yang bermutu dan berketerampilan dengan kompetensi keahlian yang cukup bagi menghadapi kehidupan yang penuh tantangan; 4. 5. Analisis SWOT Pengembangan Madrasah Unggul Sebagian besar pendidik, terutama guru PAUD dan MI banyak yang belum memenuhi kualifikasi akademik S1 atau D4 seperti persyaratan Undang- undang Nomor 14 Tahun 2005 tentang Guru dan Dosen, kecuali itu juga adanya fakta bahwa persebaran tenaga pendidik tidak merata, dan umumnya bertumpu di wilayah perkotaan; 5. Banyak madrasah, terutama di wilayah pedesaan dan madrasah swasta ter- tentu yang belum memenuhi fasilitas pembelajaran seperti laboratorium, perpustakaan dan lain-lain, guna memenuhi ketentuan Standar Nasional Pendidikan; 6. Kurangnya aksesabilitas buku pelajaran terutama di wilayah pedesaan; Agustini Buchari, Erni Moh. Saleh \| 104 7. Belum optimalnya penggunaan teknologi informasi dan masih banyak mad- rasah yang gagap dan asing dalam penggunaan e-learning, dengan disparitas yang amat mencolok antara wilayah perkotaan dan wilayah pedesaan; y g y p y p 8. Layanan pendidikan nonformal belum sepenuhnya mampu membekali warga belajar dengan berbagai jenis keterampilan yang dibutuhkan oleh pasar kerja; p j 9. Masih belum fokusnya kurikulum pendidikan madrasah, masih terus dicari implementasi yang efektif dan efisien dari proporsi 70% : 30% pendidikan umum dan pendidikan agama. 10. Diarasakan sampai saat ini pengembangan madrasah masih bersifat tambal sulam dan belum menemukan fokus dan arah yang tepat. 11. Proses pendidikan madrasah masih belum sesuai dengan visi dan misi pen- didikan madrasah sendiri, masih bersifat parsial dan belum menyentuh keseluruhan yang integratif. 12. Belum adanya cetak biru pengembangan madrasah. 12. Belum adanya cetak biru pengembangan madrasah. Selain kekuatan dan kelemahan, dalam globalisasi ini juga ada beberapa peluan opportunity) sebagai berikut: 1. Sistem perdagangan dunia yang terbuka memberikan peluang dalam me- ningkatkan mutu pendidikan pada seluruh tataran pendidikan; 2. Pendidikan mutlak diperlukan guna menopang pengembangan ekonomi ber- basis pengetahuan (education for the knowledge based economy); 3. Pembangunan pendidikan saat ini semakin disadari merupakan bagian penting dari upaya menyeluruh dan sungguh-sungguh dalam meningkatkan harkat dan martabat bangsa; 4. Peningkatan Indeks Pembangunan Manusia Indonesia disadari amat ditentu- kan oleh kualitas dan akses pendidikan, sehingga upaya untuk melaksana- kan pemberantasan buta aksara dan angka partisipasi sekolah disadari me- rupakan upaya pokok dalam mencapai IPM yang tinggi; 5. Laju perkembangan IPTEKS dunia semakin cepat, untuk mengejar ketertinggalan dalam penguasaan IPTEKS hanya perbaikan mutu pendidik- an yang terus menerus berkesinambungan dan pengembangan budaya riset di seluruh jenjang madrasah mulai dari jenjang pendidikan dasar sampai perguruan tinggi merupakan jawabannya; 6. Peluang yang dimiliki oleh pendidik dan tenaga kependidikan saat ini cukup prospektif. 5. Analisis SWOT Pengembangan Madrasah Unggul Dengan adanya Direktorat Tenaga Kependidikan, kegiatan yang berkaitan dengan usaha peningkatan mutu tenaga kependidikan akan dapat difasilitasi dengan baik. Semakin luasnya akses informasi melalui teknologi komunikasi dan informasi (ICT, Web dan sejenisnya) membuka peluang bagi pendidik dan tenaga kependidikan untuk belajar terus menerus me- ningkatkan kemampuan diri. 7. Munculnya arus yang kuat dari warga belajar dan stakeholders madrasah untuk memiliki cetak biru pengembangan madrasah berupa Rencana Induk Pengembangan (RIP) Pendidikan Madrasah, sebagai pedoman dan panduan pengembangan madrasah di Indonesia ke depan. Meskipun demikian dalam memanfaatkan peluang yang ada, juga dihadapkan pada berbagai tantangan atau kendala (threat) yang diperkirakan akan dihadapi antara lain adalah: 105 \| Agustini Buchari, Erni Moh. Saleh 1. Adanya komitmen global yang mengikat untuk memperluas akses dan meningkatkan mutu pendidikan, dalam kerangka aksi Dakkar mengenai Pendidikan untuk Semua (PUS, Education for All). 2. Penerapan pengarusutamaan gender (PUG, gender mainstream) di Indo- nesia menghadapi kendala karena pada sebagian masyarakat masih meng- anggap tidak ada perlunya bagi perempuan bersekolah tinggi-tinggi. 3. Masih adanya keengganan sejumlah satuan pendidikan yang dikelola Ya- yasan untuk menerapkan Manajemen Berbasis Madrasah/Sekolah, karena dianggap mencampuri urusan Yayasan dalam mengelola pendidikan. Sementara itu bahkan di sejumlah madrasah negeri terutama di wilayah pe- desaan masih dijumpai kegagapan dalam menerapkan Manajemen Berbasis Sekolah/Madrasah (MBS); ( ) 4. Tantangan yang dihadapi pendidik dan tenaga kependidikan dalam waktu dekat ini adalah merespon kesungguhan pemerintah untuk meningkatkan mutu pendidik dan tenaga kependidikan. Menampilkan kinerja optimal untuk bersaing dengan pendidik dan tenaga kependidikan dari negara lain yang saat ini sudah boleh bekerja di negeri ini sebagai konsekuensi dari era WTO; 5. Tantangan jangka pendek yang dihadapi adalah segera menyiapkan diri, mengubah pola pikir untuk sanggup berkompetisi secara terbuka, selalu mencari informasi baru dan memperbaharui diri, baik pengetahuan, ke- terampilan dan sikap kerja. Pada jangka menengah pendidik dan tenaga ke- pendidikan harus mampu menjadi manusia pembelajar yang selalu mening- katkan dirinya sesuai dengan tantangan perubahan. Pada jangka panjang tantangan pendidik dan tenaga kependidikan adalah kemampuan menampil- kan citra diri sebagai individu yang sadar terhadap produktivitas kerja, prestasi kerja, dan perilaku yang adaptif dalam pengembangan diri untuk menyesuaikan kemampuannya sesuai tuntutan profesionalnya. 6. Upaya dan Strategi Pengembangan Madrasah Unggul Syarat menuju pengembangan madrasah unggulan antara lain ketersediaan tenaga pendidikan yang profesional, kelengkapan sarana dan prasarana, perlu ditangani dengan sistem manajemen profesional yang modern, transparan dan demokratis, dan adanya kurikulum yang sesuai dengan kebutuhan masyarakat dan tantangan dunia modern (Burhanudin dan Afrianty, 2006, p. 42). Selain itu, madrasah juga perlu memberikan perhatian untuk senantiasa meningkatkan kualitas, mengembangkan inovasi dan kreativitas, membangun jaringan kerjasama (networking), dan memahami karakteristik pelaksanaan otonomi daerah. Pada akhirnya, keunggulan sebuah madrasah akan sangat ditentukan oleh keberhasilan peserta didik (output dan outcome) yang memiliki prestasi yang membang- gakan. Dalam konteks keberhasilan madrasah, maka keberhasilan tersebut tidak saja diukur dari nilai akademik yang tinggi, tetapi juga harus dilihat dari perilaku yang Islami (akhlaqul karimah). ( q ) Madrasah unggulan dimaksudkan sebagai center for excellence. Madrasah Unggul- an diproyeksikan sebagai wadah menampung putra-putri terbaik masing-masing daerah untuk dididik secara maksimal tanpa harus pergi ke daerah lain. Dengan demikian terjadinya eksodus SDM terbaik suatu daerah ke daerah lain dapat diperkecil, dan se- kaligus menumbuhkan persaingan sehat antara daerah dalam menyiapkan SDM mereka. Agustini Buchari, Erni Moh. Saleh \| 106 Karena menjadi center for excellence anak-anak terbaik, maka kesempatan belajar di kedua jenis madrasah ini haruslah melalui proses seleksi yang ketat dan dengan ber- bagai kententuan lainnya. Madrasah ini diperkuat oleh keberadaan majelis madrasah yang juga memiliki peran penting dalam pengembangannya. Madrasah unggul dikembangkan untuk mencapai keistimewaan dalam keluaran pendidikannya. Untuk mencapai keistimewaan tersebut, maka masukan, proses pen- didikan, guru dan tenaga kependidikan, manajemen, layanan pendidikan, serta sarana penunjangnya harus diarahkan untuk menunjang tercapainya tujuan tersebut. Dengan memerhatikan fakta-fakta yang ada, maka ciri-ciri madrasah unggul yang dapat diidentifikasi ialah (1) prestasi akademik dan nonakademik di atas rata-rata se- kolah yang ada di daerahnya; (2) sarana dan prasarana dan layanan yang lebih lengkap; (3) sistem pembelajaran lebih baik dan waktu belajar lebih panjang; (4) melakukan seleksi yang cukup ketat terhadap pendaftar; (5) mendapat animo yang besar dari masyarakat, yang dibuktikan banyaknya jumlah pendaftar dibanding dengan kepasitas kelas; (6) biaya sekolah lebih tinggi dari sekolah di sekitarnya. ( ) y gg y Kementerian Agama sebagai salah satu pelaksana program pendidikan sekolah telah mengembangkan beberapa jenis madrasah unggulan, yaitu: Madrasah Aliyah Keagamaan, Madrasah Tsanawiyah Terbuka, Madrasah Model, Madrasah Aliyah Ung- gulan dan Madrasah Aliyah Keterampilan. Pengembangan kelembagaan di lingkungan madrasah dan sekolah Islam tidak hanya berhenti pada beberapa jenis sekolah di atas, tetapi terus berkembang hingga saat ini. 6. Upaya dan Strategi Pengembangan Madrasah Unggul Wacana pengembangan sekolah terpadu dan bertaraf internasional yang saat ini banyak diminati merupkan bagian dari pengembang- an lebih lanjut dari beberapa jenis lembaga pendidikan di atas. Oleh karena itu, strategi pengembangan madrasah perlu dirancang agar mampu menjangkau jangka panjang, dan mampu menghasilkan perubahan yang signifikan, ke arah pencapaian visi dan misi lembaga, sehingga akan memiliki keunggulan komparatif dan kompetitif baik di tingkat lokal, regional, nasional bahkan internasional. Dengan demikian pengembangan madrasah tidak boleh apriori terhadap trend pendidikan yang dibawa oleh proses globalisasi, internasionalisasi dan universalisasi, misalnya kom- puterisasi, vokasionalisasi dan ekonomisasi. Namun di pihak lain pengembangan mad- rasah harus tetap tegar dengan karakteristik khas yang dimilikinya sebagai perisai dan benteng pertahanan masyarakat dari persoalan-persoalan dekadensi moral dan melemah- nya nilai-nilai spiritual. Strategi pengembangan madrasah harus mengacu kepada strategi pengembangan pendidikan nasional dan diwujudkan dalam lima strategi pokok, yakni; (1) perluasan dan pemerataan memperoleh kesempatan pendidikan di madrasah; (2) peningkatan layanan pendidikan di madrasah; (3) peningkatan mutu dan relevansi pendidikan mad- rasah; (4) pengembangan sistem dan manajemen pendidikan yang akuntabel, dan (5) pemberdayaan kelembagaan untuk meningkatkan pencitraan madrasah. Sebenarnya kelima strategi pokok tersebut tetap berfokus kepada tiga pilar pem- bangunan pendidikan nasional. Strategi peningkatan layanan pendidikan di madrasah dan strategi pemberdayaan kelembagaan untuk meningkatkan pencitraan madrasah ber- muara kepada pilar tata kelola akuntabilitas dan pencitraan publik (pilra ketiga dalam pembangunan pendidikan nasional). Strategi perluasan dan pemerataan kesempatan pendidikan di madrasah difokuskan kepada pelaksanaan wajib belajar 9 tahun, bahkan di perkotaan sudah berkembang pada wajib belajar 12 tahun. Indikator kunci keberhasilan strategi ini antara lain adalah; (1) mayoritas penduduk berpendidikan minimal MTs diwujudkan oleh meningkatnya APK 107 \| Agustini Buchari, Erni Moh. Saleh pada semua jenjang dan jenis madrasah, (2) meningkatnya budaya belajar yang di- tunjukkan dengan meningkatnya angka melek aksara; dan (3) proporsi jumlah penduduk yang kurang beruntung yang mendapat kesempatan pendidikan semakin meningkat. Strategi peningkatan layanan pendidikan di madrasah difokuskan pada upaya men- cegah peserta didik agar tidak putus sekolah dan mempertahankan mutu pendidikan agar tidak semakin menurun. Indikator kunci keberhasilan implementasi stretegi ini adalah; (1) memperkecil angka putus sekolah di madrasah; (2) meningkatnya APK dan APM, baik MI dan MTs terutama bagi peserta didik yang kurang beruntung (miskin, tinggal di wilayah terpencil, broken home dan sebagainya); (3) mendorong kembali dan memfasilitasi siswa yang telanjur putus sekolah; (4) mempertahankan dan meningkat- kan mutu proses kegiatan belajar dan mengajar di madrasah, termasuk penggunaan ICT dalam pembelajaran, meskipun dana yang terbatas. 6. Upaya dan Strategi Pengembangan Madrasah Unggul Strategi peningkatan mutu dan relevansi pendidikan di madrasah difokuskan ke- pada pengembangan empat aspek yakni kurikulum, guru dan tenaga kependidikan lain, sarana pendidikan dan kepemimpinan madrasah. Dalam kaitan ini dilaksanakan pe- ngembangan kurikulum berkelanjutan di semua jenjang dan jenis madrasah, dilakukan pembinaan profesi guru madrasah, dilaksanakan peningkatan pengadaan dan pendaya- gunaan sarana dan prasarana pendidikan di madrasah serta pengembangan kepemim- pinan di madrasah yang berlandaskan prinsip manajemen berbasis madrasah. Strategi pengembangan manajemen pendidikan madrasah berkenaan dengan upaya pengembangkan sistem manajemen madrasah sehingga secara kelembagaan madrasah memiliki kemampuan: (1) mengembangkan prakarsa dan kemampuan kreatif dalam mengelola pendidikan, (2) mengembangkan organisasi pendidikan madrasah yang lebih berorientasi profesionalisme, dan (3) layanan pendidikan yang semakin cepat, terbuka, adil, dan merata. Strategi pemberdayaan kelembagaan madrasah ditekankan kepada pemberdayaan madrasah sebagai pusat pembelajaran, pendidikan dan pembudayaan. Indikator kunci keberhasilannya antara lain adalah: (1) tersedianya madrasah yang semakin bervariasi dengan dukungan organisasi yang efektif dan efisien; (2) mutu dan sarana dan prasarana madrasah yang semakin meningkat dan iklim pembelajaran yang semakin kondusif bagi peserta didik, dan (3) tingkat kemandirian madrasah yang semakin tinggi. 7. Unsur Pendukung Pengembangan Madrasah Unggul Dalam pelaksanaannya, pengembangan madrasah unggul perlu mendapat dukungan beberapa unsur pokok yang harus terpenuhi. Idealnya kata unggulan itu memiliki per- formansi yang sebanding lurus dengan amanah yang diembannya guna memenuhi harapan dan kepercayaan dari stakeholders, orangtua siswa, masyarakat dan pemerintah. formansi yang sebanding lurus dengan amanah yang diembannya guna memenuhi harapan dan kepercayaan dari stakeholders, orangtua siswa, masyarakat dan pemerintah. Menurut Arifin (2008, pp. 322—323), unsur pendukung madrasah atau sekolah Islam berprestasi (unggul) itu setidaknya ada sembilan faktor, yaitu: p p y g y p Menurut Arifin (2008, pp. 322—323), unsur pendukung madrasah atau sekola Islam berprestasi (unggul) itu setidaknya ada sembilan faktor, yaitu: 1. Faktor sarana dan prasarana. Meliputi (a) fasilitas sekolah yang lengkap dan memadai, (b) sumber belajar yang memadai dan (c) sarana penunjang belajar yang memadai. 2. Faktor guru. Meliputi (a) tenaga guru mempunyai kualifikasi memadai, (b) kesejahteraan guru terpenuhi, (c) rasio guru-murid ideal, (d) loyalitas dan komitmen tinggi, dan (e) motivasi dan semangat kerja guru tinggi. gg ( ) g j g gg 3. Faktor murid. Meliputi (a) pembelajaran yang terdiferensiasi, (b) kegiatan intra dan ekstrakulikuler bervariasi, (c) motivasi dan semangat belajar ting- gi, (d) pemberdayaan belajar bermakna. Agustini Buchari, Erni Moh. Saleh \| 108 4. Faktor tatanan organisasi dan mekanisme kerja. Meliputi (a) tatanan organi- sasi yang rasional dan relevan, (b) program organisasi yang rasional dan re- levan, (c) mekanisme kerja yang jelas dan terorganisasi secara tepat. 4. Faktor tatanan organisasi dan mekanisme kerja. Meliputi (a) tatanan organi- sasi yang rasional dan relevan, (b) program organisasi yang rasional dan re- levan, (c) mekanisme kerja yang jelas dan terorganisasi secara tepat. ( ) j y g j g p 5. Faktor kemitraan. Meliputi (a) kepercayaan dan harapan orangtua tinggi, (b) dukungan dan peran serta masyarakat tinggi, (c) dukungan dan bantuan pemerintah tinggi. ( ) j y g j g p 5. Faktor kemitraan. Meliputi (a) kepercayaan dan harapan orangtua tinggi, (b) dukungan dan peran serta masyarakat tinggi, (c) dukungan dan bantuan pemerintah tinggi. 6. Faktor komitmen/sistem nilai. Meliputi (a) budaya lokal yang saling mendukung, (b) nilai-nilai agama yang memicu timbulnya dukungan positif. 6. Faktor komitmen/sistem nilai. Meliputi (a) budaya lokal yang saling mendukung, (b) nilai-nilai agama yang memicu timbulnya dukungan positif. 7. Faktor motivasi, iklim kerja, dan semangat kerja. Meliputi (a) motivasi ber- prestasi pada semua komunitas sekolah, (b) suasana, iklim kerja dan iklim belajar sehat dan positif, dan (c) semangat kerja dan berprestasi tinggi. 7. 7. Unsur Pendukung Pengembangan Madrasah Unggul Faktor motivasi, iklim kerja, dan semangat kerja. Meliputi (a) motivasi ber- prestasi pada semua komunitas sekolah, (b) suasana, iklim kerja dan iklim belajar sehat dan positif, dan (c) semangat kerja dan berprestasi tinggi. 8. Faktor keterlibatan Wakil Kepala sekolah dan guru-guru. Meliputi (a) ke- terwakilan kepala sekolah dalam pembuatan kebijakan dan pengimplemen- tasiannya, (b) keterwakilan kepala sekolah dan guru-guru dalam menyusun kurikulum dan program-program sekolah, dan (c) keterlibatan wakil kepala sekolah dan guru-guru dalam perbaikan dan inovasi pembelajaran. 9. Faktor kepemimpinan kepala sekolah. Meliputi (a) piawai memanfaatkan nilai religio-kultural, (b) piawai mengkomunikasikan visi, inisiatif, dan kreativitas, (c) piawai menimbulkan motivasi dan membangkitkan se- mangat, piawai memperbaiki pembelajaran yang terdiferensiasi, (e) piawai menjadi pelopor dan teladan, dan (f) paiwai mengelola administrasi sekolah. j p p , ( ) p g Dalam rangka mewujudkan pengembangan madrasah dan sekolah Islam unggulan memerlukan langkah dan upaya yang fisibel dan kredibel (Mujtahid, 2011, p. 286). Oleh karena itu, madrasah dan sekolah Islam unggulan harus bersaing dengan beberapa lembaga pendidikan yang sedang mencanangkan program rintisan madrasah bertaraf internasional (RMBI) atau rintisan sekolah bertaraf internasional (RSBI). Perencanaan (pengembangan) membutuhkan langkah strategis untuk mengembangkan keunggulan madrasah dan sekolah Islam unggulan. Penguatan keunggulan lembaga tersebut melalui cara membangun cita dan kultur akademik yang kokoh. Cita-cita didirikannya madrasah dan sekolah Islam adalah sangat mulia, yaitu ingin melahirkan lulusan yang unggul di bidang akademik, spiritual dan moral. Selama ini, hanya ada dua lembaga pendidikan yang melahirkan identitas ilmuwan yang berbeda, yaitu pondok pesantren yang ingin melahirkan ulama (ahli agama) dan sekolah umum yang ingin melahirkan kaum intelek- tual (akademis). Madrasah dan sekolah Islam unggulan selama ini sesungguhnya ber- cita-cita ingin meraih kedua corak tersebut, yakni mencetak calon ulama sekaligus inte- lek atau intelek yang sekaligus ulama. Visi dan misi yang ideal tersebut harus diper- juangkan dan diwujudkan melalui pembenahan berbagai aspek, baik terkait dengan konsep bangunan keilmuannya (kurikulum), sumber daya manusia, pengembangan sarana dan prasarana, kelembagaan maupun leadership dan managerialnya (Mujtahid, 2011, p. 286). Gambar di bawah ini akan menjelaskan skema pengembangan madrasah unggul dan menjadi rujukan layanan pendidikan bagi masyarakat. 109 \| Agustini Buchari, Erni Moh. Saleh Gambar 1: Skema Pengembangan Madrasah Unggul. Adaptasi Bafadhal (2003) dan Depag RI (2004) Madrasah Unggul Lingkungan Sosial Input Latar Belakang Keluarga Guru Profesional Kelas Representatif Sarana memadai Pembelajaran efektif Siswa Unggul Mutu Berkualitas Inovasi Kurikulum 109 \| Agustini Buchari, Erni Moh. Saleh Mutu Berkualitas Gambar 1: Skema Pengembangan Madrasah Unggul. 7. Unsur Pendukung Pengembangan Madrasah Unggul Adaptasi Bafadhal (2003) dan Depag RI (2004) Dilihat dari gambar di atas, dapat dijelaskan bahwa adanya madrasah unggul perlu ditunjang dengan berbagai aspek di antaranya adanya input yang unggul, guru yang pro- fesional, sarana yang memadai, kurikulum yang inovatif, ruang kelas atau pembelajaran yang representatif, sehingga dapat mendorong terciptanya pembelajaran yang efektif dan efisien akhirnya dapat menghasilkan out put yang unggul dan berkualitas. Hal se- nada diungkapkan oleh Bafadhal (2003, p. 28) dan Depag RI (2004, pp. 1—5) bahwa- sannya untuk mencapai madrasah yang unggul dituntut adanya tenaga, fasilitas, dan dana yang memadai, dan tidak semua sekolah/madrasah dapat memenuhinya, serta secara teknis, pengembangan madrasah unggulan menuntut adanya tenaga yang pro- fesional dan fasilitas yang memadai. Berdasarkan uraian di atas dapat dikemukakan bahwa langkah strategis yang perlu dilakukan untuk mengembangkan madrasah unggul dan menjadi rujukan layanan pen- didikan bagi masyarakat antara lain: 1. Membangun Pola Pikir Kolektif. Untuk mengembangkan mutu madrasah unggulan membutuhkan pandangan, cita-cita, imajinasi, nilai-nilai keyakin- an yang kuat dan kolektif. Aspek kepentingan institusi harus dibangun se- cara kolektif dengan orientasi yang sama. Kepentingan institusi harus dikedepankan daripada kepentingan individu. Sedangkan, pola pikir (min- dset) yang perlu dibangun pada lembaga pendidikan madrasah unggulan adalah menanamkan keyakinan dan tekad bersama kepada seluruh warga se- kolah atau madrasah. Seluruh komponen madrasah haris digerakkan untuk memperjuangkan keunggulan institusi, dengan cara mengimplementasikan visi, misi, tradisi, dan orientasi madrasah. Mindset secara kolektif tersebut menjadi modal sosial (social capital) bagi pengembangan kultur akademik di madrasah unggulan ke depan. Madrasah unggulan membutuhkan ling- kungan akademik yang handal dan tekad bersama. Inspirasi dan semangat inilah yang harus dibangun dan dikembangkan untuk meningkatkan mutu akademik dan institusi madrasah. 2. Menciptakan Inovasi secara Terus Menerus. Keunggulan lembaga mad- rasah sesungguhnya terletak pada inovasinya. Inovasi merupakan usaha dan kerja nyata untuk mencari dan membuat hal baru demi meraih kemajuan dan keunggulan bagi lembaga pendidikan madrasah secara berkelanjutan. Inovasi harus didasarkan pada kebutuhan agar lembaga madrasah dan se- Agustini Buchari, Erni Moh. Saleh \| 110 kolah Islam itu terus maju dan berkembang. Inovasi secara terus-menerus harus digerakkan untuk memacu kualitas dan daya saing yang tinggi. Ino- vasi tidak saja diperlukan untuk selalu menyempurnakan kondisi madrasah, tetapi juga penting untuk membangun keutuhan (holistika) tujuan pendidik- an madrasah dan sekolah Islam. Usaha dan kerja nyata itu ditempuh secara serentak, menyeluruh dan integratif di antara beberapa elemen yang ada di madrasah. 7. Unsur Pendukung Pengembangan Madrasah Unggul Bentuk inovasi itu misalnya, perbaikan atau penambahan sarana fisik, akademik, tenaga guru dan karyawan, perekrutan siswa dan seluruh aspek yang ada. Inovasi lainnya misalnya menciptakan kultur madrasah ber- basis bilingual, mentradisikan hafalan Alquran, menggerakkan pusat seni dan olah raga, dan seterusnya. Modal seperti inilah yang harus dituangkan dalam visi dan orientasi madrasah unggul itu. Dengan cara seperti itulah madrasah unggulan dapat menawarkan sesuatu yang baru, yang khas dan memiliki keunikan yang diperhitungkan oleh masyarakat. Tugas ini mem- butuhkan seorang pemimpin yang efektif dan didukung oleh warga mad- rasah yang dedikatif dan dapat bekerja secara extra role. 3. 3. Memanfaatkan Teknologi Informasi. Untuk memajukan madrasah dan se- kolah Islam yang merata dan berkualitas membutuhkan energi pikiran, tenaga dan usaha yang konsisten dan berkelenjutan. Sudah saatnya mad- rasah mengembangkan pembelajaran berbasis digital, selain yang sudah ada, guna mengefektifkan program dan kegiatan pendidikan yang lebih mak- simal. Pendidikan madrasah tidak boleh tertinggal di bidang teknologi informasinya. Dengan pemanfaatan teknologi informasi tersebut para siswa dapat belajar lebih intensif, di samping melalui sistem reguler dan kurikuler. Teknologi informasi perlu dimanfaatkan sebagai sumber belajar yang mudah dan berjangkauan luas, tanpa hambatan waktu dan tempat. Untuk menciptakan mutu layanan akademik, menurut hemat penulis dapat dikembangkan sistem digital di madrasah. Hampir semua aktivitas akade- mik melibatkan internet, sehingga program-program madrasah dapat ber- jalan secara sinergis antara unit satu dengan unit-unit lainnya. Melalui prog- ram digitalisasi ini, upaya untuk memajukan madrasah sangatlah mudah diukur dan dirasakan oleh para pengguna. 4. Membangun Kepemimpinan yang Terbuka dan Berprinsip. Dalam rangka pengembangan madrasah unggul dibutuhkan sistem manajemen yang terbuka (open management) termasuk di antaranya adalah perilaku ke- pemimpinan. Diperlukan kepemimpinan yang efektif: kepemimpinan yang lebih interaktif dan visioner—yang konsisten memobilisasi cita-cita, nilai- nilai, dan transformasi gagasan dan institusi—; kepemimpinan yang dijalankan tidak berdasarkan naluri, intuisi, dan nalar subjektif, tetapi yang dijalankan berdasarkan logika dan akal pikiran yang sehat, serta dibangun di atas kerangka objektivitas-akademik yang tinggi; dan kepemimpinan yang dijalankan haruslah dapat merefleksikan tumbuhnya suasana akademik yang sehat, komunikasi, koordinasi, dan interaksi yang efektif serta mengimple- mentasikan praktik-praktik baik (good practices) yang berkembang secara dinamis dalam institusi. 4. Membangun Kepemimpinan yang Terbuka dan Berprinsip. Dalam rangka pengembangan madrasah unggul dibutuhkan sistem manajemen yang terbuka (open management) termasuk di antaranya adalah perilaku ke- pemimpinan. 7. Unsur Pendukung Pengembangan Madrasah Unggul Diperlukan kepemimpinan yang efektif: kepemimpinan yang lebih interaktif dan visioner—yang konsisten memobilisasi cita-cita, nilai- nilai, dan transformasi gagasan dan institusi—; kepemimpinan yang dijalankan tidak berdasarkan naluri, intuisi, dan nalar subjektif, tetapi yang dijalankan berdasarkan logika dan akal pikiran yang sehat, serta dibangun di atas kerangka objektivitas-akademik yang tinggi; dan kepemimpinan yang dijalankan haruslah dapat merefleksikan tumbuhnya suasana akademik yang sehat, komunikasi, koordinasi, dan interaksi yang efektif serta mengimple- mentasikan praktik-praktik baik (good practices) yang berkembang secara dinamis dalam institusi. 5. Meningatkan Kualitas Tata Kelola Madrasah. Madrasah yang unggul adalah madrasah yang secara terus-menerus mempebaiki sisitem dan tata kelola organisasinya. Langkah awalnya ialah penataan struktur organisasi, 5. Meningatkan Kualitas Tata Kelola Madrasah. Madrasah yang unggul adalah madrasah yang secara terus-menerus mempebaiki sisitem dan tata kelola organisasinya. Langkah awalnya ialah penataan struktur organisasi, 5. Meningatkan Kualitas Tata Kelola Madrasah. Madrasah yang unggul adalah madrasah yang secara terus-menerus mempebaiki sisitem dan tata kelola organisasinya. Langkah awalnya ialah penataan struktur organisasi, 111 \| Agustini Buchari, Erni Moh. Saleh melakukan analisis jabatan secara tepat, membuat uraian tugas, menyusun instruksi kerja, dan membuat standar operasional prosedur (SOP). Melalui langkah ini, maka diharapkan semua komponen dapat bekerja secara efektif sehingga dapat memberikan layanan pendidikan yang maksimal. melakukan analisis jabatan secara tepat, membuat uraian tugas, menyusun instruksi kerja, dan membuat standar operasional prosedur (SOP). Melalui langkah ini, maka diharapkan semua komponen dapat bekerja secara efektif sehingga dapat memberikan layanan pendidikan yang maksimal. melakukan analisis jabatan secara tepat, membuat uraian tugas, menyusun instruksi kerja, dan membuat standar operasional prosedur (SOP). Melalui langkah ini, maka diharapkan semua komponen dapat bekerja secara efektif sehingga dapat memberikan layanan pendidikan yang maksimal. 6. Meningatkan Kualitas Sistem Rekruitmen Siswa Baru. Siswa adalah komponen input madrasah. Untuk mendapatkan output yang baik, diperlu- kan sistem rekruitmen yang baik dan berkulitas. Permasalahan yang ada se- lama ini ialah sistem rekruitmen siswa baru kurang konsisten menerapkan prosedur yang telah ditetapkan. Untuk mengembangkan madrasah yang unggul, dibutuhkan proses seleksi atau pola rekruitmen yang standar sebagaimana telah diterapkan di beberapa madrasah atau sekolah unggulan. 7. Meningatkan Kuantititas dan Kualitas Kegiatan Ekstrakurikuler. Pengem- bangan minat dan bakat siswa perlu mendapat perhatian. Proses pembelajar- an di madrasah tidak hanya berpusat pada pemenuhan kompetensi kognitif melalui pembelajaran di kelas, tetapi juga kegiatan di luar kelas yang ber- sifat ekstrakurikuler untuk pemenuhan kompetensi atau keterampilan hidup (life sklills). Program-program pengembangan minat dan bakat siswa harus juga menjadi perhatian. 8. 7. Unsur Pendukung Pengembangan Madrasah Unggul Meningkatkan Kegiatan Pembinaan dan Pengembangan Profesi Keguruan. Orientasi pembinaan dan pengembangan profesi keguruan harus bertumpu pada tiga kegiatan utama yaitu pengembangan diri, publikasi ilmiah, dan karya inovatif. Pengembangan diri dapat dilakukan melalui kegiatan pen- didikan dan pelatihan, seminar, dan workshop tentang praktik pendidikan dan pembelajaran. Publikasi ilmiah dapat dilakukan dengan mendorong dan memfasilitasi para guru melakukan penelitian baik berupa penelitian tindak- an kelas maupun penelitian eksperimen. Hasil-hasil penelitian itu dapat difasilitasi untuk dipublikasi melalui jurnal-jurnal ilmiah. Oleh karena itu, program-program literasi bagi guru perlu ditingkatkan. Demikian pula, karya inovatif guru perlu didorong terutama karya-karya inovatif dalam bidang pembelajaran. 8. Kesimpulan Kunci untuk membangun madrasah unggulan harus memenuhi beberapa kriteria, yaitu ketersediaan tenaga pendidikan yang profesional, kelengkapan sarana dan pra- sarana, sistem manajemen profesional yang modern, transparan dan demokratis, dan adanya kurikulum yang sesuai dengan kebutuhan masyarakat dan tantangan dunia modern. Selain itu, madrasah juga perlu memberikan perhatian untuk senantiasa me- ningkatkan kualitas, mengembangkan inovasi dan kreativitas, dan membangun jaringan kerjasama (networking), sehingga input dan output menjadi baik. Dengan kata lain, untuk mengembangkan madrasah unggulan memerlukan daya dukung yang efektif dan fungsional, seperti sumber daya manusia yang unggul, sarana- prasarana (ruang belajar yang representatif, perpustakaan dan laboratorium), fasilitas penunjang (boarding/ma’had, masjid atau mushala). Madrasah unggulan harus di- rancang sesuai dengan visi-misi dan tujuan kelembagaan, analisis kebutuhan sistem akademik dan kelembagaan, dan memahami konteks geografis dan budaya. Sedangkan, pengembangannya memerlukan kebersamaan dan maindset secara kolektif, inovasi secara terus menerus, dan memanfaatkan teknologi informasi, membangun kepemim- pinan yang terbuka dan berprinsip, meningatkan kualitas tata kelola madrasah, me- Agustini Buchari, Erni Moh. Saleh \| 112 ningatkan kualitas sistem rekruitmen siswa baru, meningatkan kuantititas dan kualitas kegiatan ekstrakurikuler, dan meningkatkan kegiatan pembinaan dan pengembangan profesi keguruan. 9. Daftar Pustaka Arifin, I. (2008). 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https://openalex.org/W4226176770	https://revistaseletronicas.pucrs.br/index.php/teo/article/download/41450/27438	Portuguese	null	A interpretação simbólica de 666 face a Ap 7,4-8 e aos Doze Apóstolos	Teocomunicação	2,022	cc-by	11,827	1 Este artigo se trata de texto atualizado, corrigido e reformulado – contendo acréscimos, gráficos e imagens – do artigo “El Número 666 y las Doce Tribus de Israel”, publicado na Revista Bíblica 68/3-4 (2006), p. 191-214, PPC Editorial, Buenos Aires, Argentina. A publica- ção brasileira foi autorizada pelo diretor da Revista Bíblica, Jorge Blunda, em 5 de abril de 2021. Dedicatória de Adylson Valdez: dedico este trabalho a São Thomas More, meu intercessor, e a Pierre Prigent, meu incentivador. 2 Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brasil. 3 Pesquisador autônomo Santos SP Brasil Palavras-chave: 666. Doce Tribus. Apostasía. Apóstoles. OPEN ACCESS OPEN ACCESS Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 e-ISSN: 1980-6736 \| ISSN-L: 0103-314X Recebido em: 3 ago. 2021. Aprovado em: 11 out. 2021. Publicado em: 8 abr. 2022. 3 Pesquisador autônomo, Santos, SP, Brasil. Artigo está licenciado sob forma de uma licença Creative Commons Atribuição 4.0 Internacional. SEÇÃO: ARTIGOS LIVRES A interpretação simbólica de 666 face a Ap 7,4-8 e aos Doze Apóstolos1 The symbolical interpretation of 666 in the face of Rev. 7:4-8 and the Twelve Apostles La interpretación simbólica de 666 frente al Ap 7,4-8 y a los Doce Apóstoles 4 Além das duas interpretações apresentadas neste texto existem mais duas outras: uma que procura soluções meramente matemá- ticas, com base em sistemas pitagóricos ou em outros sistemas. É o caso de van Eysinga, que propõe a fórmula n (n + 1): 2 = 1 + 2 + 3... + n, para achar-se o número triangular de 8 e depois o número 666, o qual, portanto, refere-se ocultamente ao oitavo rei de Ap 17,11 (VAN EYSINGA, 1912, p. 293-305). A outra interpretação é de natureza cronológica, que considera 666 a duração do domínio da Besta ou do poder anticristão. Nela existem três vertentes: a) 666 anos é a duração do paganismo até o imperador romano Constantino; b) a cifra corresponde à duração do período muçulmano; c) e o número representa a duração do papado. Introdução O império romano foi, durante um grande período de paz (31 a.C./235 d.C.), favorável ao comércio, às trocas culturais e, por isso mesmo, à difusão dos seguidores de Jesus de Nazaré. Paulo de Tarso, o apóstolo dos gentios, mostra bem isso, ao ser um dos responsáveis pela divulgação da chamada Boa Nova, ou Evangelho, muito além de tradição judaica, para antigos politeístas. Mas, ao mesmo tempo, o Império Romano apresentava-se como um reino (basileia) fundado no culto imperial, como se o governante, abençoado (sebastos, em grego, tradução de “augusto”), fosse um deus ou ao menos divinizado. Dominador, gerava revolta entre muitos submetidos. Era, pois, uma situação contraditória. Um imperador como Nero, em meados do primeiro século, era tanto popular em certos grupos, inclusive populares, como odiado por senadores e, mais ainda, por subjugados diversos. Os seguidores de Jesus, logo chamados de cristãos, em diversas ocasiões perseguidos, tinham os perseguidores como inimi- gos, como no caso notável e precoce do imperador Nero. Os cristãos podiam ser perseguidos por serem considerados como perturbadores da ordem social e da paz dos deuses (pax deorum), causadores de catástrofes, como incêndios ou doenças. Podiam ser considerados ímpios, ao não prestar culto aos deuses, ateus, e mesmo revolucionários. Este é o contexto de redação do Apocalipse, obra de datação discutível, mas que reflete esse contexto histórico mais amplo. Não obstante estes esforços, pode-se observar em ambas as interpretações muitas dificuldades e enganos, de maneira que é necessário rever-se os seus sistemas para se descobrir nelas algum mérito com relação às suas propostas. Este artigo procura justamente proceder a esta re- visão, observando a possibilidade da conjugação das duas interpretações, assim como trazer um indício da influência do simbolismo particular e tipológico do número 666 na formação da lista das Doze Tribos de Israel, encontrada em Ap 7,4-8, levando-se ainda em consideração a lista dos Doze Apóstolos de Jesus. Pedro Paulo Abreu F i2 1, p. 1-17, jan.-dez. 2022 \| e-41450 enigmática uma qualidade simbólica do ponto de vista bíblico. Pedro Paulo Abreu F i2 Resumo: Diante do problema do número 666 (Ap 13,18), a maioria dos intér- pretes modernos opta pela explicação gemátrica ou simbólica. Este artigo expõe as dificuldades internas, os erros e os acertos destas explicações. Como resultado da análise se conclui que a conjugação de ambas as interpretações é uma solução viável, não só pelo fato de que o autor do Apocalipse utiliza a cifra para ocultar um nome, mas também porque o número 666 indica claramente os antagonistas dos verdadeiros cristãos. A análise da lista das Doze Tribos de Israel (Ap 7,4-8), em conjunto com a lista dos Doze Apóstolos, confirma este padrão de pensamento simbólico e tipológico. orcid.org/0000-0003-0183-7622 ppfunari@uol.com.br Adylson Valdez3 orcid.org/0000-0002-0002-0701 adylv@lbm.com.br Palavras-chave: 666. Doze Tribos. Apostasia. Apóstolos. Abstract: Faced with the problem of the number 666 (Rev. 13:18), most modern interpreters opt for the gematric or symbolic explanation. This article exposes the internal difficulties, errors and successes of these explanations. As a result of the analysis, it is concluded that the combination of both interpretations is a viable solu- tion, not only because the author of the Apocalypse uses the cipher to hide a name, but also because the number 666 clearly indicates the antagonists of true Christians. The analysis of the list of the Twelve Tribes of Israel (Rev 7,4-8), together with the list of the Twelve Apostles, confirms this pattern of symbolic and typological thinking. Keywords: 666. Twelve Tribes. Apostasy. Apostles Recebido em: 3 ago. 2021. Aprovado em: 11 out. 2021. Publicado em: 8 abr. 2022. Keywords: 666. Twelve Tribes. Apostasy. Apostles Resumen: Ante el problema del número 666 (Ap 13,18), la mayoría de los in- térpretes modernos opta por la explicación gemátrica o simbólica. Este artículo expone las dificultades internas, errores y aciertos de estas explicaciones. Como resultado del análisis, se concluye que la combinación de ambas interpretacio- nes es una solución viable, no solo porque el autor del Apocalipsis usa la cifra para ocultar un nombre, sino también porque el número 666 indica claramente los antagonistas de los verdaderos cristianos. El análisis de la lista de las Doce Tribus de Israel (Ap 7,4-8), junto con la lista de los Doce Apóstoles, confirma este patrón de pensamiento simbólico y tipológico. Artigo está licenciado sob forma de uma licença Creative Commons Atribuição 4.0 Internacional. 2/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 Teocomunicação, Porto Alegre, v. 52, n. 1.1 A interpretação pela gematria A vertente interpretativa de que vamos tratar nes- te item defende a posição de que 666 é o resultado da soma dos números correspondentes às letras do nome da Besta (Ap 13,17; 14,11; 15,2), conforme o antigo sistema de numeração utilizado pelos gregos e judeus. Esta operação chama-se gematria. A primeira notícia da utilização desta forma de interpretação entre os cristãos encontramos em Ireneu (séc. II d.C.), o qual recomendou aguardar o cumprimento da profecia do Apocalipse para se saber qual é o nome correspondente ao número 666, pois uma quantidade enorme de palavras pode conter a cifra. Não obstante isto, ele pro- pôs os nomes gregos Euanthas (“florações” ou “botões”, acusativo plural de euanthes), Lateinos (“Latino”, referindo-se ao império romano) e Teitan (“Titã”, para indicar um rei tirano) (IRENEU, 5.30.3). Ireneu ainda menciona uma variante da cifra, 616, encontrada em alguns manuscritos, conside- rando-a uma falha dos copistas (IRENEU, 5.30.1). Mais tarde, Vitorino de Pettau concorda com Ireneu, repetindo Teitan, mas acrescentou os Sem dúvida, a passagem mais enigmática do livro do Apocalipse se encontra nos versículos 17 e 18 do seu capítulo 13, na qual o autor nos revela o número do nome da Besta, que é 666. Através dos tempos, os estudiosos da Bíblia procu- raram um nome que correspondesse a este número, levando em conta não só sistemas alfabéticos de numeração como também o aspecto anticristão de um determinado personagem proeminente da História. Paralelamente a este modo de interpretação, outra linha de pesquisadores vislumbrou na cifra Pedro Paulo Abreu Funari • Adylson Valdez A interpretação simbólica de 666 face a Ap 7,4-8 e aos Doze Apóstolos 3/17 nomes Antemos (do grego, “Contrário”) e Gen- serikos (forma grega do gótico gaisa-reik, “Lan- ça-Rei”), bem como as letras latinas DICLVX (correspondentes a Teitan, conforme o sistema numérico romano) (VITORINO DE PETTAU, cap. 13). Tendo em vista as perseguições feitas contra o cristianismo nos primeiros séculos da Era Cristã, as propostas de gematria que se seguem através da História representam nomes e títulos impe- riais romanos, sendo muitas e variadas. Entre elas, com relação a 666, podemos mencionar: Oulpios, sugerida por Grotius, forma grega do latim Ulpius, vocábulo designativo do clã do imperador Trajano (GROTIUS, 1630, p. 368); Diocles Augustus, referente ao imperador Diocleciano, utilizando-se as letras DICLVVV conforme o sistema numérico romano, criada por Bossuet (1689, p. 1.1 A interpretação pela gematria 302); e as primeiras letras e sílabas (marcadas em itálico) de Autokra- tor Kaisar Dometianos Sebastos Germanikos, título imperial de Domiciano, em grego, encontrado em uma antiga inscrição, proposta por Stauffer (1947, p. 237-241). conforme o sistema numérico hebraico usado no século I d.C., que era limitado até o número 400). Como esta teoria reafirma a lenda do Nero redivivus, a proposta ganhou a aceitação da grande maioria dos especialistas, apesar de adotar uma gematria de letras hebraicas (o Apocalipse foi escrito em grego e se destinou a pessoas de fala grega; porém, deve-se lembrar que o autor pensava em hebraico, pois utilizou hebraísmos e cometeu muitos erros gramaticais). Contrapôs-se a esta gematria que a forma Qsr é defeituosa, sendo que a correta é Qysr (com a letra iod). Ewald observou, em con- trapartida, que Qsr é encontrada em inscrições e documentos siríacos descobertos em Palmira, datados do século III d.C. (EWALD, 1862, p. 263). Charles argumentou que o dicionário de Marcus Jastrow (1903, p. 1365) admite a forma Qsr,e ainda citou a possibilidade de que 616 corresponde- ria à mesma gematria sem a letra nun final de Nrwn (conforme uma transliteração a partir da forma latina do nome) (CHARLES, 1920, p. 367). Por fim, na década de 1950, foi descoberto entre os manuscritos do Mar Morto um docu- mento em aramaico com Qsr, datado do se- gundo ano do reinado de Nero, fato que aca- bou confirmando a probabilidade da teoria. Ver as figuras 1 e 2 a seguir. Para a variante 616, sugeriram-se: Caesar Romae (em latim, “César, em Roma”), conforme Ewald (1828, p. 237); Kaisar Theós (em grego, “César Deus”), ima- ginada por Deissmann (1910, p. 344); Gaios Kaisar, referindo-se a Calígula, gematria grega sugerida por Spitta (1889, p. 134-136, 369-371); e o título hebraico Qeysar Dwmytyanus (“César Domiciano”), sugerido por Hartingsveld (1978, p. 191-201). Figura 1 – Frente e verso do documento encontrado no ano de 1951 em Wadi Murabba’at, com NRWN QSR (“Nero César”) no fim da primeira linha Podemos citar, ainda, a gematria hebraica tehom qadmoniyyah (“o caos primordial”), para 666, con- forme Gunkel (1895, p. 377); assim como he latinè basileía (do grego, “o reino latino”) para 666 e he italè basileía (“o reino italiano”) para 616, criadas por Clemen (1901, p. 109-114; 1910, p. 204-223; 1921, p. 144-148). Entre 1831 e 1837, os estudiosos alemães Frit- zsche (1831, p. 1.1 A interpretação pela gematria 59-60), Benary (1836, p. 205-206), Hitzig (1837, p. 3) e Reuss (1837, p. 520) propuseram para 666, de forma independente, Nrwn Qsr (uma transliteração do grego para o hebraico do nome “Nero César”, formada por nun = 50, resh = 200, vav = 6, nun = 50, kof = 100, sâmek = 60 e resh = 200, 4/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 4/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 além dos trechos de Ireneu, é comum mencionar Gn 1,26-31 (a criação do homem no sexto dia da primeira semana) e 1Sm 17,4.7 (a altura do gigante Golias, de seis côvados e um palmo, e o peso da ponta de sua lança, de 600 siclos de ferro). Com frequência, também encontramos a menção de 2Sm 21,20-21 (o gigante de Gat, com seis dedos em cada mão e pé, derrotado por um parente de Davi). Fonte: Discoveries in the Judaean Desert (DJD) II, n. 18, tav. XXIX; a imagem do manuscrito foi fornecida pela Tyndale House Library, Cambridge, Inglaterra. Na busca do número 666, os estudiosos en- contraram 1Rs 10,14 (666 talentos de ouro levados anualmente a Salomão; 2Cr 9,13), por sugestão de Beda (PL 93,172.D), e Esd 2,13 (a quantidade dos filhos de Adonicam), sugerida por Hengstenberg (1852, p. 52). Fonte: Discoveries in the Judaean Desert (DJD) II, n. 18, tav. XXIX; a imagem do manuscrito foi fornecida pela Tyndale House Library, Cambridge, Inglaterra. Na tentativa de achar os significados que dima- nam destas passagens, chegou-se à conclusão de que o número seis simboliza o homem, o qual, por não ser indicado pelo número sete, que se refere a Deus (Gn 2,1-3), lembra que jamais poderá atingir a perfeição. Assim, o número 666, por ser o resultado de seis multiplicado por 1, 10 e 100, ou, por ser composto de três seis seguidos, significa um homem imperfeito, completamente iníquo e perdido, arrogante e com a pretensão de ser deus, partidário do Mal, remetendo a 2Ts 2,3-8 e especial- mente a Dn 7,8.11.25-27 e Ap 13,1-8; 17,8; 19,19-20. Figura 2 – Reconstituição da primeira linha (lê-se da direita para a esquerda) Fonte: Discoveries in the Judaean Desert (DJD) II, n. 18, tav. XXIX; a imagem do manuscrito foi fornecida pela Tyndale House Library, Cambridge, Inglaterra. Fonte: Discoveries in the Judaean Desert (DJD) II, n. 18, tav. XXIX; a imagem do manuscrito foi fornecida pela Tyndale House Library, Cambridge, Inglaterra. 1.1 A interpretação pela gematria Sem dúvida, pode-se extrair dos textos várias relações, como, por exemplo, os gigantes derrotados por Davi e por seu parente, lembrando Jesus, o descendente de Davi, derrotando o Anticristo, um homem poderoso; o dilúvio, destruição da humanidade, rememorando antecipadamente o Juízo Final, combinando-se com Mt 24,37-39 e Lc 17,26-30; e a sensação de fragilidade e caricatura na figura do homem forte, gigantesco, inimigo estrangeiro, que remete aos antigos gigantes de Gn 6,4, a ser derrotado facilmente pelo Messias com um sopro, como diz Paulo (2Ts 2,8). Figura 2 – Reconstituição da primeira linha (lê-se da direita para a esquerda) 2 A análise das duas interpretações Outrossim, não existe nenhuma evidência no Apocalipse de que João tenha se baseado nas pas- sagens apontadas por esta explicação e as tenha interpretado no modo como ela as interpreta. Na realidade, o número seis, em suas formas cardinal e ordinal, do ponto de vista dos contextos, não possui, na Bíblia, os significados que a interpre- tação simbólica utiliza. Como se verá a seguir, as passagens de Gn 1,26-31; 1Rs 10,14-20.23; Dn 3,1.7; 1Sm 17,4.7; 2Sm 21,20-21; e Gn 7,6.11 não possuem a intenção primária objetiva de expressar o sím- bolo distintivo do homem, a idolatria, a inimizade ao Messias, a recapitulação da iniquidade, o Mal, a apostasia e a imperfeição. Também deve ser observado que o número seis não é exclusivo da figura do homem, mas também, às vezes, é usado para animais, e, por outro lado, pode ter tanto uma conotação positiva como negativa. Como podemos verificar, as duas interpre- tações possuem fundamentos aparentemente convincentes dentro da lógica de seus sistemas interpretativos. No entanto, ambas apresentam algumas dificuldades. Com relação à interpretação pela gematria, embora ela pareça ser simples e óbvia perante o contexto histórico e a literatura apocalíptica, o problema se encontra no fato de que a descoberta do nome tem de enfrentar um processo mental altamente complexo. Deve-se lembrar que o Apocalipse, ao contrário do apócrifo Oráculos Sibilinos, não dá nenhuma equação de letras, e as únicas coordenadas que nele se encontram para se descobrir o nome são: a frase arithmòs gàr anthrópou estín (“pois é número de homem”; tradução de Adylson Valdez); e a revelação do número correspondente à Besta e ao seu nome. Admitindo-se a teoria de que o nome possa cor- responder à gematria Nrwn Qsr e de que o autor pensava na lenda do Nero redivivus, o leitor, tendo somente em mãos as coordenadas daquela frase e da revelação do número, teria que, em primeiro lugar, deduzir que o Anticristo seria uma pessoa; depois, conhecendo previamente a lenda, con- cluir, pela análise dos capítulos 13 e 17, que João a adotou, e que as sete cabeças da Besta e as sete montanhas são imperadores romanos; e, por fim, já sabendo que o Anticristo era Nero, imaginar uma transliteração do grego para o hebraico que correspondesse ao título “Nero César”, mas sem a letra iod. 1.2 A interpretação simbólica Após a escrita do Apocalipse, Ireneu foi o primei- ro de que se tem notícia a apontar um significado simbólico bíblico para 666, embora em conjugação com a gematria, ao relacioná-lo à recapitulação da iniquidade, referindo-se aos 600 anos de Noé, data do dilúvio (Gn 7,6.11), e às medidas da estátua erigi- da pelo rei babilônio Nabucodonosor, 60 côvados de altura e 6 de largura (Dn 3,1.7) (IRENEU, 5.29.2). Devido à menção destas passagens do Antigo Testamento por Ireneu, os adeptos do simbolismo procuraram outras passagens onde surgem os números 6, 60, 600 e 666. Geralmente, as suges- tões dos outros trechos se baseiam nas relações que existem entre as circunstâncias relatadas ou características das passagens e o conteúdo humano e tipológico que possam vir a ter. Assim, Um outro fundamento para a interpretação simbólica é a utilização de múltiplos de seis no Apocalipse (42 e 1.260: Ap 11,2-3.9.11; 12,6.14; 13,5), formando um padrão de raciocínio matemático apocalíptico, que está relacionado ao tempo do governo da Besta (um padrão que deriva de Dn 7,25; 8,14; 9,24-27; 12,11-12). Pedro Paulo Abreu Funari • Adylson Valdez A interpretação simbólica de 666 face a Ap 7,4-8 e aos Doze Apóstolos 5/17 Pedro Paulo Abreu Funari • Adylson Valdez A interpretação simbólica de 666 face a Ap 7,4-8 e aos Doze Apóstolos Pedro Paulo Abreu Funari • Adylson Valdez 5/17 2 A análise das duas interpretações A complexidade deste processo denota que ele não poderia ser empreendido por um leitor comum despreparado, mas sim por uma pessoa iniciada nos códigos de João ou que se propusesse a fazer todo este exercício mental por um estudo bastante cuidadoso do gênero apocalíptico, de suas regras peculiares e da literatura respectiva existente na época, assim como teria de ser um judeu ou um conhecedor da língua hebraica. Quanto à interpretação simbólica ape- O número seis originalmente está conectado com a semana de sete dias, indicando o término de uma atividade ou de um período necessário e suficiente para se completar uma determinada situação. Assim, o escravo hebreu deve servir por seis anos e no sétimo será libertado (Gn 31,41; Ex 21,2); o homem trabalhará durante seis dias e des- cansará no sétimo (Ex 16,26; 20,9-11); e a terra será semeada por seis anos, mas deve permanecer em paz no sétimo ano (Ex 23,10-11). Enquanto o número sete significa liberdade, descanso, feli- cidade, paz e inatividade, bem como dedicação ao culto de Deus, com justeza e respeito à lei, o número seis remete ao sofrimento, ao trabalho, à escravidão, à construção da existência para haver uma liberação ou o gozo pleno em uma próxima fase. Em decorrência deste significado básico, o número seis também passa a ter o conteúdo de cumprimento, totalidade perfeita, completeza, construtivismo e acabamento. A influência do princípio sétuplo gera no número seis o sen- tido de limite máximo, de um ápice alcança- do. Este significado parece ser uma decorrência do fato de que seis é um número par, o que in- duz à ideia de perfeição de uma obra ou ação. É o caso da criação do homem no sexto dia, que completa definitivamente toda a obra divina (Gn Quanto à interpretação simbólica, ape- sar de fazer correspondência com trechos bí- blicos onde se encontram os números 6, 60, 600 e 666, ela ignora o fato de que o núme- ro 666 oculta obrigatoriamente um nome. 6/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 a lenda do Nero redivivus era bastante conhe- cida na Ásia Menor (João endereça seu livro a esta região), justamente na ocasião da escrita do livro, quando surgiram falsos Neros, conforme testemunhos de Tácito e Suetônio, a qual faci- litaria a identificação das cabeças e montanhas de Ap 13 e 17 com os imperadores romanos. 2 A análise das duas interpretações Também, a complexidade para se achar o nú- mero parece estar de acordo com o intuito do autor, pois ele nunca revela claramente o meio de se achar o significado de suas alegorias e sím- bolos, exigindo um conhecimento prévio da Sa- grada Escritura que somente um judeu ou um estudioso acurado e detalhista poderiam ter. Esta exigência parece ser bem evidente nas se- guintes frases de Ap 13,18: Hòde he sofía estín; ho ékhon noûn psefisáto tòn arithmòn toû theríou; ou seja, somente alguém que tivesse a ca- pacidade de compreender e que estives- se preparado com sofía (“sabedoria”) pode- ria indicar as letras para se chegar ao número. A interpretação simbólica procura uma base bíblica para fundamentar sua proposição, o que é abso- lutamente válido, pois o autor do Apocalipse está bastante influenciado por várias passagens bíblicas, especialmente por trechos do livro de Daniel. Os significados de iniquidade, perdição, apostasia, idolatria e de relação com o Mal estão presentes na aplicação que João faz para o número, já que 666 marca os antagonistas de Deus, de Jesus e dos cristãos, bem como os cristãos apóstatas. Mas estes significados de 666 emanam exclusivamente do texto do Apocalipse, não dos textos do resto da Bíblia, em sua intenção original. É possível deduzir, no entanto, que João possa ter sido inspirado pelas passagens em que os inimigos de Davi se apresen- tam com uma força física gigantesca, demonstrada pelo número seis multiplicado por cem e por quatro, a qual é poderosa apenas aparentemente, pois eles são derrotados facilmente pelo rei e por seus heróis (conforme mencionado anteriormente na alínea c). Isto porque há uma relação tipológica bastante clara entre os inimigos de Davi e o Anticristo, que é o antagonista de Jesus. A relação com a idolatria também é possível diante de Dn 3,1.7 e Ap 13,14-15. 1,26-31); da queda da muralha de Jericó após seis dias (Js 6,3-5.14-15); do aniquilamento total do ini- migo com o lançamento simbólico de seis flechas (2Rs 13,19); e das medidas perfeitas do templo de Deus na visão de Ezequiel (Ez 40,5.12; 41,1.3.5.8). 2 A análise das duas interpretações Hipoteticamente poderíamos dizer que a gematria Nrwn Qsr teria sido imaginada sem a letra iod justamente para que o número a ser obtido fosse 666: uma cifra feita deliberadamente desta forma com o fim de remeter ao significado simbólico e talvez tipológico do Anticristo de maneira perfeita e veemente. No capítulo 7, o autor do Apocalipse relaciona as Doze Tribos de Israel conforme a seguinte orde- nação: Judá, Rubem, Gad, Aser, Neftali, Manassés, Simeão, Levi, Issacar, Zabulon, José e Benjamin. Esta lista pode ser estudada de acordo com: a) os grupos de filhos de cada esposa de Jacó: Judá-Rubem, filhos de Lia; Gad- Aser, filhos de Lia, por meio da sua escra- va Zelfa (Gn 30,9-13); Neftali-Manassés, respectivamente, filho de Raquel, por meio da sua escrava Bala (Gn 30,3.7- 8), e neto de Raquel, por meio do seu filho José (Gn 46,27; 48,1); Simeão-Levi- Issacar-Zabulon, filhos de Lia; e José- Benjamin, filhos de Raquel; Naturalmente que o conteúdo simbólico par- ticular de 666 pode ter gerado um padrão de pensamento que tenha influenciado o autor na elaboração de outras passagens do Apocalipse, considerando-se também a possível relação tipológica do número. b) os grupos geograficamente próxi- mos: Judá-Rubem-Gad (sul, sudeste e este); Aser-Neftali (norte e nordeste); Manassés, isolado no centro, sem formar um grupo; Simeão-Levi (sul e sudeste); Issacar-Zabulon (centro-norte e nordes- te); e José-Benjamin (centro e sudeste); A busca de outras passagens, especialmente aquelas que remetem a múltiplos de seis, revela que, entre elas, a lista das Doze Tribos de Israel, contida em Ap 7,4-8, parece apontar sutilmente para este padrão simbólico e tipológico de pen- samento, tendo em vista a evidente substituição da tribo de Dã pela de Manassés no sexto lugar da lista, por razões visivelmente teológicas. c) o conteúdo histórico, político e teoló- gico de cada tribo, tendo por base textos bíblicos, não canônicos e patrísticos; Por este motivo, iremos analisar aquele trecho e realçar um indício interno que, devido ao seu possível conteúdo tipológico e bíblico, pode con- firmar a função não só gemátrica como simbólica do número 666. d) a comparação com as demais listas existentes na Bíblia e na literatura judaica antiga; e) uma seleção de listas que apresentem as sequências que compõem Ap 7,4-8. 2 A análise das duas interpretações Outros significados extensivos do significado de limite máximo surgem ao longo da Bíblia: a) imponência, opulência e grandiosidade: na medida do comprimento do templo de Salomão (60 côvados: 1Rs 6,2), na quantida- de de sua riqueza (666 talentos e 600 siclos de ouro: 1Rs 10,14-20.23) e nas medidas da estátua erigida por Nabucodonosor (60 côvados de altura e 6 de largura: Dn 3,1.7); b) numerosidade, abundância e força, nos números altos: um exemplo singelo é Ct 3,7 (60 guerreiros), um mediano é Jz 18,11 (600 homens; cf. 1Sm 13,15; 23,13) e um outro ex- tremo é Ex 12,37 (600.000 homens; cf. Nm 11,21 e 1Sm 13,5). Também encontramos o sentido de numerosidade para a quan- tidade de animais (Nm 7,88: 60 carneiros, 60 bodes e 60 cordeiros). Nos evangelhos, a fração de 60 por 1 indica abundância, grande produção (Mt 13,8.23; Mc 4,8.20); c) enorme força física guerreira que está sujeita à fácil derrota e ao ridículo: encon- tramos este sentido nos 600 melhores car- ros do Faraó, cujos guerreiros foram afoga- dos (Ex 14,6-7.27-28); na altura de Golias, de seis côvados e um palmo, e no peso da ponta de sua lança, de 600 siclos de ferro, o qual foi derrotado por Davi (1Sm 17,4.7); e nos seis dedos do gigante de Gat, morto por um parente de Davi (2Sm 21,20-21); d) marcação de um momento crucial, final e dramático: é a data escatológica do dilúvio nos 600 anos de Noé (Gn 7,6.11); e as trevas da sexta hora na agonia de Jesus (Mt 27,45; Mc 15,33; Lc 23,44; Jo 19,14). Por outro lado, apesar das dificuldades e en- ganos, ambas as interpretações são admissíveis, considerando-se alguns pontos. A primeira interpretação harmoniza-se com o contexto histórico da época de João, no qual a gematria era largamente usada entre os gregos, judeus e romanos, havendo vários testemunhos literários dentro do gênero apocalíptico. De fato Pedro Paulo Abreu Funari • Adylson Valdez A interpretação simbólica de 666 face a Ap 7,4-8 e aos Doze Apóstolos Pedro Paulo Abreu Funari • Adylson Valdez Devido a estes pontos, a conjugação parece ser consequente, pois o texto de João permite a aplicação de ambas as interpretações ao mesmo tempo. 2 A análise das duas interpretações Do ponto de vista dos grupos de filhos de cada esposa de Jacó, podemos notar que Lia se faz representar no início das primeira e segunda me- tades da lista, havendo uma separação entre os grupos Judá-Rubem e Simeão-Levi-Issacar-Za- bulon — que são tribos oriundas dos seus filhos legítimos — mediante o grupo Gad-Aser, gerado da sua escrava Zelfa, e o grupo Neftali-Manassés, que pertence a Raquel. Esta sucede a Lia com os grupos subsequentes em cada metade: Neftali, tribo que provém de sua escrava Bala; Manassés, advinda do primogênito de José (portanto, neto de Raquel); e José-Benjamin, grupo derivado dos seus filhos legítimos. Assim, no que concerne às mães, é escolhida uma ordem alternada Lia- -Raquel-Lia-Raquel, começando sempre pela primeira esposa de Jacó, sendo que, no caso 3 O simbolismo do número seis em Ap 7,4-8 3.1 Estudo analítico da formação de Ap 7,4-8 e de seus objetivos símbolo-teológicos 3.1 Estudo analítico da formação de Ap 7,4-8 e de seus objetivos símbolo-teológicos O presente estudo terá como objetivo de- monstrar que o autor do Apocalipse tinha em mente um significado simbólico de apostasia e antagonismo ao Messias para o número seis, considerando-se que este mesmo significado é encontrado ocultamente no nome do sexto lugar da lista de Ap 7,4-8, e que esta foi manipulada por João para que houvesse a aplicação deste simbolismo sobre aquela posição. Este simbolis- mo teria sido aplicado por causa da influência do próprio número 666, devido à sua relação com o Anticristo. Então, tendo esta premissa em mente, vamos proceder à análise. 8/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 8/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 28,1.3; Jz 8,1-3; 12,1-7; 2Cr 25,10; 30,10). Na litera- tura não canônica e patrística também encontra- mos uma conotação negativa com relação a Dã. Para a nossa pesquisa, é importante já verificar que Manassés surge em sexto lugar, uma posição talvez indicadora da existência de um princípio simbólico na formação da lista, no sentido de assinalar a eliminação dos cristãos apóstatas do meio do Israel espiritual (2Cor 11,3; 2Ts 2,11-12), mediante a substituição de Dã por Manassés. A comparação da lista de Ap 7,4-8 com as de- mais listas existentes na Bíblia e na literatu- ra judaica antiga pode nos auxiliar na desco- berta dos princípios formadores da lista de João, considerando-se preliminarmente que nenhuma outra lista é igual à de Ap 7,4-8. As demais listas são as seguintes, enumeradas por grupos de tipos de listas similares, sendo que os nomes das mães estão abreviados pelas iniciais: desta, a sua escrava surge posteriormente a ela (Gad-Aser após Judá-Rubem), e, no caso de Raquel, a escrava respectiva surge anteriormente (Neftali antes de Manassés e José-Benjamin). A análise do ponto de vista da proximidade geográfica nos ajuda a perceber que o posiciona- mento dos grupos ocorre por saltos distanciados desconexos: sul, este, norte, centro, sul, norte e sul; dando a entender que a lista não tem um princípio geográfico na sua formação. Mas é importante notar o isolamento de Manassés no centro, que parece indicar uma posição especial. 3 O simbolismo do número seis em Ap 7,4-8 3.1 Estudo analítico da formação de Ap 7,4-8 e de seus objetivos símbolo-teológicos Com relação ao conteúdo histórico, político e teológico das tribos, temos a constatação da preocupação de João em colocar a tribo de Judá em primeiro lugar, ao invés da tribo de Rubem (que se originou do primogênito de Lia: Gn 49,3-4), notadamente não só por causa de sua importân- cia política e religiosa, já que dela originou-se o rei Davi, ou por aparecer em primeiro lugar em algumas listas do Antigo Testamento (Nm 2,3-31; 7,12-83; 10,14-27; 34,19-28; Js 21,4-7; 21,9-40; Jz 1; 1Cr 4-7; 6,39-48; 6,49-66; 12,24-38), mas também porque representa Jesus Cristo, o leão da tribo de Judá (Ap 5,5; Gn 49,9), cabeça da Igreja (Cl 1,15-18; Ef 1,22-23). Outra constatação é a eliminação da tribo de Dã, a qual deveria estar presente para formar um grupo com Neftali, pois originou-se de Dã, filho de Bala, escrava de Raquel. A sua substituição pela tribo de Manassés é ilógica, uma vez que José, tribo do pai de Manassés, e Levi aparecem na oitava e na décima primei- ra posição (José e Levi desaparecem das listas quando os dois filhos de José são colocados). 1) Gn 29,31-35; 30,1-24; 35,16-18 (tradições E-JP do Pentateuco; sécs. VIII, IX-V a.C.): Rubem-Simeão-Levi-Judá (L), Dã-Neftali (R-B), Gad-Aser (L-Z), Issacar-Zabulon (L), José-Benjamin (R). Similares: Demétrio, o cronografista, fragmento 2,3-5.8.10.17-18 (três listas; III a.C.); Jubileus 28,11-24; 32,3 (II a.C.); F. Josefo, Antiguidades Judaicas 1.19.8; 1.21.3 (93 d.C.). 1) Gn 29,31-35; 30,1-24; 35,16-18 (tradições E-JP do Pentateuco; sécs. VIII, IX-V a.C.): Rubem-Simeão-Levi-Judá (L), Dã-Neftali (R-B), Gad-Aser (L-Z), Issacar-Zabulon (L), José-Benjamin (R). Similares: Demétrio, o cronografista, fragmento 2,3-5.8.10.17-18 (três listas; III a.C.); Jubileus 28,11-24; 32,3 (II a.C.); F. Josefo, Antiguidades Judaicas 1.19.8; 1.21.3 (93 d.C.). 2) Gn 35,23-26 (P Pent.; V a.C.): Rubem- Simeão-Levi-Judá-Issacar-Zabulon (L), José-Benjamin (R), Dã-Neftali (R-B), Gad- Aser (L-Z). Similares: Jubileus 33,22 (II a.C.); Pseudo-Fílon 8,6 (71 d.C.); F. Josefo, Ant. 2.7.4 (93 d.C.). Uma observação interessante é a preferência por Manassés em detrimento de Efraim. 3 O simbolismo do número seis em Ap 7,4-8 3.1 Estudo analítico da formação de Ap 7,4-8 e de seus objetivos símbolo-teológicos Os motivos da supressão, da substituição e da escolha pa- recem estar vinculados: à representação de Dã pela figura da serpente, que remete à imagem do Dragão (Ap 12,9; Gn 49,17; 3,1.13; Is 27,1; 2Cor 11,3; Sb 2,24); ao fato de que Manassés foi oca- sionalmente a favor de Davi e de Deus (1Cr 12,19- 21.31.37-38; 2Cr 30,1.10-11.18; 31,1); e ao histórico de idolatria, traição e apostasia atribuído a Dã e a Efraim (Jz 17,3-6; 18,15-17.30-31; Am 8,14; 1Rs 12,25-30; Jz 5,17; Jr 8,16; Os 4,17; 7,11; 8,9; 12,1-2; Is 3) Gn 46,9-27 (P Pent.; V a.C.): Rubem- Simeão-Levi-Judá-Issacar-Zabulon (L), Gad-Aser (L-Z), José-Benjamin (R), Dã- Neftali (R-B). Similar: Jubileus 44,11-30 (II a.C.). 4) Gn 49,3-27 (J-P Pent.; IX-V a.C.): Rubem- Simeão-Levi-Judá-Zabulon-Issacar (L), Dã (R-B), Gad-Aser (L-Z), Neftali (R-B), José- Benjamin (R). 5) Ex 1,1-5 (P Pent.; V a.C.): Rubem-Simeão- Levi-Judá-Issacar-Zabulon (L), Benjamin (R), Dã-Neftali (R-B), Gad-Aser (L-Z). Pedro Paulo Abreu Funari • Adylson Valdez Pedro Paulo Abreu Funari • Adylson Valdez A interpretação simbólica de 666 face a Ap 7,4-8 e aos Doze Apóstolos 9/17 6) Nm 1,5-15 (P Pent.; V a.C.): Rubem- Simeão-Judá-Issacar-Zabulon (L), Efraim- Manassés (R-J), Benjamin (R), Dã (R-B), Aser-Gad (L-Z), Neftali (R-B). 6) Nm 1,5-15 (P Pent.; V a.C.): Rubem- Simeão-Judá-Issacar-Zabulon (L), Efraim- Manassés (R-J), Benjamin (R), Dã (R-B), Aser-Gad (L-Z), Neftali (R-B). 17) 1Cr 2,1-2 (IV a.C.): Rubem-Simeão-Levi- Judá-Issacar-Zabulon (L), Dã (R-B), José- Benjamin (R), Neftali (R-B), Gad-Aser (L-Z). 18) 1Cr 4-7 (IV a.C.): Judá-Simeão-Rubem (L), Gad (L-Z), Manassés (R-J; meia-tribo), Levi-Issacar (L), Benjamin (R), Neftali (R-B), Manassés-Efraim (R-J), Aser (L-Z). 17) 1Cr 2,1-2 (IV a.C.): Rubem-Simeão-Levi- Judá-Issacar-Zabulon (L), Dã (R-B), José- Benjamin (R), Neftali (R-B), Gad-Aser (L-Z). 18) 1Cr 4-7 (IV a.C.): Judá-Simeão-Rubem (L), Gad (L-Z), Manassés (R-J; meia-tribo), Levi-Issacar (L), Benjamin (R), Neftali (R-B), Manassés-Efraim (R-J), Aser (L-Z). 18) 1Cr 4-7 (IV a.C.): Judá-Simeão-Rubem (L), Gad (L-Z), Manassés (R-J; meia-tribo), Levi-Issacar (L), Benjamin (R), Neftali (R-B), Manassés-Efraim (R-J), Aser (L-Z). 18) 1Cr 4-7 (IV a.C.): Judá-Simeão-Rubem (L), Gad (L-Z), Manassés (R-J; meia-tribo), Levi-Issacar (L), Benjamin (R), Neftali (R-B), Manassés-Efraim (R-J), Aser (L-Z). 7) Nm 1,20-43 (P Pent.; V a.C.): Rubem- Simeão (L), Gad (L-Z), Judá-Issacar- Zabulon (L), Efraim-Manassés (R-J), Benjamin (R), Dã (R-B), Aser (L-Z), Neftali (R-B). Similar com a inversão Manassés- Efraim: Nm 26,5-50. 19) 1Cr 6,39-48 (IV a.C.): Judá (L), Benjamin (R), Manassés (R-J; meia-tribo), Issacar (L), Aser (L-Z), Neftali (R-B), Manassés (R-J; meia-tribo), Rubem (L), Gad (L-Z), Zabulon (L). 3 O simbolismo do número seis em Ap 7,4-8 3.1 Estudo analítico da formação de Ap 7,4-8 e de seus objetivos símbolo-teológicos 8) Nm 2,3-31 (P Pent.; V a.C.): Judá-Issacar- Zabulon-Rubem-Simeão (L), Gad (L-Z), Efraim-Manassés (R-J), Benjamin (R), Dã (R-B), Aser (L-Z), Neftali (R-B). Similares: Nm 7,12-83; Nm 10,14-27. 20) 1Cr 6,49-66 (IV a.C.): Judá-Simeão (L), Benjamin (R), Efraim-Manassés (R-J; Manassés: duas meia-tribos), Issacar (L), Aser (L-Z), Neftali (R-B), Zabulon-Rubem (L), Gad (L-Z). 20) 1Cr 6,49-66 (IV a.C.): Judá-Simeão (L), Benjamin (R), Efraim-Manassés (R-J; Manassés: duas meia-tribos), Issacar (L), Aser (L-Z), Neftali (R-B), Zabulon-Rubem (L), Gad (L-Z). 9) Nm 13,4-15 (P Pent.; V a.C.): Rubem- Simeão-Judá-Issacar (L), Efraim (R-J), Benjamin (R), Zabulon (L), Manassés (R-J), Dã (R-B), Aser (L-Z), Neftali (R-B), Gad (L-Z). 9) Nm 13,4-15 (P Pent.; V a.C.): Rubem- Simeão-Judá-Issacar (L), Efraim (R-J), Benjamin (R), Zabulon (L), Manassés (R-J), Dã (R-B), Aser (L-Z), Neftali (R-B), Gad (L-Z). 21) 1Cr 12,24-38 (IV a.C.): Judá-Simeão-Levi (L), Benjamin (R), Efraim-Manassés (R-J; Manassés: meia-tribo), Issacar-Zabulon (L), Neftali-Dã (R-B), Aser (L-Z), Rubem (L), Gad (L-Z), Manassés (R-J; meia-tribo). 10) Nm 34,19-28 (P Pent.; V a.C.): Judá- Simeão (L), Benjamin (R), Dã (R-B), Manassés-Efraim (R-J), Zabulon-Issacar (L), Aser (L-Z), Neftali (R-B). 22) 1Cr 27,16-22 (IV a.C.): Rubem-Simeão- Levi-Judá-Issacar-Zabulon (L), Neftali (R- B), Efraim-Manassés (R-J; Manassés: duas meia-tribos), Benjamin (R), Dã (R-B). 22) 1Cr 27,16-22 (IV a.C.): Rubem-Simeão- Levi-Judá-Issacar-Zabulon (L), Neftali (R- B), Efraim-Manassés (R-J; Manassés: duas meia-tribos), Benjamin (R), Dã (R-B). 11) Dt 27,12-13 (D; VII a.C.): Simeão-Levi- Judá-Issacar (L), José-Benjamin (R), Rubem (L), Gad-Aser (L-Z), Zabulon (L), Dã-Neftali (R-B). 23) Ez 48,1-29 (VI a.C.): Dã (R-B), Aser (L- Z), Neftali (R-B), Manassés-Efraim (R-J), Rubem-Judá (L), Benjamin (R), Simeão- Issacar-Zabulon (L), Gad (L-Z). 12) Dt 33,6-24 (E Pent.; VIII a.C.): Rubem- Judá-Levi (L), Benjamin-José (R), Zabulon- Issacar (L), Gad (L-Z), Dã-Neftali (R-B), Aser (L-Z). 12) Dt 33,6-24 (E Pent.; VIII a.C.): Rubem- Judá-Levi (L), Benjamin-José (R), Zabulon- Issacar (L), Gad (L-Z), Dã-Neftali (R-B), Aser (L-Z). 24) Ez 48,30-35 (VI a.C.): Rubem-Judá-Levi (L), José-Benjamin (R), Dã (R-B), Simeão- Issacar-Zabulon (L), Gad-Aser (L-Z), Neftali (R-B). 13) Js 13-19 (P de Js; V a.C.): Rubem (L), Gad (L-Z), Manassés (R-J; meia-tribo), Judá (L), Efraim-Manassés (R-J), Benjamin (R), Simeão-Zabulon-Issacar (L), Aser (L-Z), Neftali-Dã (R-B). 25) Testamentos dos Doze Patriarcas (II a.C.-I d.C.): Rubem-Simeão-Levi-Judá- Issacar-Zabulon (L), Dã-Neftali (R-B), Gad- Aser (L-Z), José-Benjamin (R). Similares: Jubileus 34,20 (II a.C.); Pseudo-Fílon 26,10- 11 (71 a.C.). 25) Testamentos dos Doze Patriarcas (II a.C.-I d.C.): Rubem-Simeão-Levi-Judá- Issacar-Zabulon (L), Dã-Neftali (R-B), Gad- Aser (L-Z), José-Benjamin (R). Similares: Jubileus 34,20 (II a.C.); Pseudo-Fílon 26,10- 11 (71 a.C.). 3 O simbolismo do número seis em Ap 7,4-8 3.1 Estudo analítico da formação de Ap 7,4-8 e de seus objetivos símbolo-teológicos 14) Js 21,4-7 (P de Js; V a.C.): Judá-Simeão (L), Benjamin (R), Efraim (R-J), Dã (R-B), Manassés (R-J; meia-tribo), Issacar (L), Aser (L-Z), Neftali (R-B), Manassés (R-J; meia- -tribo), Rubem (L), Gad (L-Z), Zabulon (L). 15) Js 21,9-40 (P de Js; V a.C.): Judá-Simeão (L), Benjamin (R), Efraim (R-J), Dã (R-B), Manassés (R-J; duas meia-tribos), Issacar (L), Aser (L-Z), Neftali (R-B), Zabulon- Rubem (L), Gad (L-Z). 14) Js 21,4-7 (P de Js; V a.C.): Judá-Simeão (L), Benjamin (R), Efraim (R-J), Dã (R-B), Manassés (R-J; meia-tribo), Issacar (L), Aser (L-Z), Neftali (R-B), Manassés (R-J; meia- -tribo), Rubem (L), Gad (L-Z), Zabulon (L). 26) Testamento de Judá 25,1-2 (II a.C.): Levi- Judá-Simeão-Rubem-Issacar-Zabulon (L), José-Benjamin (R), Dã-Neftali (R-B), Gad-Aser (L-Z). 15) Js 21,9-40 (P de Js; V a.C.): Judá-Simeão (L), Benjamin (R), Efraim (R-J), Dã (R-B), Manassés (R-J; duas meia-tribos), Issacar (L), Aser (L-Z), Neftali (R-B), Zabulon- Rubem (L), Gad (L-Z). 27) Jubileus 38,5-8 (II a.C.): Judá (L), Neftali (R-B), Gad (L-Z), Levi (L), Dã (R-B), Rubem- Issacar-Zabulon-Simeão (L), Benjamin (R), Henoc (filho de Rubem). 16) Jz 1 (VIII-V a.C.): Judá-Simeão (L), Benjamin (R), Manassés-Efraim (R-J), Zabulon (L), Aser (L-Z), Neftali-Dã (R-B). 10/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 7 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 28) 11QTemple, col. 24.10-16 (II a.C.): Judá (L), Benjamin (R), Efraim-Manassés (R- J), Rubem-Simeão-Issacar-Zabulon (L), Gad-Aser (Z). mas se especializa pela ordenação das tribos das escravas em ordem respectiva (Lia-Zelfa, Raquel-Bala, ao invés de Raquel-Bala, Lia-Zelfa) e pelo posicionamento delas na primeira metade. Isto permite a suspeita de que nela também há um motivo particular. 29) 11QTemple, col. 39.12-13 (II a.C.): Simeão-Levi-Judá-Rubem (L), José- Benjamin (R), Issacar-Zabulon (L), Gad (L-Z), Dã-Neftali (R-B), Aser (L-Z). 29) 11QTemple, col. 39.12-13 (II a.C.): Simeão-Levi-Judá-Rubem (L), José- Benjamin (R), Issacar-Zabulon (L), Gad (L-Z), Dã-Neftali (R-B), Aser (L-Z). A comparação estatística determina que os tipos se contrapõem a Ap 7,4-8 nos seguintes detalhes: 30) 11QTemple, col. 39.14-16; 40.15; 41 (II a.C.): Simeão-Levi-Judá-Issacar-Zabulon (L), Gad (L-Z), Dã-Neftali (R-B), Aser (L-Z). 30) 11QTemple, col. 39.14-16; 40.15; 41 (II a.C.): Simeão-Levi-Judá-Issacar-Zabulon (L), Gad (L-Z), Dã-Neftali (R-B), Aser (L-Z). a) a tribo de Judá aparece no início junto e antes da tribo de Rubem apenas em uma lista (Pseudo-Fílon 25,4); 31) 11QTemple, col. 44 (II a.C.): Simeão- Judá-Levi-Rubem (L), Efraim-Manassés (R-J), Benjamin (R), Issacar (L). 3 O simbolismo do número seis em Ap 7,4-8 3.1 Estudo analítico da formação de Ap 7,4-8 e de seus objetivos símbolo-teológicos b) Gad, Aser, Dã e Neftali aparecem muito mais na segunda metade, conforme as seguintes frequências: Gad vinte e seis vezes, Aser trinta vezes, Dã vinte e quatro vezes e Neftali trinta vezes; na primeira metade prevalecem aparições isoladas: Gad cinco vezes, Aser uma vez, Dã quatro vezes e Neftali uma vez, contra os grupos Dã-Neftali (duas vezes), Gad-Aser (uma vez), Dã-Aser-Neftali (uma vez) e Neftali- Gad-Dã (uma vez); 32) 4Q554, frag. 1, cols. 1-2 (I d.C.): Simeão- Levi-Judá (L), José-Benjamin (R), Rubem- Issacar-Zabulon (L), Gad (L-Z), Dã (R-B), Aser (L-Z), Neftali (R-B). 33) Fílon, De Somniis 2.5.34-40 (30 d.C.): Rubem-Simeão-Levi-Judá-Issacar- Zabulon (L), Dã (R-B), Gad-Aser (L-Z), Neftali (R-B), Benjamin (R). 33) Fílon, De Somniis 2.5.34-40 (30 d.C.): Rubem-Simeão-Levi-Judá-Issacar- Zabulon (L), Dã (R-B), Gad-Aser (L-Z), Neftali (R-B), Benjamin (R). 34) Pseudo-Fílon 8,11-14 (71 d.C.): Rubem- Simeão-Levi-Judá-Issacar-Zabulon (L), Dã-Neftali (R-B), Gad (L-Z), Efraim- Manassés (R-J), Benjamin (R). c) a ordem Dã-Neftali surge na grande maioria das listas, ao invés de Neftali-Dã (esta somente aparece em três tipos de listas: Js 13-19; Jz 1; 1Cr 12,24-38); 35) Pseudo-Fílon 10,3 (71 d.C.): Rubem- Issacar-Zabulon-Simeão (L), Gad-Aser (L-Z), Dã-Neftali (R-B), Levi-Judá (L), José- Benjamin (R). d) o mais frequente é a colocação de Gad-Aser após Dã-Neftali (oito tipos contra três; além disso, os dois grupos aparecem intercalados entre si em sete tipos, e também ocorre a alternância das tribos em outros sete tipos); 36) Pseudo-Fílon 25,4 (71 d.C.): Judá- Rubem-Simeão-Levi-Issacar-Zabulon (L), Gad-Aser (L-Z), Manassés-Efraim (R-J), Benjamin (R). e) Dã vem antes de Neftali e Gad-Aser em dezessete tipos de listas; 37) Pseudo-Fílon 25,9-13 (71 d.C.): Rubem- Levi-Issacar-Zabulon (L), Dã-Neftali (R-B), Gad-Aser (L-Z), Manassés-Efraim (R-J), Benjamin (R). 37) Pseudo-Fílon 25,9-13 (71 d.C.): Rubem- Levi-Issacar-Zabulon (L), Dã-Neftali (R-B), Gad-Aser (L-Z), Manassés-Efraim (R-J), Benjamin (R). f) Dã aparece apenas uma vez no sexto lugar (Ez 48,30-35); f) Dã aparece apenas uma vez no sexto lugar (Ez 48,30-35); g) com relação à tribo de Manassés, deve-se dizer que das vinte vezes em que ela surge, por dezesseis vezes ela se apresenta junto da de Efraim, sendo que em doze vezes, contra oito, ela está na segunda metade; quando ambas as tribos aparecem, a de José fica ausente; e a de Manassés sempre substitui a de Levi (que acaba sendo suprimida de algumas Os modelos básicos das listas são os tipos 1, 2 e 3, os quais sofrem modificações devido a mo- tivos territoriais, geográficos, políticos, militares, teológicos e redacionais. Esta observação denota a importância do livro de Gênesis para o fator de influência. A lista de Ap 7,4-8 também obedece aos princípios daqueles modelos, como o de co- locar em primeiro lugar Lia e em segundo Raquel; Pedro Paulo Abreu Funari • Adylson Valdez A interpretação simbólica de 666 face a Ap 7,4-8 e aos Doze Apóstolos 11/17 Pedro Paulo Abreu Funari • Adylson Valdez A interpretação simbólica de 666 face a Ap 7,4-8 e aos Doze Apóstolos 11/17 listas porque não possuía território) ou naturalmente a de José, nunca a de Dã; listas porque não possuía território) ou naturalmente a de José, nunca a de Dã; h) as demais tribos surgem frequente- mente na primeira metade, praticamente em trinta tipos de listas, embora às vezes parcialmente. sés, a qual aparece nas demais listas somente quando a de Levi não aparece). O deslocamento de Judá para antes de Rubem, provocado por motivos teológicos, forma a sequência de Ap 7,4-8, ocorrendo, no entanto, a sua fragmenta- ção com o núcleo Gad-Aser-Neftali-Manassés, posto entre Rubem e Simeão. Este núcleo não existe em nenhuma lista, a não ser de maneira fragmentária em Gad-Aser, Gad-Aser-Neftali e Aser-Neftali-Manassés ou Aser-Neftali-Dã. h) as demais tribos surgem frequente- mente na primeira metade, praticamente em trinta tipos de listas, embora às vezes parcialmente. Estes detalhes acabam demonstrando que João não segue um pensamento lógico perante os princípios internos das demais listas, o que torna Ap 7,4-8 bastante peculiar. A ocorrência de Gad-Aser e Aser-Neftali-Manas- sés na primeira metade somente se dá em duas listas, enquanto Gad-Aser-Neftali e a alternativa Aser-Neftali-Dã sempre surgem na segunda me- tade, o que denota a falta de lógica de Ap 7,4-8 perante a maioria das listas. f) Dã aparece apenas uma vez no sexto lugar (Ez 48,30-35); A possibilidade esta- tística de se colocar Manassés após Neftali é pra- ticamente nula, pois a sequência acontece apenas em Ez 48,1-29. A colocação de Manassés no sexto lugar nas demais listas acima citadas não devem ser consideradas, porque se trata de um isolamento particular, independente das sequências. Por fim, devemos também estudar Ap 7,4-8 do ponto de vista das semelhanças nas sequências com outras listas, para descobrirmos se João tende para alguma lista em particular. Selecio- nemos então estas listas: a) Judá-Rubem no início: Pseudo-Fílon 25,4; invertida Rubem-Judá em Dt 33,6- 24 e Ez 48,30-35; b) Gad-Aser na primeira metade: somente em Pseudo-Fílon 10,3; c) Gad-Aser-Neftali: ocorre somente na segunda metade: Gn 49,3-27; De Somniis 2.5.34-40; Nm 1,5-15 (com inversão Aser- Gad-Neftali); e Ez 48,30-35; O que se pode deduzir da sequência Gad-A- ser-Neftali-Manassés é que a sua própria frag- mentação indica que João não levaria em conta o padrão de nenhuma das listas coincidentes a ela, porque seria um trabalho muito complexo de montagem, na busca de listas esparsas. Assim, a sequência é peculiar somente de Ap 7,4-8. d) Aser-Neftali-Manassés na primeira metade: somente em Ez 48,1-29; a alter- nativa Aser-Neftali-Dã: somente aparece na segunda metade em Js 13-19 e Jz 1; e) Manassés no sexto lugar: Js 13-19 (meia- -tribo); 21,4-7; 21,9-40 (meia-tribo); 1Cr 12,24-38 (meia-tribo); e 11QTemple, col. 44; Poderia ser que o início de Ap 7,4-8 tivesse uma inspiração geográfica com a sequência Rubem-Gad, como vemos em Js 13-19, dando motivação para se colocar os grupos Gad-Aser e Neftali-Manassés no início. Mas, como já vimos, uma inspiração geográfica deve ser descartada. f) Simeão-Levi-Issacar-Zabulon: ocorre apenas na primeira metade: Gn 46,9-27 (Judá entre Levi e Issacar); f) Simeão-Levi-Issacar-Zabulon: ocorre apenas na primeira metade: Gn 46,9-27 (Judá entre Levi e Issacar); g) Simeão-Levi-Issacar-Zabulon-José- Benjamin: somente aparece na primeira metade: Gn 35,23-26 (Judá entre Levi e Issacar). g) Simeão-Levi-Issacar-Zabulon-José- Benjamin: somente aparece na primeira metade: Gn 35,23-26 (Judá entre Levi e Issacar). Disto tudo podemos concluir que Ap 7,4-8 tende para Gn 35,23-26. No entanto, Gn 46,9-27 transparece de forma subsidiária, devido à adoção da ordem respectiva Lia-Zelfa, Raquel-Bala. Desta maneira, ao utilizar-se destas listas modelares, a montagem tornava-se mais simples e acessível para João no processo de busca e ordenação. 3.2 Objeções ao simbolismo de Ap 7,4-8 Mas esta possibilidade se desfaz perante os seguintes fatores fortemente determinantes: a preferência por colocar os grupos das escravas na primeira metade da lista, causando a fragmenta- ção ilógica do grupo de Lia; a escolha da ordem respectiva Lia-Zelfa, Raquel-Bala, propiciando a colocação da sequência Neftali-Manassés nos quinto e sexto lugares; o significado negativo que 666 e a figura da serpente atribuem res- pectivamente ao número seis e à tribo de Dã; e a própria substituição de Dã por Manassés, que cria uma relação do número seis com a apostasia e o Anticristo. Ademais, caso a sua intenção não fosse marcar o sexto lugar, João poderia ter po- sicionado as tribos das escravas no final da lista, deixando Manassés no décimo segundo lugar, ou, em qualquer outro lugar; e, também, não se pode dizer que a ordenação de Ap 7,4-8 seja aleatória, considerando-se que a inspiração geral do autor pelo Antigo Testamento permite deduzir a exis- tência de uma lista prévia veterotestamentária. A segunda objeção é a de que não poderia haver intenção simbólica numérica, tendo em vista que, se assim ocorresse, todos os números da lista teriam de ganhar significados conforme a tipo- logia de cada tribo. É visível que as outras tribos não atribuem um significado específico para os números respectivos. Mas observe-se que em todo o texto do Apocalipse a preocupação do autor é a de realçar principalmente duas figuras: a do leão de Judá e a da Besta. Desta forma, de seu ponto de vista simbólico, as demais tribos não eram importantes. Os deslocamentos da tribo de Judá e dos grupos das escravas indicam quais eram as tribos especiais que ele precisava assinalar, levando-se em conta os princípios determinantes de Ap 5,1-5; 2,8; e 13,18.i A primeira objeção é a de que Manassés aca- bou ficando em sexto lugar por mera consequên- cia do desejo de João de dispor as três tribos das escravas uma perto da outra (Gad-Aser-Neftali). 12/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 12/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 3.2 Objeções ao simbolismo de Ap 7,4-8 de um modo tal, que permitisse o surgimento de Manassés no sexto lugar, em substituição de Dã. Se realmente foi assim, fica confirmado que em Ap 7,4-8 há princípios teológicos na sua formação, assinalados por dois deslocamentos: o de Judá e o dos grupos das escravas; e, notadamente, a semelhança de Ap 7,4-8 com as duas listas citadas demonstra que a tribo de Manassés é uma ver- dadeira intrusa e nunca deveria estar ali presente: sua colocação foi uma decisão deliberada de João. A constatação do deslocamento ilógico dos grupos Gad-Aser e Dã-Neftali para a primeira metade da lista indica que João desejava dispor os grupos das escravas em conjunto. Isto teria acontecido porque ficaria sem sentido deslocar a tribo de Dã sem des- locar os grupos das escravas, aos quais ela pertence por uma questão de ordenação óbvia (como vemos em Gn 35,23-26). Assim, devido a esta observação e à preferência pela inclusão de Levi e José, podemos perceber que João não pretendia substituir nenhuma outra tribo a não ser a de Dã. Naturalmente, quando João dispôs os grupos das escravas entre Rubem e Simeão, ele não quis colocar Manassés antes de Nef- tali, que é a posição original de Dã em Gn 46,9-27, e a preferida na maioria das listas; mas sim após Neftali, na sexta posição, ferindo completamente a lógica. A intenção desta modificação seria de rememorar a Besta pelo número seis, sem dúvida por causa de Ap 13,18, e indicar os seus seguidores, os apóstatas, que foram eliminados do meio do Israel espiritual. Esta eliminação se fez pela substituição simbólica de Dã — a figura do apóstata — por Manassés. P i li t t i i l ã t Para a teoria apresentada pode-se opor três objeções. A primeira objeção é a de que Manassés aca- bou ficando em sexto lugar por mera consequên- cia do desejo de João de dispor as três tribos das escravas uma perto da outra (Gad-Aser-Neftali). 3.2 Objeções ao simbolismo de Ap 7,4-8 Mas esta possibilidade se desfaz perante os seguintes fatores fortemente determinantes: a preferência por colocar os grupos das escravas na primeira metade da lista, causando a fragmenta- ção ilógica do grupo de Lia; a escolha da ordem respectiva Lia-Zelfa, Raquel-Bala, propiciando a colocação da sequência Neftali-Manassés nos quinto e sexto lugares; o significado negativo que 666 e a figura da serpente atribuem res- pectivamente ao número seis e à tribo de Dã; e a própria substituição de Dã por Manassés, que cria uma relação do número seis com a apostasia e o Anticristo. Ademais, caso a sua intenção não fosse marcar o sexto lugar, João poderia ter po- sicionado as tribos das escravas no final da lista, deixando Manassés no décimo segundo lugar, ou, em qualquer outro lugar; e, também, não se pode dizer que a ordenação de Ap 7,4-8 seja aleatória, considerando-se que a inspiração geral do autor pelo Antigo Testamento permite deduzir a exis- tência de uma lista prévia veterotestamentária. A segunda objeção é a de que não poderia haver intenção simbólica numérica, tendo em vista que, se assim ocorresse, todos os números da lista teriam de ganhar significados conforme a tipo- logia de cada tribo. É visível que as outras tribos não atribuem um significado específico para os números respectivos. Mas observe-se que em todo o texto do Apocalipse a preocupação do autor é a de realçar principalmente duas figuras: a do leão de Judá e a da Besta. Desta forma, de seu ponto de vista simbólico, as demais tribos não eram importantes. Os deslocamentos da tribo de Judá e dos grupos das escravas indicam quais eram as tribos especiais que ele precisava assinalar, levando-se em conta os princípios determinantes de Ap 5,1-5; 2,8; e 13,18. Por fim, a terceira objeção é a de que a intenção simbólica de Ap 7,6 não poderia existir porque não Para visualizar esta teoria e a manipulação entre as listas de Gn 35,23-26 e Ap 7,4-8, veja o Gráfico 1 abaixo. Gráfico 1 – Deslocamento das tribos Fonte: Elaborado por Adylson Valdez (2009). Gráfico 1 – Deslocamento das tribos f) Dã aparece apenas uma vez no sexto lugar (Ez 48,30-35); Aparentemente, a modificação que ele fez, por meio da coloca- ção dos grupos das escravas entre Rubem e Si- meão, deve ter tido o objetivo de conformar a lista A sequência da linha g acima demonstra que Gn 35,23-26 serve como lista básica de formação, pois contém originalmente a mesma sequência, mas com Rubem antes e Judá entre Levi e Issa- car (perceba-se que João inclui a tribo de Levi em sua lista, o que confirma sua tendência para Gn 35,23-26, mesmo que coloque a de Manas- 12/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 Gráfico 1 – Deslocamento das tribos Por fim, a terceira objeção é a de que a intenção simbólica de Ap 7,6 não poderia existir porque não Fonte: Elaborado por Adylson Valdez (2009). Pedro Paulo Abreu Funari • Adylson Valdez A interpretação simbólica de 666 face a Ap 7,4-8 e aos Doze Apóstolos 13/17 Pedro Paulo Abreu Funari • Adylson Valdez A interpretação simbólica de 666 face a Ap 7,4-8 e aos Doze Apóstolos 13/17 irmãos juntos, sendo que André surge em segundo lugar após Pedro; d) Lucas muda o nome de Tadeu para Judas, irmão de Tiago, filho de Alfeu. irmãos juntos, sendo que André surge em segundo lugar após Pedro; d) Lucas muda o nome de Tadeu para Judas, irmão de Tiago, filho de Alfeu. irmãos juntos, sendo que André surge em segundo lugar após Pedro; é imediatamente perceptível para o leitor. Porém, deve-se lembrar que a característica principal do gênero apocalíptico é a de dificultar o entendi- mento de sua mensagem por meio de códigos que não podem ser compreendidos de imediato. Muitos trechos do Apocalipse até hoje não têm explicação, e, em todo o seu texto há coisas im- perceptíveis, como, por exemplo, a existência de sete bem-aventuranças espalhadas através dele (Ap 1,3; 14,13; 16,15; 19,9; 20,6; 22,7.14). Além do mais, a própria passagem de Ap 13,18 não deixa dúvidas de que João não quis colocar tudo em evidência. O evangelho de João nomeia Natanael, que não está nas listas dos sinóticos, identificado pela tradição com Bartolomeu. Nos Atos, a relação de apóstolos é bem diversa das outras relações: João passa para o segundo lugar, entre Pedro e Tiago; Tomé para o sexto, en- tre Felipe e Bartolomeu; e Simão para o décimo, entre Judas (Tadeu) e Tiago. Nas listas dos evangelhos sinóticos, Judas Is- cariotes, o traidor, aparece sempre no décimo se- gundo lugar. Mais tarde, é substituído por Matias, o qual teve de concorrer com José Barsabás, o Justo (At 1,23-26). Já neste fato podemos perceber uma possível relação com a lista do Apocalipse: o autor substitui o contrário ao Messias — Dã, a serpente, ou Judas Iscariotes, que foi inspirado pelo Diabo — por um a seu favor: Manassés ou Matias. Assim como Manassés concorre com Efraim, Matias concorre com José Barsabás, o Justo. 4 Comparação de Ap 7,4-8 com as listas dos apóstolos Como João relaciona as tribos de Israel aos doze apóstolos de Jesus (Ap 21,12.14), pode haver a possibilidade de que ele, em Ap 7,4-8, tenha desejado sutilmente remeter à lista nominal dos apóstolos e à sua história. No Novo Testamento existem quatro listas dos apóstolos, encontradas em Mt 10,2-4; Mc 3,13-19; Lc 6,12-16; e At 1,13. O quarto evangelho nos dá apenas citações esparsas (Jo 1,35-51; 13,26; 14,5.8; 21,2). Este último cita nominalmente André, Pe- dro, Felipe, Natanael, Judas Iscariotes e Tomé, e possivelmente mais seis, não nomeados, ou seja, o discípulo que Jesus amava, os filhos de Zebe- deu e três incógnitos (Jo 1,40; 13,23; 19,26; 20,2; 21,2.7.20). Em Jo 14,22, o autor do quarto evangelho menciona outro Judas, que deve ser um dos três incógnitos, pois chama os discípulos de “os Doze”. Entre as listas dos três primeiros evangelhos, chamados sinóticos (porque podem ser lidos quase que ao mesmo tempo), há variações: Para tentarmos relacionar Ap 7,4-8 com a lista dos apóstolos, de maneira que Dã oculto este- ja paralelo a Judas Iscariotes, deveremos nos basear na lista de Gn 35,23-26, relacionando as tribos em grupos, começando pelas mulheres legítimas. Também, para se fazer a comparação, a lista dos apóstolos deve ser harmonizada, ou seja, ser montada conforme a verificação das posições mais estáveis entre as listas do Novo Testamento. Assim, desta forma encontraremos as disposições constantes do Gráfico 2: Gráfico 2 – Comparação entre as tribos e os após- tolos Gráfico 2 – Comparação entre as tribos e os após- tolos a) Mateus coloca o próprio Mateus em oitavo lugar, enquanto Lucas e Marcos no sétimo; a) Mateus coloca o próprio Mateus em oitavo lugar, enquanto Lucas e Marcos no sétimo; a) Mateus coloca o próprio Mateus em oitavo lugar, enquanto Lucas e Marcos no sétimo; Fonte: Elaborado por Adylson Valdez (2009). b) Lucas passa Simão para o décimo lugar, enquanto Mateus e Marcos o co- locam no décimo primeiro; c) Marcos coloca André em quarto lugar, intercalando Pedro e André (que é irmão de Pedro) com os irmãos Tiago e João, enquanto Mateus e Lucas colocam os Fonte: Elaborado por Adylson Valdez (2009). 14/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 14/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 14/17 Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 Nesta comparação entre as listas, vemos que João coloca Gad e Aser antes de Neftali, e Dã, ou Manassés, para depois de Neftali. Tendo em vista a lista dos apóstolos, parece que tais mo- dificações foram intencionais, para que Dã ou Manassés se direcionem para Judas Iscariotes ou Matias. Após estas mudanças, João teria desloca- do o grupo das escravas para depois de Rubem, deixando Dã ou Manassés, por consequência, Judas Iscariotes e Matias, para o sexto lugar, e, assim, simbolizá-los com o número 6. Procedendo-se à revisão das duas principais interpretações sobre o número 666, pôde-se averiguar que o significado simbólico desta cifra somente deve ser extraído do próprio texto do Apocalipse, muito embora possa ter havido uma influência tipológica sobre o autor advinda de passagens do Antigo Testamento, como as de 1Sm 17,4.7; e 2Sm 21,20-21. No entanto, não se pode negar a possibilida- de do emprego da gematria por parte de João, considerando-se que 666 representa obrigato- riamente um nome, sendo resultado evidente de uma operação matemática, apesar de o autor não revelar a sua equação. Gráfico 2 – Comparação entre as tribos e os após- tolos Há outras coincidências: Pedro, chefe dos doze apóstolos, está no primeiro lugar, como Judá, chefe das doze tribos; existem quatro filhos principais de Lia e quatro apóstolos principais; Tiago, filho de Alfeu, e Tadeu, como Gad e Aser, são irmãos; os apóstolos Tiago e João foram chamados “Boanerges” ou “filhos do trovão”, talvez por causa de suas naturezas impetuosas;5 enquanto Simeão e Levi tinham caráter violento em Gn 49,5-7. Estas coincidências confirmam um possível paralelismo entre as listas. Desta forma, parece ser conveniente a conju- gação de ambas as interpretações. O padrão de pensamento simbólico e tipo- lógico do autor sobre o número 666 pode ser confirmado pela análise da formação da lista das Doze Tribos de Israel, que se encontra em Ap 7,4-8. A sua disposição permite entrever uma manipulação com o intuito de fazer surgir a tribo de Manassés no sexto lugar, indicando que ali havia a tribo de Dã, que teria sido marcada pri- mitivamente com o número seis, devido à sua relação com a apostasia e o Anticristo. AUNE, David E. Revelation. Word Biblical Commentary, 52B. Dallas: Word, 1997. v. 2, p. 462. Considerações finais Como vimos neste artigo, os primeiros segui- dores de Jesus, os cristãos primitivos, estavam muito centrados na tradição hebraica, mesmo quando ao provir e atuar no âmbito de pessoas oriundas do politeísmo. Os representantes do poder imperial romano, divinizado e opressivo, podiam ser entendidos como escatológicos, a prenunciar o fim do tempo de injustiça. Nero, nes- te contexto, pode ser um imperador a prenunciar em tudo: adorado por tantas pessoas, o que podia ser considerado pelos seguidores de Jesus, como o supremo sacrilégio; perseguidor de cristãos, podia ser interpretado como prenunciador do fim da iniquidade e do reino de Deus. Mesmo que o texto do Apocalipse possa ser posterior a Nero, contemporâneo a outros imperadores romanos divinizados e perseguidores dos cristãos, não seria descabido considerar que Nero foi o grande e inicial referente, como o que Ernest Renan viria a chamar de Anticristo. Por fim, a possível relação da lista das Doze Tribos de Israel com a lista dos Doze Apóstolos e a sua história (substituição de Judas Iscariotes por Matias, entre outras coincidências) nos permite concluir que o autor do Apocalipse também es- taria fazendo um paralelo simbólico e tipológico entre o Antigo e o Novo Testamento, o que fecha a sua cadeia de significados ocultos apocalípticos e proféticos, totalmente contida e sintetizada no número 666. BAUCKHAM, Richard. The List of the Tribes in Revela- tion 7 Again. Journal for the Study of the New Testament (JSNT), Thousand Oaks, n. 42, p. 99-115, 1991. 5 Mc 3,17; Lc 9,54. BAUCKHAM, Richard. The Climax of Prophecy: Studies on the Book of Revelation. Edingbourgh: T. & T. Clarke, 1993. Referências AUNE, David E. Revelation. Word Biblical Commentary, 52B. Dallas: Word, 1997. v. 2, p. 462. BAUCKHAM, Richard. The Climax of Prophecy: Studies on the Book of Revelation. Edingbourgh: T. & T. Clarke, 1993. 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São Paulo: Edições Loyola, 1993. Doutor em Arqueologia pela Universidade de São Paulo (USP), em São Paulo, SP, Brasil. Pós-Doutorado pela Illinois State University (ILSU), EUA; pela University College London (UCL), Grã-Bretanha; pela Universitat de Barcelona (UB), Espanha; pela Université de Paris X (UPX), França; pela Durham University (DURHAM), Inglaterra; pela Stanford University (STANFORD), EUA. Livre-Docência pela Universidade Estadual de Cam- pinas (UNICAMP), em Campinas, SP, Brasil. Professor titular da Universidade Estadual de Campinas (UNI- CAMP), em Campinas, SP, Brasil. PSEUDO-FÍLON. Disponível em: https://www.sacred- -texts.com/bib/bap/index.htm. Acesso em: 3 ago. 2021. REUSS, Eduard W. E. Allgemeine Literatur-Zeitung, die Intelligenzblätter, vol. V, 62, IV, Halle, Sept., 1837, p. 520. Tradução de Adylson Valdez em Academia.edu. Disponível em: https://www.academia.edu/32966601/ Ferdinand_Hitzig_1837_Eduard_Wilhelm_Eugen_ Reuss_1837_deutsch_English_Portugu%C3%AAs. 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Schöpfung und Chaos in Urzeit und Endzeit. Göttingen: Vandenhoeck und Ruprecht, 1895. Disponível em: http://www.etana.org/sites/default/ files/coretexts/14497.pdf. Acesso em: 3 ago. 2021. CLEMEN, Carl. Die Zahl 666, ein Hinweis auf Trajan? Protestantische Monatshefte 25, p. 144-148, 1921. HARTINGSVELD, Lodewijk van. Die Zahl des Tieres, die Zahl eines Menschen: Apokalypse XIII.18. Miscellanea Neotestamentica 2, p. 191-201, 1978. CLEMEN, Carl. Die Zahl des Tieres Apc 13:18. Zeitschrift für die neutestamentliche Wissenschaft (ZNW), v. 2, p. 109-114, 1901. HENGSTENBERG, Ernst W. The Revelation of St. John Expounded for those who search the Scriptures. Edin- gbourg: Mack Publishing,1852. CLEMEN, Carl. Nochmals die Zahl des Tieres. Zeitschrift für die neutestamentliche Wissenschaft (ZNW) v. 11, p. 204-223, 1910. HILLERS, Delbert R. Revelation 13, 18 and a Scroll from Murabba’at. Bulletin of the American Schools of Oriental Research (BASOR), v. 170, p. 65, 1963. Disponível em: https://www.journals.uchicago.edu/toc/basor/current. Acesso em: 3 ago. 2021. CORPUS THOMISTICUM (commentaria biblica, super Apocalypsim). Disponível em: https://www.corpus- thomisticum.org/iopera.html. Acesso em: 3 ago. 2021. DEISSMANN, Gustav A. Light from the Ancient East. London: Hodder and Stoughton, 1910. Disponível em: https://openlibrary.org/books/OL6527179M/Light_ from_the_ancient_East. Acesso em: 3 ago. 2021. HITZIG, Ferdinand. Ostern und Pfingsten: Heidelberg, 1837, p. 3. Tradução de Adylson Valdez em Acade- mia.edu. Disponível em: https://www.academia. edu/32966601/Ferdinand_Hitzig_1837_Eduard_Wi- lhelm_Eugen_Reuss_1837_deutsch_English_Portu- gu%C3%AAs. Acesso em: 3 ago. 2021. ENCYCLOPAEDIA BIBLICA. Disponível em: http://on- linebooks.library.upenn.edu/webbin/book/lookupi- d?key=olbp14092. Acesso em: 3 ago. 2021. IRENEU. Adversus Haereses (Adv. Haer.) 1.24.7, 5.28.2, 5.28.3, 5.29.2, 5.30.1, 5.30.2, 5.30.3. Disponível em: ht- tps://www.newadvent.org/fathers/0103.htm. Acesso em: 3 ago. 2021. ENCYCLOPEDIA BRITANNICA. Verbete Abbreviation. 11. ed. 1911. Disponível em: https://en.wikisource.org/ wiki/1911_Encyclop%C3%A6dia_Britannica/Abbrevia- tion. Acesso em: 3 ago. 2021. Teocomunicação, Porto Alegre, v. 52, n. 1, p. 1-17, jan.-dez. 2022 \| e-41450 SPITTA, Friedrich. Die Offenbarung des Johannes unter- sucht. Halle: Weisenhauses, 1889. v. XII. JASTROW, Marcus. Adylson Valdez Adylson Valdez Graduado em Direito pela Universidade Católica de Santos (UNISANTOS), em Santos, SP, Brasil. Graduado em Direito pela Universidade Católica de Santos (UNISANTOS), em Santos, SP, Brasil. SELLIN, Ernst; FOHRER, George. Introdução ao Antigo Testamento. Tradução de D. Mateus Rocha. São Paulo: Edições Paulinas, 1983. SKA, Jean Louis. Introdução à leitura do Pentateuco: chaves para a interpretação dos cinco primeiros livros da Bíblia. 3. ed. Tradução de Aldo Vannucchi. São Paulo: Edições Loyola, 2003. SMITH, Christopher R. The Portrayal of the Church as the New Israel in the Names and Order of the Tribes in Revelation 7.5-8. Journal for the Study of the New Testament (JSNT) 39, p. 111-118, 1990. Disponível em: https://journals.sagepub.com/home/jnt. Acesso em: 3 ago. 2021. Endereços para correspondência Pedro Paulo Abreu Funari Universidade Estadual de Campinas Departamento de História Cidade Universitária Zeferino Vaz 13.083-970 Campinas, SP, Brasil Adylson Valdez Rua Benjamin Constant, n. 154, ap. 32 11.040-140 Santos, SP, Brasil Adylson Valdez Os textos deste artigo foram revisados e submetidos para validação do(s) autor(es) antes da publicação.
https://openalex.org/W2429084198	https://www.shs-conferences.org/articles/shsconf/pdf/2016/06/shsconf_rptss2016_01002.pdf	English	null	Social and economic well-being in the conditions of the urban space: the evolution of methodological approaches in the historical urban studies	SHS web of conferences	2,016	cc-by	3,500	a Corresponding author: agilalex@mail.ru Social and economic well-being in the conditions of the urban space: the evolution of methodological approaches in the historical urban studies Ilya Ageev1,a, Anastasia Nikolaeva1, Peter Rumyantsev2 1 National Research Tomsk Polytechnic University, Lenin Avenue, 30, Tomsk 634050, Russia 2 National Research Tomsk State University Lenin Avenue, 36, Tomsk 634050, Russia Abstract. A city as a type of a human settlement is characterized by high population density, well- developed infrastructure, comfortable living conditions. However, a city is a source of social problems due to high population density, limited resources and conflicts between indigenous population and newcomers. The article analyzes the development of research about the city, provides an assessment of the scope of the historical urban studies in the development of solutions to contemporary problems of urban space. Methodological resource of historical urban studies allows fully exploring the city as a set of historically interconnected spaces and social processes. The analysis of the problem field of historical urban studies at various stages of its formation allowed tracing the evolution of ideas about the city as an object of scientific knowledge, to identify future prospects of research on conditions of Russian urban development, to improve the comfort of living in them. the proximity of raw materials and resources, the availability of labor force. 1 Introduction Cities are diverse types of human settlements, transforming under the influence of economic and technical progress on such parameters as the shape, function, a legal status and a social structure of the population. At all times, cities have differed from the surrounding area with their high density and other kind of activity of the population. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). , Web of Conferences 01002 (2016) SHS 28 RPTSS 2015 , Web of Conferences 01002 (2016) SHS 28 RPTSS 2015 , Web of Conferences 01002 (2016) SHS 28 DOI: 10.1051/shsconf/20162801002 3 Results and discussion Historical urban studies, as a part of urban studies, consider issues of urban development and functioning in the historical perspective. D.Ya. Rezun was one of the first Russian researchers who used the term “urban studies” as applied to historical research. He considered it as all city historical research and historiography [3, 22]. This definition eliminates economic and geographical component of urban studies object, which was the inherent generic concept formulated in the socio-economic knowledge. The reason for this difference is likely to become the abundance of accumulated historical knowledge of the city that required a terminological clarification. In contrast to urban history, the consideration of the city in the context of historic urban studies suggests that the formation of appearance and structure of cities to a greater extent is under the influence of objective economic and social processes rather than historical persons. Unlike the urban history, historical urban studies translate the idea of exploring the city as a social phenomenon - a specially formed space for life and human activity. In addition to urban studies, historical science and the city interact in such a rapidly developing scientific field, as the history of the city. It is relevant to compare the methodological practices of historical urban studies and the history of the city with a view to distinguishing their subject areas. This city is not just a home to the society, it effects intensively the society, creating a different model of interpersonal relationships and changing lifestyles. Modern researchers consider the city as a possible area for their studies, but do not concretize the possible impact of cities on the socio-economic and political relations [15]. The history of the city involves the study of the city as a community of the most active people of their era (managers, politicians, scientists), united by a common space of the city and as a result of the application of their creative and business efforts. Thus, the local history of the city pays the greatest attention to the activities of people living in a town or place, whereby the leading method of this study becomes a locally-historical method [4; 5]. In addition, the specificity of the city as an object of historical urban studies is determined by other factors. Not only the economic and social process but increasing volumes of scientific knowledge about the city have a direct impact on urban development. 3 Results and discussion Another type of urban history, in which attempts are made to go away from the local history component, is the research demonstrating involvement of the city’s history in the history of the state, achievements of residents in the implementation of public tasks, their “contribution to the common cause” [10-12]. Identifying the causes of today’s urban problems and search for possible solutions are in the research area of special scientific discipline - urban studies. The term comes from the economic geography [1], where it means the analysis and study of the problems associated with the functioning of cities. As the area of socio- humanitarian knowledge, urban studies aim at identifying the most common features and urbanization trends, formation of shape and structure of cities, their existence and transformations as a result of technological revolutions and social change. Finally, the third research experience of urban history is microhistory that reflects global historical patterns in the development of society by using local examples, extracted by the historical specification of the city or locality [13]. In all these algorithms, the subject of scientific research is the human community, while the city defines only the geographical scope of the particular historical work. At the same time, questions about how the city developed in a particular historical period, in terms of the internal connections - both engineering and social, - remain without answer. Environment description is undoubtedly present, but it often has the character of fixing the city’s status at a particular time. Urban studies are an interdisciplinary science. Researchers from different disciplines bring to the urban studies their methodology, but consider the city in the narrow sense - only in the context of their scientific field, without referring to the experience of other research models. This problem is typical not only for the interdisciplinary discourse but for intra-historical one [2]. The qualitative breakthrough in urban planning will be possible in the case of integrated research of Cities. Description of the living conditions provided by the city, most states full or partial unavailability of infrastructure, lack or low qualification of personnel who are responsible for its development [14]. Thus, the modern historical studies of Russian cities, because of the specificity of the subject and methodology, do not analyze the issues which are in the focus of historical urban studies. , Web of Conferences 01002 (2016) SHS 28 RPTSS 2015 in the development of solutions of contemporary problems of urban space. and the socio-political phenomena that occur in it [6]. City, a worker’s settlement or village, turn into a point on the map, which localize a historical event or phenomenon [7; 8]. Today studies of Russian cities largely are realized in three types of historical works. Writing works on urban history most often leads to the popularization of a particular city, demonstrating its uniqueness in the galaxy of other ones [9]. These form- factors are often used by ethnographers. The main historiographical sources for the research were works on urban planning, published in Russia and abroad. The methodology is traditional for the historiographical research. It involves the use of comparative analysis and a historical-genetic method. 2 Materials and method The scientific significance of the article is to find a new aspect of the urban studies, which maintains the continuity of the traditional research on cities but suggests investigation of the processes that determine the interaction of the urban space and the urban community. For example, the research object should include the work of municipal services, city improvement projects, investment in urban infrastructure, creating the conditions for development of the city’s economy and private business, reducing costs arising from the poor quality of the urban environment, etc. Research relevance of the problems of modern urban spaces is connected with the need for timely solutions of such issues as overcrowding of cities, overloaded communications, ethnic and social conflicts, high cost of living, low efficiency of management, lack of resources. However, at each previous stage of development inhabitants of the cities also had to solve problems to improve urban living conditions. By eliminating the infrastructure or the social problems from their cities, their residents received new ones instead. The results of scientific research should be the formation of complex knowledge about the historical development of cities, the factors that determine the quality of infrastructure, investment attractiveness and other factors, for which the city is estimated at the present time. The novelty of this study is the summarizing the theoretical models of modern foreign and domestic research on urban spaces as well as the identification of heuristic resource of this models for the scientific understanding of contemporary urban issues. The effects of modernization and economic transformations are added to a range of these issues. As a result, the cities with similar geographic and economic potential become the holders of unequal indicators of information, investment and tourist attractiveness, different development levels of engineering and social infrastructure. In post-industrial society, these indicators of the city’s economic potential assessment are undeniably more significant than previously used such as The purpose of this article is to identify the methodological resource for the historical urban studies DOI: 10.1051/shsconf/20162801002 , Web of Conferences 01002 (2016) SHS 28 RPTSS 2015 , Web of Conferences 01002 (2016) SHS 28 RPTSS 2015 Rezun well noted the Soviet style of urban historiography. He called it a “production for production’s sake”, meaning scholastic disputes about “the moment of transition from the manufactory to the small-scale production”, etc. instead of focusing on the customer and the market [18]. Only some works of Soviet historians of cities focused on the study of the city structure and not characteristic of its external parameters [19]. heaps of engineering systems, industrial space exceeding residential development in cities. This practice increased the distance between the man and the nature, leading to environmental problems. By the 1930s, in the European and American industrial city there was no more comfortable life. The increase in the growth rate of urban population has given rise to social conflicts. In the 1930s, American sociologists from the University of Chicago (Nels Anderson, Ernest Burgess, Ruth Shonle Cavan) among the first initiated studies on the social development in a rapidly growing city. Their joint activities received generalized name of “Chicago School” and laid the foundations of historical urban studies. The first works in this direction were historical reviews on the development of Chicago contemporary to them [16; 17]. Currently, urban issues experience the growth of popularity in Russian and international science due to the growth of cities and the expansion of various topics and methodologies. A number of researchers study the history of individual groups of cities in the socio- economic or socio-political context [20; 21]. Soviet historians did not conduct special studies, entering the problem field of historical urban studies. But that does not mean the absence of urban studies in the USSR. A separate area of urban studies, demonstrated the diversity of urban development and replaced urban research in the Soviet Union, was the concept of a socialist city [22]. A further solution of urban problems was interrupted by the events of the World War II. In the second half of the 20th century, the population solved independently these problems in accordance with their financial capabilities. Representatives of the middle class to bought private cars and moved to the suburbs. Big businessmen and investors transferred their plants in the neighborhood of cities, on which authorities imposed fewer requirements for the eco-friendliness of production. The vacated districts have undergone the gentrification and revitalization. Local authorities have contributed, increasing environmental fees and taxes on transport. This concept developed by Soviet urban planning theorist N. A. , Web of Conferences 01002 (2016) SHS 28 RPTSS 2015 Milyutin, exerted a decisive influence on the research and development of Soviet cities, starting from the 1930s. In the postwar period, there was the transfer of socialist city ideas (“Sotsgorod”) in the People’s Democracies. The concept evolved during the 20th century, but the main idea remains the same: to achieve financial and social equality in the city. Social equality, that was standardization and regulation of urban consumption of goods, should be ensured on an equal basis with the smooth operation of public transport and the supply of drinking water to the population. The result was a partial unloading of the largest centers of European and American cities from industrial pollution. Cities began to transform from industrial to office and business centers. Specific phenomena of that time were suburbanization, the creation of green areas, integrated public transport systems, the functional evolution of urban areas. Thus, the gentrification process, which began in the middle of the 20th century, involving the scientific community of ecologists and psychologists in the solution of urban problems. The socialist city concept has been designed to solve the same problems that were considered by Western urbanists: to create favorable conditions for the distribution of population; to meet their needs in the residential, cultural services and social communication; to minimize social conflicts. First of all, a socialist city should have a place as close as possible to the labor settlement of industrial facilities i.e. to jobs, in the shortest possible time and with minimal costs, but with the greatest possible convenience. However improving the quality of citizens’ life as well as the reorientation of the urban employment to the office or intellectual work has brought a new wave of migration in the late 20th - early 21st century. Since then, immigrants were not the villagers from the suburbs, but citizens of other countries and representatives of other cultures. Conflict of identities once again led to an increase in tension in the cities. Today street safety issues, urban and national identity, tolerance are relevant than ever. The result of urban policy of “socialist city” was the emergence of cities that meet all these requirements, but the key feature of the socialist city was ignoring the individual characteristics and needs of population in specific Soviet cities, their historical development and the surrounding landscape. 3 Results and discussion At the beginning of the 20th century the significant infrastructure improvement of the largest cities contributed inevitably to their further growth. However, the completion of the industrial revolution provided the The identification of regional historical specificity eliminates the value of the settlement type, as the historian’s research interest is not intended at the place 2 DOI: 10.1051/shsconf/20162801002 , Web of Conferences 01002 (2016) SHS 28 , Web of Conferences 01002 (2016) SHS 28 RPTSS 2015 , Web of Conferences 01002 (2016) SHS 28 RPTSS 2015 The history of the urban environment modernization demonstrates the oscillation amplitude of the comfort level of city life with the increase in population. Complex measures for the improvement of urban infrastructure almost inevitably led to an increase in urban population and the consequent reduction in the level of urban life comfort. Due to a lack of time, the selection of sites for districts in relation to industrial buildings was carried out without the long-term measurements of the wind rose, the number of water sources and their quality. In terms of districts construction the priority was given to visual harmony and ease, rather than rationality. Construction of streets and roads was carried out without regard to parking spaces required for all vehicles that these streets and roads can accommodate. Placing social objects did not take into account a real demand on the population of the city or district. Passenger traffic of public transport focused on the connection of the region with the city Historical urban studies as the original area of scientific knowledge gained an impulse of the development in international historical science in the first half of the 20th century. Scientific, including historical interpretation of cities also developed in Soviet science in this period but in a different direction. D. Ya. 3 , Web of Conferences 01002 (2016) SHS 28 DOI: 10.1051/shsconf/20162801002 , Web of Conferences 01002 (2016) SHS 28 RPTSS 2015 center and the main enterprise, which employs residents of the region, while the inter-regional connections were not established. • Establishing the degree of intensity and the radius of influence of the city’s economic and social development on the surrounding rural areas. Changes in the Russian socio-economic system led to the aggravation of all these shortcomings. Houses of Culture, built in Soviet times, came to desolation, and cases of their conversion within the urban space were a rarity. The places of people’s attraction changed in the cities, but the public urban transport could not reorient and began to decline. Road infrastructure, despite the apparent sufficiency, was not prepared to increase the number of private cars. The set of the circumstances and conditions of formation and functioning of modern Russian cities requires scientific studies using the experience of Western historical urban studies, in particular methodological tools of the Chicago School of Sociology. 4 Conclusion 4. A. Mazanik, Urban History, 42(3), 509-520. (2015) 5. H.T. Graef, Zeitschrift fur historische forschung, 41(3), 450-451 (2014) Modern Russian city arose in the transition between the successive political regimes and different types of socio- economic relations. Historical urban studies involve the study of the processes in the cities that are relevant and problematic, but this methodological resource in the study of Russian urban space is now only partially involved. 6. V.V. Ageeva, I.A. Ageev, A.M. Nikolaeva, Z.N. Levashkina, The European Proceedings of Social & Behavioural Sciences EpSBS, VII, 167-172 (2016) 7. V.P. Zinoviev, Siberian historical research, 2, 33-41 (2013) 8. P.P. Rumyantsev, Bulletin of the Tomsk State University. History, 1, 18-21 (2010) The methodological complexity of solving the issues of historical urban studies is that the emergence of urban issues that require scientific judgment is fluctuating in nature. The imbalance of the urban development or an uncomfortable increase in population density at a certain point, is usually due to the lack of integrated solutions of various socio-economic difficulties at the past stage of formation of urban space. In addition, the formation of cities and foundation for their modern development took place in different historical periods under very different circumstances: it was without taking into account scientific development of those problems, which have acceded to solving problems of cities at the later stages. y y ( ) 9. X. Meng, Science, 343(6167), 138-139 (2014) 10. P. Capobianco, Cultural Geographies, 23(1), 174- 175 (2016) 11. V. Das, M. Walton, Current Anthropology, 56, 44- 54 (2015) 12. V.P. Zinoviev, Bylye Gody, 29(3), 42-46 (2013) 13. I.A. Ageev, V.V. Ageeva, Procedia - Social and Behavioral Sciences, 166, 24-29 (2015) 14. C. Evans, Planning Perspectives, 30(1), 165-170 (2015) 15. P. Hadfield, Urban Studies, 52(3), 606-616 (2015) 16.E.W. Burgess. The urban community: Selected papers from the proceedings of the American sociological society (Chicago, 1927) The problem field of historical urban studies is vast and very promising for a modern Russian researcher: 17. . Weber, The city. (Free Press, 1966) • The identification of historical factors that determine the position of the city in the hierarchy of other cities, as well as the efficiency of resource accumulation for the successful development of cities - demographic, economic and infrastructure. Search of reasons for the explosive growth or the inevitable degradation of individual cities. 18. K. Wiest, Urban Geography, 33(6), 829-849 (2012) 19. M.B. , Web of Conferences 01002 (2016) SHS 28 RPTSS 2015 Changing the external characteristics of the city as the aggregate results of its population activities, there should be a study showing the city from the inside, as a social and infrastructural organism. Methods of urban studies in the Western pattern were not applied in planning and development of Soviet cities. Therefore, the modern inhabitants of post-socialist cities have to not only minimize the direct impact of industrial construction (environmental pollution and disposal of vacant industrial sites), but also expend resources for the elimination of many errors that occur from the use of the Socialist city concept. References 1. L. Fernandez, Journal of urban history, 41(4), 566- 571 (2015) 2. C. Nicholson, Series Historical urban studies. Slovo, 22(2), 125-126 (2010) 3. D.Ya. Rezun, Essays explored the history of the Siberian city: the 18th century (Novosibirsk, 1991) 4 Conclusion Katz, Journal of urban history, 41(4), 560-566 (2015) 20. G. Bridge, International journal of urban and regional research, 38(5), 1644-1659 (2014) 21. J. Lavrinec, Filosofija-Sociologija, 21(1), 54-63 (2010) • Modeling schemes of social interaction in the urban environment in accordance with its economic orientation, social systems and strategic importance in specific regional and historical realities. The diagnosis of the compliance level of the urban infrastructure and the pace of economic and social processes taking place on its territory. 22. K. Ki, Journal of North-East Asian Cultures, 1(34), 469-488 (2013) 4
https://openalex.org/W4361234922	https://figshare.com/ndownloader/files/39863946	English	null	Supplemental Figures 1-9 from Amplification of Oncolytic Vaccinia Virus Widespread Tumor Cell Killing by Sunitinib through Multiple Mechanisms	null	2,023	cc-by	2,184	Supplemental Figure 1. Three versions of mouse prototypes of JX-594 (mpJX-594) differing by the insertion cassette used to disrupt vaccinia thymidine kinase gene. All viruses were based on the Western Reserve (WR) strain, a mouse-adapted Wyeth strain vaccinia that was isolated through serial passage in mouse brain. WR-TK(-)-eGFP/YFP vaccinia viruses that express transgenes for (A) human GM-CSF, (B) mouse GM-CSF, or (C) mCherry instead of GM-CSF were generated by disrupting the vaccinia thymidine kinase gene (Tk) with an insertion cassette containing eGFP (enhanced green fluorescent protein) or YFP (yellow fluorescent protein) driven by a synthetic early/later promoter (pSel) and human or mouse GM-CSF or mCherry transgene driven by the p7.5 early/late promoter. J1R, gene for virion protein required for morphogenesis; J3R, gene for multifunctional poly-A polymerase subunit, cap methyltransferase, and transcription elongation factor. Supplemental Figure 2. mpJX-594 uptake from vasculature and replication in RIP-Tag2 Supplemental Figure 2. mpJX-594 uptake from vasculature and replication in RIP-Tag2 tumors. A, B: Confocal micrographs of vaccinia immunoreactivity (green) in RIP-Tag2 tumors. No vaccinia is present after iv injection of vehicle, but 6 hours after mpJX-594, faint vaccinia is uniformly visible in blood vessels (B, CD31, red). Some vessels have strong staining (arrows). At 1 day, vaccinia is also located in small groups of cells near tumor vessels (arrow). At 5 days, larger patches of vaccinia are present (arrows). C, D: Yellow fluorescent protein (YFP, green) is absent after vehicle, but is visible in tumor vessels (D, CD31, red) at 6 hours after mpJX-594 (arrows), and in vessels and extravascular cells (arrows) at 1 and 5 days. Scale bar in D applies to all images: 100 µm in A, B and 50 µm in C, D. YFP Vaccinia YFP Vaccinia 5 days after mpJX-594 6 hours after mpJX-594 Liver YFP 5 days after mpJX-594 1 day after mpJX-594 6 hours after mpJX-594 Vehicle Spleen Vaccinia 5 days after mpJX-594 1 day after mpJX-594 6 hours after mpJX-594 Vehicle Spleen C B A Transient vaccinia but not YFP in spleen and liver after intravenous mpJX-594 5 days a 1 day after mpJX 594 6 hours after mpJX 594 cle Spleen Transient vaccinia but not YFP in spleen and liver after intravenous mpJX-59 Spleen Transient vaccinia but not YFP in spleen and liver after intravenous mpJX-594 1 day after mpJX-594 6 hours after mpJX-594 Vehicle Spleen Vaccinia 1 day after mpJX-594 6 hours after mpJX-594 Vehicle Spleen B A Transient vaccinia but not YFP in spleen and liver after intravenou 1 day afte 6 hours after mpJX-594 Spleen 1 day afte 6 hours after mpJX-594 Spleen nsient vaccinia but not YFP in spleen and liver a 5 days after mpJX-594 5 days after mpJX-594 A 6 hours after mpJX-594 Sp 1 day after mpJX-594 een 1 day after mpJX-594 n YFP Vehicle B B 5 days after mpJX-594 5 days after mpJX-594 YFP Vaccinia YFP Vaccinia 5 days after mpJX-594 6 hours after mpJX-594 Liver C Liver 5 days after mpJX-594 Vaccinia 6 hours after C C Vaccinia y YFP Vaccinia Supplemental Figure 3 Supplemental Figure 3. mpJX-594: transient vaccinia but not YFP in spleen and liver. Confocal micrographs of spleen or liver of RIP-Tag2 mice after iv injection of vehicle or mpJX-594; tissue prepared 6 hours, 1 day, or 5 days later. A: Spleen stained for vaccinia immunoreactivity (red). No vaccinia is evident after injection of vehicle as expected, but vaccinia is conspicuous (arrows) in splenic red pulp at 6 hours after mpJX-594, but little or none is present at 1 or 5 days. B: Staining of spleen for yellow fluorescent protein (YFP, red) as an indication of mpJX-594 viral replication. No YFP staining is evident at any time point. C: Liver stained for vaccinia (green) and YFP (red). Faint vaccinia staining is evident in liver at 6 hours (C left, arrows) but not at 5 days (C right). No YFP is visible at either time point. Scale bar in C applies to all images: 160 µm. Supplemental Figure 4. mpJX-594: effects on extravasation of fibrin and erythrocytes in RIP-Tag2 tumors. A, B: Regions of extravasated fibrin/fibrinogen (red, A) and erythrocytes (TER119 staining, red, B) are scattered in RIP-Tag2 tumors of control (vehicle) mice but are much greater and more widespread at 5 days after mpJX-594. Most erythrocytes in the vehicle-treated tumor are concentrated in blood lakes, which are separate from the vasculature (CD31, green) and are not washed free of blood by vascular perfusion of fixative (4). C: Comparison of tumor vasculature co-stained for CD31 and VEGFR-2 at 5 days after iv injection of vehicle or mpJX-594. The tumor vasculature is similar when assessed by CD31 or VEGFR-2. Tumors are highly vascular after vehicle and less vascular after mpJX-594. D: Measurements of tumors co-stained for CD31 and VEGFR-2 show similar tumor densities assessed under baseline conditions (vehicle) and similar reductions at 5 days after mpJX-594. ANOVA. P < 0.05 for difference compared to vehicle (n = 5 mice/group). Scale bar in D applies to all images: 200 µm. Supplemental Figure 4. mpJX-594: effects on extravasation of fibrin and erythrocytes in RIP-Tag2 tumors. A, B: Regions of extravasated fibrin/fibrinogen (red, A) and erythrocytes (TER119 staining, red, B) are scattered in RIP-Tag2 tumors of control (vehicle) mice but are much greater and more widespread at 5 days after mpJX-594. Most erythrocytes in the vehicle-treated tumor are concentrated in blood lakes, which are separate from the vasculature (CD31, green) and are not washed free of blood by vascular perfusion of fixative (4). Supplemental Figure 3. mpJX-594: transient vaccinia but not YFP in spleen and liver. C: Comparison of tumor vasculature co-stained for CD31 and VEGFR-2 at 5 days after iv injection of vehicle or mpJX-594. The tumor vasculature is similar when assessed by CD31 or VEGFR-2. Tumors are highly vascular after vehicle and less vascular after mpJX-594. D: Measurements of tumors co-stained for CD31 and VEGFR-2 show similar tumor densities assessed under baseline conditions (vehicle) and similar reductions at 5 days after mpJX-594. ANOVA. P < 0.05 for difference compared to vehicle (n = 5 mice/group). Scale bar in D applies to all images: 200 µm. Supplemental Figure 5. Antitumor effects of mpJX-594 variants expressing human GM-CSF, mouse GM-CSF, or no GM-CSF and dose-response. A: Confocal micrographs of apoptotic cells (activated caspase-3, red) in tumors of RIP-Tag2 mice at 5 days after iv injection of mpJX-594 variants that express human GM-CSF, mouse GM-CSF, or no GM-CSF (mCherry). B: Measurements of activated-casepase-3 staining in tumors show similar increases in apoptotic cells at 5 days after all three mpJX-594 variants compared to vehicle-treated controls. ANOVA. * P < 0.05 compared to vehicle (n = 4-5 mice/group). C: Differences in area densities of activated caspase-3 (red) and vaccinia (green) in RIP-Tag2 tumors at 5 days after doses of 1, 3, or 6 times the standard dose (107 pfu) of mpJX-594/mGM-CSF. ANOVA. P < 0.05 compared to corresponding value for * vehicle or # vaccinia (vehicle, n = 3; 1x mpJX-594, n = 4; 3x mpJX-594, n = 5; 6x mpJX-594, n = 3). Scale bar in A applies to all images: 200 µm. Supplemental Figure 5. Antitumor effects of mpJX-594 variants expressing human Supplemental Figure 6. JX-594: effects on U87 glioma xenografts. A: Confocal micrographs show the amount and distribution of vaccinia staining (green) in and around the vasculature (CD31, red) of human U87 tumor xenografts in nude mice at 6 hours, 1 day, or 5 days after iv injection of JX-594. Control tumor (vehicle) without vaccinia shown for comparison. Arrows point to vaccinia staining in blood vessels. B: Little extravasated fibrin (red) in a U87 tumor at baseline compared to conspicuous perivascular fibrin at 1 day and widespread fibrin at 5 days after JX-594. C: One apoptotic cell (activated caspase-3, red, arrow) near a blood vessel (CD31, green) in a vehicle-treated U87 tumor compared to numerous apoptotic cells in or near blood vessels in a U87 tumor 1 day after JX-594. D: Blood vessels (CD31, red) of U87 tumor at baseline compared to 5 days after JX-594. Tumor vessels are sparse and narrow or fragmented after JX-594. E: Reduction of vascularity of U87 tumors from 1 to 5 days after JX-594. ANOVA P < 0 05 for differences compared to * vehicle or to # 2 days or less after JX 594 (n ANOVA. P < 0.05 for differences compared to * vehicle or to # 2 days or less after JX-594 (n = 5 mice/group). Scale bar in D applies to all images: 100 µm for A, C, D; 400 µm for B. Supplemental Figure 7. Sunitinib amplification of mpJX-594 antitumor actions on tumors independent of GM-CSF. A: Confocal micrographs comparing sparse apoptotic cells (activated caspase-3, red) in RIP-Tag2 tumors at 5 days after vehicle or sunitinib with extensive apoptosis after mpJX-594/mGM-CSF (mouse GM-CSF) and even more widespread apoptosis after the virus plus sunitinib. B: Measurements of treatment-related differences in activated-caspase-3 staining in tumors from the four groups of mice shown in A. ANOVA. P < 0.05 compared to * vehicle, to # sunitinib alone, or to § virus alone (vehicle, n = 4; virus, n = 8; sunitinib, n = 3; virus plus sunitinib, n = 7). C: Weight of 4T1 mouse mammary carcinomas implanted subcutaneously in BALB/c mice after treatment with vehicle, mpJX-594/mCherry (no GM-CSF), sunitinib, or virus plus sunitinib over 5 days. One dose of virus was injected iv and daily sunitinib was started on day 0. 4T1 tumors treated with virus plus sunitinib were significantly smaller than the controls. ANOVA. Supplemental Figure 5. Antitumor effects of mpJX-594 variants expressing human * P < 0.05 compared to vehicle (n = 5 mice/group). Scale bar in A: 200 µm. Supplemental Figure 8. mpJX-594 and sunitinib: effects of administration sequence. A: Treatment regimens used to compare sequence of mpJX-594 and sunitinib administration on efficacy in RIP-Tag2 mice: Simultaneous (a), mpJX-594-first (b), and Sunitinib-first (c). B-D: Area densities of vaccinia (B), CD31 (C), and activated caspase-3 (D) are expressed as percentages of the corresponding value for mpJX-594 alone for 10 days (Simultaneous and mpJX-594-first) or 5 days (Sunitinib-first). E-G: Treatment-related differences in area density of vaccinia (E), CD31 (F), and activated caspase-3 (G) in tumors after vehicle (-/-), mpJX-594 alone, or sunitinib alone for 5 or 10 days. P < 0.05 in B-D compared to * Sunitinib-first (group c) or to # mpJX-594 alone for 5 or 10 days. P < 0.05 in E-G compared to * 10-day mpJX-594 group, # vehicle and sunitinib groups, § vehicle group, or ¶ 5-day sunitinib group. Student’s t-test comparison of group (b) in panel C and of 10-day mpJX-594 group in panel E to other groups; otherwise ANOVA (n = 4-5 mice/group). H: Measurements of tumor burden, assessed as mean sectional areas of RIP-Tag2 tumors in four treatment groups, show smaller values after mpJX-594 plus sunitinib regardless of administration sequence. ANOVA. * P < 0.05 compared to vehicle (n = 4-5 mice/group). Supplemental Figure 9. Sunitinib compared to DC101 in combination with mpJX-594. A: Supplemental Figure 9. Sunitinib compared to DC101 in combination with mpJX-594. A: Treatment protocols for administration of sunitinib or DC101 with mpJX-594 to RIP-Tag2 mice over 5 days. mpJX-594 injected iv on day 0. Sunitinib given by gavage daily on days 1 through 5. DC101 given on days 1 and 3 (2x) or as a priming dose on day -1 followed by doses on days 1 and 3 (3x). B: Confocal micrographs of tumors show patches of strong vaccinia staining (green) after mpJX-594 alone and more widespread vaccinia after mpJX-594 plus sunitinib. By Supplemental Figure 9. Sunitinib compared to DC101 in combination with mpJX-594. A: Treatment protocols for administration of sunitinib or DC101 with mpJX-594 to RIP-Tag2 mice over 5 days. mpJX-594 injected iv on day 0. Sunitinib given by gavage daily on days 1 through 5. DC101 given on days 1 and 3 (2x) or as a priming dose on day -1 followed by doses on days 1 and 3 (3x). B: Confocal micrographs of tumors show patches of strong vaccinia staining (green) after mpJX-594 alone and more widespread vaccinia after mpJX-594 plus sunitinib. By comparison, vaccinia is weaker after mpJX-594 plus DC101 (2x or 3x). Tumor vessels marked by CD31 (red). C: Measurements of vaccinia staining in tumors after mpJX-594 show 3-fold increase after sunitinib but not after DC101. ANOVA. P < 0.05 compared to * mpJX-594 alone or to # mpJX-594 plus sunitinib (n = 4 mice/group). D: Measurements of tumor vascular density at 5 days after vehicle, sunitinib, or DC101, with or without mpJX-594. Only sunitinib plus mpJX-594 resulted in greater reduction in vascular density than other treatments. P < 0.05 compared to * vehicle or # all other groups (n = 3-6 mice/group). E: Treatment-related differences in activated caspase-3 in tumors. ANOVA. P < 0.05 compared to * mpJX-594 alone or # mpJX-594 plus sunitinib (n = 4 mice/group). F: Linear regression shows significant correlation between vaccinia and activated caspase-3 staining in tumors from the four treatment groups with consistently greater staining for activated caspase-3 than vaccinia. Scale bar in B applies to all images: 200 µm.
https://openalex.org/W4206504757	https://strathprints.strath.ac.uk/79040/7/Kieswetter_etal_JAC_2021_Intranasally_administered_S_MGB_364_displays_antitubercular_activity.pdf	English	null	Intranasally administered S-MGB-364 displays antitubercular activity and modulates the host immune response to <i>Mycobacterium tuberculosis</i> infection	The journal of antimicrobial chemotherapy/Journal of antimicrobial chemotherapy	2,022	cc-by	7,683	Received 16 September 2021; accepted 20 December 2021 Background: Previously, we evaluated the intracellular mycobactericidal activity of the minor groove binder, S-MGB-364 against the clinical Mycobacterium tuberculosis (Mtb) strain HN878 in macrophages. Objectives: To assess the mycobactericidal activity of S-MGB-364 in Mtb-infected mice. Further, we investigated a plausible DNA binding mechanism of action of S-MGB-364. Methods: The anti-TB and host immune effects of intranasal S-MGB-364 or S-MGB-364 encapsulated in non- ionic surfactant vesicles (NIV) were assessed in Mtb-infected mice by cfu enumeration, ELISA, histology, and ﬂow cytometry. DNA binding was examined using native mass spectrometry and UV-vis thermal melt deter- mination. S-MGB interference with DNA-centric biological events was assessed using a representative panel of Mtb and human topoisomerase I, and gyrase assays. Results: S-MGB-364 bound strongly to DNA as a dimer, signiﬁcantly increasing the stability of the DNA:S-MGB complex compared with DNA alone. Moreover, S-MGB-364 inhibited the relaxation of Mtb topoisomerase I but not the human form. In macrophages, S-MGB-364 or S-MGB-364-NIV did not cause DNA damage as shown by the low γ-H2AX expression. Importantly, in the lungs, the intranasal administration of S-MGB-364 or S-MGB- 364-NIV formulation in Mtb-infected mice was non-toxic and resulted in a ≏1 log cfu reduction in mycobacter- ial burden, reduced the expression of proinﬂammatory cytokines/chemokines, altered immune cell recruitment, and importantly reduced recruitment of neutrophils. Conclusions: Together, these data provide proof of concept for S-MGBs as novel anti-TB therapeutic bacteria.4 Their mechanism of action involves engagement at multiple DNA sites in the pathogen, and interference with normal protein synthesis at promoter sites in the case of Gram-positive bacteria.5 One S-MGB, MGB-BP-3, is ready for Phase 3 clinical trials for the treatment of Clostridioides difﬁcile infection, having shown clinical potential.6 Although binding to AT-rich sites in dsDNA has been shown to be a key component of the mechanism of J Antimicrob Chemother https://doi.org/10.1093/jac/dkac001 © The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 of 11 Nathan S. Kieswetter1,2†, Mumin Ozturk1,2†, Lerato Hlaka1,2†, Julius Ebua Chia1,2, Ryan J. O. Nichol3, Jasmine M. Cross3, Leah M. C. McGee3, Izaak Tyson-Hirst3, Rebecca Beveridge3, Frank Brombacher1,2,4, Katharine C. Carter5, Colin J. Suckling3, Fraser J. Scott3 and Reto Guler1,2,4* 1International Centre for Genetic Engineering and Biotechnology, Cape Town Component, Cape Town 7925, South Africa; 2Department of Pathology, University of Cape Town, Institute of Infectious Diseases and Molecular Medicine (IDM), Division of Immunology and South African Medical Research Council (SAMRC) Immunology of Infectious Diseases, Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa; 3Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, Scotland; 4Wellcome Centre for Infectious Diseases Research in Africa (CIDRI-Africa), Institute of Infectious Disease and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa; 5Strathclyde Institute of Pharmacy of Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, Scotland Corresponding author. E-mail: reto.guler@uct.ac.za †These authors contributed equally to this work. Corresponding author. E-mail: reto.guler@uct.ac.za †These authors contributed equally to this work. Corresponding author. E-mail: reto.guler@uct.ac.za †These authors contributed equally to this work. Intranasally administered S-MGB-364 displays antitubercular activity and modulates the host immune response to Mycobacterium tuberculosis infection Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 Nathan S. Kieswetter1,2†, Mumin Ozturk1,2†, Lerato Hlaka1,2†, Julius Ebua Chia1,2, Ryan J. O. Nichol3, Jasmine M. Cross3, Leah M. C. McGee3, Izaak Tyson-Hirst3, Rebecca Beveridge3, Frank Brombacher1,2,4, Katharine C. Carter5, Colin J. Suckling3, Fraser J. Scott3 and Reto Guler1,2,4 Introduction Minor groove binders (MGBs) have great potential as anti-infective agents.1 In particular, analogues of distamycin and netropsin synthesized at the University of Strathclyde, Strathclyde Minor Groove Binders (S-MGBs) have been shown to possess in vivo and in vitro activity against fungi,2 parasites,3 and Gram-positive Kieswetter et al. action,4,5 the key to selectivity, and therefore, utility as a drug, re- lies upon the physicochemical properties of MGBs. 100 μM KCl, and 100 μM S-MGB, 1% DMSO. DNA solutions containing no S-MGB including 1% DMSO were used as controls. Native mass spectrometry (nMS) experiments were carried out on a Synapt G2-Si instrument (Waters, Manchester, UK) with a nanoelectros- pray ionization source (nESI). Mass calibration was performed by a sepa- rate infusion of NaI cluster ions. Solutions were ionized from a thin-walled borosilicate glass capillary (i.d. 0.78 mm, o.d. 1.0 mm, (Sutter Instrument Co., Novato, CA, USA) pulled in-house to nESI tip with a Flaming/Brown micropipette puller (Sutter Instrument Co., Novato, CA, USA). A negative potential in the range of 1.0–1.2 kV was ap- plied to the solution via a thin platinum wire (diameter 0.125 mm, Goodfellow, Huntingdon, UK). The following instrument parameters were used for the DNA:S-MGB-364 complex: capillary voltage 1.2 kV, sample cone voltage 80 V, source offset 110 V, source temperature 40° C, trap collision energy 3.0 (V), trap gas 4 mL/min. For DNA:S-MGB-176 complex: capillary voltage 1.1 kV, sample cone voltage 90 V, source off- set 110 V, source temperature 40°C, trap collision energy 3.0 (V), trap gas 4 mL/min was used. For DNA with no S-MGB present, a capillary voltage of 1.0 kV was applied to the sample. Sample cone voltage 80 V, source offset 95 V, source temperature 40°C, trap collision energy 3.0 (V) and trap gas 4.0 mL/min was used. Data were processed using Masslynx V4.2 and OriginPro 2021, and ﬁgures were produced using Chemdraw. We previously reported that S-MGBs have direct mycobac- tericidal activity against Mycobacterium tuberculosis (Mtb).2,7 Signiﬁcantly, most of the active compounds contained S-MGBs with amidine tail groups. Ethics All animal experiments were conducted in accordance with the Animal Research Ethics Committee of South African National Standard (SANS 10386:2008). The protocol (019/24) was approved by the Animal Ethics Committee, Faculty of Health Sciences, University of Cape Town, South Africa. M. tuberculosis and human topoisomerase I relaxation assay 1 U of Mtb and human topoisomerase I (topo I) was incubated with 0.5 μg supercoiled plasmid DNA (pBR322) in a 30 μL reaction at 37°C for 30 min under the following conditions: 40 mM Tris-HCl (pH7.6), 20 mM NaCl, 1 mM EDTA, 5 mM MgCl2 and 0.05 mg/mL BSA for Mtb topo I and 20 mM Tris HCl (pH 7.5), 200 mM NaCl, 0.25 mM EDTA and 5% glycerol plus 5% DMSO for human topo I. Each reaction was stopped using 30 μL chloroform/iso-amyl alcohol (24:1) and 30 μL Stop Dye be- fore being loaded on a 1.0% TAE gel run at 90 V for 2 h. Topoisomerase and gyrase inhibition Male C57BL/6 mice were purchased from Jackson Laboratories (USA) and housed in a biosafety level 3 containment facility as previously described.9 The activity of each enzyme was determined, and 1 U enzyme was re- quired to fully supercoil or relax the substrate. Compounds ranging from 0.01 μM to 100 μM were added to the reaction before the addition of the enzyme. The ﬁnal DMSO concentration in the assays was 1% (v/v). /advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 M. tuberculosis gyrase supercoiling assay 1 U of DNA gyrase was incubated with 0.5 μg of supercoiled pBR322 DNA in a 30 μL reaction at 37°C for 30 min under the following conditions: 50 mM HEPES, KOH (pH 7.9), 6 mM magnesium acetate, 4 mM DTT, 1 mM ATP, 100 mM potassium glutamate, 2 mM spermidine and 0.05 mg/mL BSA. Each reaction was stopped using 30 μL chloroform/iso- amyl alcohol (24:1) and 20 μL Stop Dye (40% sucrose, 100 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.5 μg/mL bromophenol blue), before being loaded on a 1.0% TAE (Tris-acetate 0.04 mM, EDTA 0.002 mM) gel run at 80 V for 3 h. Introduction In particular, S-MGB-362 and S-MGB-364 had intracellular activity against Mtb in macrophages.7 This activity was enhanced by incorporating S-MGBs in non-ionic surfactant vesicles (NIV).7 Previous stu- dies have shown that NIV can increase the efﬁcacy of drugs by different routes, including inhalation, where NIV increased the activity of amphotericin B against Aspergillus in a murine model.8 Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac0 In this study, we demonstrate that intranasally administered S-MGB-364 in mice has anti-Mtb activity and that S-MGB-364 did not induce genotoxicity. Mechanistically, we demonstrated that S-MGB-364 strongly binds to DNA as a dimer and inhibits the action of Mtb topoisomerase I relaxation. UV-Vis DNA thermal melting experiments Salmon genomic DNA (gDNA; D1626, Sigma–Aldrich) at 1 mg/mL in 1 mM phosphate buffer (pH 7.4) containing 0.27 mM KCl and 13.7 mM NaCl (P4417, Sigma–Aldrich) was annealed at 90°C for 10 min. S-MGBs at 10 mM in DMSO were diluted with the same phosphate buffer to yield a single sample with 10 μM S-MGB and 0.02 mg/mL gDNA in 1 mM phosphate buffer containing 0.27 mM KCl and 13.7 mM NaCl. Control samples containing only S-MGB or gDNA were prepared, respec- tively. Samples were melted at a rate of 0.5°C/min from 45°C to 90°C with spectra recorded at 260 nm on a UV-1900 UV-vis spectrophot- ometer ﬁtted with a Peltier temperature controller (Shimadzhu) using LabSolutions (Tm Analysis) software. The melting temperatures (Tms) of the S-MGB:DNA complexes were determined by ﬁtting a sigmoidal function using a Boltzmann distribution in OriginPro. Two independent experiments were carried out with values quoted with an error no worse than +1°C. M. tuberculosis gyrase supercoiling assay Cytokine and chemokine determine in lung homogenates The hyper-virulent Beijing Mtb strain (HN878) was cultured, titrated, and stored as previously described.9 Lung homogenates were centrifuged, and the supernatants were col- lected. Filtered cell-free lung homogenates were used to detect IL-1α, IGF-1, IL-13, CCL3, CCL5, CXCL1, CXCL2 (R&D Systems), IL-2, TGF-β, IL-4, IL-5, IL-6, IL-12p40, IFN-γ, CCL2 (B&D Biosciences), IL-10, IL-17, IFN-β, TNF and GM-CSF (Biolegend) by enzyme-linked immunosorbent assay (ELISA). S-MGB-364 interacts strongly with DNA 24 h post infection, S-MGB-364, S-MGB-364-NIV, and the genotoxicity- inducing agent (10 μM H2O2) were prepared in DMEM media supple- mented with 10% FCS and added to Mtb-infected BMDMs. 4 and 24 h post treatment, BMDMs were harvested and assessed for geno- toxicity by ﬂow cytometry using the following antibodies: CD11b (Clone M1/70, PercP-Cy5.5); mouse/human anti-phosphohistone SER-139 H2AX (Clone: H5912-AF488); F4/80 (Clone: BM8-AF647); MHCII (Clone-M5/114.15.2-AF700). Intranuclear expression of γH2AX was detected using BD Pharmingen Transcription Factor Buffer Set (BD Biosciences). Previously, we observed that S-MGBs bearing an amidine tail group were signiﬁcantly more active against Mtb than analogues bearing a weakly basic morpholine.2,7 Speciﬁcally, whilst S-MGB-364 had been identiﬁed as a hit compound in our intracel- lular in vitro assay, S-MGB-176, its morpholine tail analogue, was inactive against Mtb.7 Consequently, the interaction of both S-MGB-364 and S-MGB-176 with DNA was investigated. Native mass spectrometry (nMS) was used to demonstrate that S-MGB-364 and S-MGB-176 bind to dsDNA, using a short DNA oligo with an AT-rich binding site (5′-CGCATATATGCG-3′). nMS of the DNA in the presence of S-MGB-364 revealed that it is bound to dsDNA exclusively as a dimer [DS+2M], observed in charge states 5−and 4−(Figure 1, Tables S1 and S2). Similar results were ob- tained for S-MGB-176 (Figure S3, Tables S3 and S4). Secondly, a DNA thermal melting experiment in the presence and absence of S-MGBs was carried out, using salmon DNA as an example gen- omic DNA (gDNA) target (Figure 2 and Figure S4). A ΔTm for the DNA:S-MGB-364 complex of 16°C indicated that S-MGB-364 read- ily binds to and stabilizes DNA. The ΔTm for the DNA:S-MGB-176 complex of only 2.9°C suggests a much weaker interaction with gDNA for S-MGB-176 compared with S-MGB-364 (Table S5). Taken together, the DNA thermal melting and nMS experiments provide conclusive evidence that S-MGB-364 can bind to and sta- bilize DNA. Moreover, it indicates that the tail group amidine signif- icantly enhances DNA binding. oup.com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 Generation of murine bone marrow-derived macrophages (BMDMs) and Mtb infection BMDMs were generated from 8–12 week old C57BL/6 mice as described previously.10 BMDMs were cultured overnight for adherence into 96-well plates (Nunc, Denmark) (5 × 104 cells per well). A single-cell suspension of Mtb HN878, from frozen stock, was prepared in DMEM media. 24 h post adherence, BMDMs were infected with Mtb HN878 at MOI 1:5 cfu per well. At 4 h post infection, BMDMs were washed once with culture media to remove extracellular bacteria and lysed. Lysates were plated on 7H10 agar plates containing 10% OADC and 0.5% glycerol for cfu counting to determine bacilli uptake. Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dka Intranasal S-MGB-364 has in vivo anti-TB activity S-MGB-364 inhibits Mtb, but not human, topoisomerase I S MGB 364 inhibits Mtb, but not human, topoisomerase I Whilst S-MGB-364 binding to DNA was conﬁrmed, it was neces- sary to demonstrate that this could inhibit DNA-centric biological events. To do this, the ability of S-MGB-364 to inhibit the relaxa- tion action of human and Mtb topoisomerase I, and the super- coiling action of Mtb gyrase was evaluated (Figure 3a and b). S-MGB-364 inhibits Mtb topoisomerase I relaxation with an IC50 of 10.3 μM, which is more effective than the control com- pound mAMSA, 17.4 μM. There was no evidence from these ex- periments that S-MGB-364 inhibits human topoisomerase I relaxation or Mtb gyrase supercoiling. These data conﬁrm the po- tential of S-MGB-364 to inhibit DNA-centric biological events through binding to DNA. In vivo Mtb infection and cfu analysis 8–12 week old C57BL/6 mice were anaesthetized and infected intrana- sally with either 100 cfu/mouse or 1000 cfu/mouse of Mtb HN878 in ster- ile saline (25 μL per nostril). Mtb-infected mouse lungs were homogenized to measure lung cfu at 5 weeks post infection and to de- termine bacilli uptake at 1 day post infection as previously described.9 Statistical analysis of data All experimental data were analysed using Graph-Pad Prism 8.0.2. Data were calculated as mean+SEM and the Student’s t-test or the one-way ANOVA was used to test for signiﬁcance. from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 Janu Flow cytometry Single-cell suspensions were achieved as previously described.9 The staining panel was composed of MerTK (Clone: 2B10C42-BV786, BioLegend), CD64 (Clone: X54-5/7-PeCy7, BioLegend), Ly6C (Clone: AL-21-PerCPCy5.5, BD Biosciences), CD11b (Clone: M1/70-V450, BD Biosciences), MHCII (Clone: M5/114.15.2-AF700, BioLegend), CD103 (Clone: M290-PE, BD Biosciences), CD11c (Clone: HL3-APC, BD Biosciences), SiglecF (Clone: E5-2440-APCCy7, BD Biosciences), Ly6G (Clone: 1A8-FITC, BD Biosciences), F4/80 (Clone: BM8-PeCy7, eBiosciences), CD4 (Clone: RM4-5-BV510, BD Biosciences), CD44 (Clone: IM7-PE, BD Biosciences), CD3 (Clone: 500A2-AF700, BD Biosciences), CD62L (Clone: MEL-14-V450, BD Biosciences), CD19 (Clone: 1D3-PerCPCy5.5, BD Biosciences) and CD8 (Clone: 53-6.7-APC, BD Biosciences). Acquisition of samples was conducted using BD LSR Fortessa, and gating strategies are outlined in the Supplementary data (Figure S1 and S2, available at JAC Online). Data analysis was per- formed with FlowJo v10 software (Treestar, Ashland, OR, US). Native mass spectrometry S-MGB-364 was reconstituted in DMSO and then diluted in 1×PBS or DMEM to yield a ﬁnal concentration of 10 mg/kg (S-MGB-364) per mouse or 10 μM in macrophages. Freeze-dried NIVs were prepared as previously described and rehydrated in PBS or DMEM with 10% FCS (Gibco, Thermoﬁsher Scientiﬁc, USA) to a ﬁnal NIV concentration range of 30 μM in formulation with S-MGB or empty NIV. 25 μL of S-MGB-364, S-MGB-364-NIV, or saline was administered to each nostril (intranasally) of the anaesthetized mice. The purity of lyophilized DNA oligonucleotide sequence 5′-CGCATATATGCG-3′ (Alpha DNA, Canada) was conﬁrmed by NMR. 100 μM DNA stock solutions were prepared with 150 mM ammonium acetate buffer solution and 2 mM KCl solution (Fisher Scientiﬁc, Loughborough, Leicestershire, UK). This solution was annealed at 90°C for 10 min and allowed to cool to room temperature. 10 mM S-MGB stocks in 100% DMSO were diluted to 1 mM S-MGB solution with 150 mM ammonium acetate to yield ﬁnal concentrations of 9 μM DNA, 2 of 11 2 of 11 Histology and alveolar space assessment Mtb-infected murine lungs were excised, ﬁxed using 4% phosphate- buffered formalin solution, and stained with haematoxylin and eosin, iNOS, MPO, ARG-1, and anti-CD3, as previously published.11,12 Analysis of lung sections and assessment of alveolar space were performed using NIS advanced software on a Nikon (Tokyo, Japan) 90i microscope. 3 of 11 Kieswetter et al. Figure 1. Characterization of S-MGB-364 binding to double-stranded DNA as a dimer by nMS. nMS of DNA sequence 5′-CGCATATATGCG-3′ (9 μM DNA, 100 μM KCl, 1% DMSO) sprayed from ammonium acetate (150 mM, pH 7) in the absence (a) and presence (b) of 100 μM s-MGB. (a) Single-stranded DNA (denoted [SS]) are present in charge states 4−and 3−, and double-stranded DNA (denoted [DS]) are present in charge states 5−and 4−. (b) [SS] is present in charge state 3−. Each [DS] molecule is seen to bind 2×S-MGB molecules (denoted [DS+2 M]) and is present in charge states 5−and 4−. Figure 1 will appear online in colour and black and white in print. p p j j Downloaded from https://academic.oup.com/jac/advance-article/doi/1 Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001 Figure 1. Characterization of S-MGB-364 binding to double-stranded DNA as a dimer by nMS. nMS of DNA sequence 5′-CGCATATATGCG-3′ (9 μM DNA, 100 μM KCl, 1% DMSO) sprayed from ammonium acetate (150 mM, pH 7) in the absence (a) and presence (b) of 100 μM s-MGB. (a) Single-stranded DNA (denoted [SS]) are present in charge states 4−and 3−, and double-stranded DNA (denoted [DS]) are present in charge states 5−and 4−. (b) [SS] is present in charge state 3−. Each [DS] molecule is seen to bind 2×S-MGB molecules (denoted [DS+2 M]) and is present in charge states 5−and 4−. Figure 1 will appear online in colour and black and white in print. Figure 2. DNA melt curve conﬁrms the ability of S-MGB-364 to bind DNA. (a) Exemplar melt curve from one experimental repeat, visually representing the different melt curves of DNA and the DNA:S-MGB-364 complex. Data has been ﬁtted with a Boltzmann distribution. (b) Melting temperatures of DNA and DNA:S-MGB-364 complex calculated from ﬁtted Boltzmann distributions using OriginPro 2021. All values are an average for n=4 experimen- tal repeats and quoted with an error of +1°C. Figure 2. DNA melt curve conﬁrms the ability of S-MGB-364 to bind DNA. (a) Exemplar melt curve from one experimental repeat, visually representing the different melt curves of DNA and the DNA:S-MGB-364 complex. S-MGB-364 and S-MGB-364-NIV are non-genotoxic in macrophages To extend this ﬁnding, S-MGB-364 and encapsulated S-MGB-364- NIV were administered intranasally to Mtb-infected mice at 1, 2, 3, and 4 weeks post infection (Figure 4a). Importantly, and in agree- ment with our in vitro data, treatment comprising of either S-MGB-364 alone or S-MGB-364-NIV was less toxic (Figure 4f) and resulted in a signiﬁcant reduction (≏1 log) in the lung HN878 burden relative to the control saline group during high dose and a more standard dose of HN878 infection (Figure 4b and c, respectively). Taken together, these data provide in vivo proof of concept for S-MGB-364 as a potential anti-TB agent. DNA binding drugs could provide safety concerns for mutageni- city and genotoxicity. We used the sensitive molecular marker, γ-H2AX, to assess drug-induced genotoxicity.13 We observed no difference in the expression of γ-H2AX in the S-MGB-364 and S-MGB-364-NIV groups relative to the non-treated control at 4 and 24 h post treatment (Figure 3c). Together, these data indi- cate that S-MGB-364 and S-MGB-364-NIV do not induce geno- toxicity in macrophages. Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 Intranasal S-MGB-364 has in vivo anti-TB activity Histology and alveolar space assessment Data has been ﬁtted with a Boltzmann distribution. (b) Melting temperatures of DNA and DNA:S-MGB-364 complex calculated from ﬁtted Boltzmann distributions using OriginPro 2021. All values are an average for n=4 experimen- tal repeats and quoted with an error of +1°C. Figure 2. DNA melt curve conﬁrms the ability of S-MGB-364 to bind DNA. (a) Exemplar melt curve from one experimental repeat, visually representing the different melt curves of DNA and the DNA:S-MGB-364 complex. Data has been ﬁtted with a Boltzmann distribution. (b) Melting temperatures of DNA and DNA:S-MGB-364 complex calculated from ﬁtted Boltzmann distributions using OriginPro 2021. All values are an average for n=4 experimen- tal repeats and quoted with an error of +1°C. 4 of 11 Intranasal S-MGB-364 and S-MGB-364-NIV administration results in decreased lung burden in Mtb-infected mice To assess the activity of S-MGB-364 on lung inﬂammation, mice were infected with 100 cfu of HN878. After the establish- ment of Mtb infection, and treatment with S-MGB-364 and Previously, we showed that treatment of HN878-infected macro- phages with S-MGB-364 reduced the intracellular growth of Mtb.7 phages with S-MGB-364 reduced the intracellular growth of Mtb. ment of Mtb infection, and treatment with S-MGB-364 and Figure 3. S-MGB-364/S-MGB-364-NIV is non-genotoxic to macrophages and inhibits topoisomerase I. (a) Inhibition of topoisomerases and gyrases by S-MGB-364. Camptothecin, mAMSA, and ciproﬂoxacin are controls for the human topoisomerase I, Mtb topoisomerase I, and Mtb gyrase enzymes, respectively. These values are presented as mean+SD with n=2. (b) Example gel illustrating the inhibition of Mtb topoisomerase I relaxation by S-MGB-364. (c) CD11b+F4/80+MHCII+ bone-marrow-derived macrophages were treated with S-MGB-364 (10 μM), S-MGB-364-NIV (10 μM), and the positive control H2O2 (10 μM) for 4 and 24 h. Intracellular expression of γ-H2Ax expression was measured by ﬂow cytometry. (a) Data show mean +SEM of triplicates. ND, not detected. Two-tailed Student’s t-test, P,0.01 compared with non-treated. demic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 Figure 3. S-MGB-364/S-MGB-364-NIV is non-genotoxic to macrophages and inhibits topoisomerase I. (a) Inhibition of topoisomerases and gyrases by S-MGB-364. Camptothecin, mAMSA, and ciproﬂoxacin are controls for the human topoisomerase I, Mtb topoisomerase I, and Mtb gyrase enzymes, respectively. These values are presented as mean+SD with n=2. (b) Example gel illustrating the inhibition of Mtb topoisomerase I relaxation by S-MGB-364. (c) CD11b+F4/80+MHCII+ bone-marrow-derived macrophages were treated with S-MGB-364 (10 μM), S-MGB-364-NIV (10 μM), and the positive control H2O2 (10 μM) for 4 and 24 h. Intracellular expression of γ-H2Ax expression was measured by ﬂow cytometry. (a) Data show mean +SEM of triplicates. ND, not detected. Two-tailed Student’s t-test, P,0.01 compared with non-treated. 5 of 11 Kieswetter et al. e 4. S-MGB-364 and S-MGB-364-NIV treatment is non-toxic and reduced the mycobacterial lung burden in HN878 Mtb-infected mice. (a) C57BL/ e (n=6 per group) were infected with Mtb HN878 via intranasal challenge. At 1, 2, 3 and 4 weeks post-infection, mice were intranasally treated 10 mg/kg of S-MGB-364, S-MGB-364-NIV, or saline. Mice were sacriﬁced at 5 weeks post-infection and lungs were isolated and homogenized for numeration. Bacterial load (cfu) measured in lungs of mice infected with an infective dose of (b) 1000 cfu and (c) 100 cfu Mtb HN878 and in- sally treated with S-MGB-364, S-MGB-364-NIV, or saline. S-MGB-364 alters the recruitment of host lymphoid cells, reduces neutrophils, and recruits interstitial macrophages in the lung during Mtb infection in mice and does not affect mouse lung histopathology We further investigated the effect of S-MGB-364 on immune cell recruitment in the lungs during HN878 infection using ﬂow cyto- metry. At 5 weeks post infection, S-MGB-364 increased the re- cruitment of interstitial macrophages whilst decreasing the frequency of neutrophils within the lungs (Figure 5a). Additionally, S-MGB-364 treatment resulted in an increased per- centage of B cells as well as a decreased percentage of effector CD8+ cells in the lung when compared with the saline-only group (Figure 5c). However, when calculated for total cell numbers, only the reduction in neutrophils and CD8+ effector cells were signiﬁcantly reduced (Figure 5b and d). Histopathological assess- ment of the lung sections showed that the percentage free al- veolar space, iNOS, CD3, and Arg1 levels were similar to the control group (Figure 6a–d). In contrast, the neutrophilic marker myeloperoxidase (MPO) showed a decreased trend in the S-MGB-364-NIV group (P=0.515) (Figure 6e). Together, these data indicate the possible immunomodulation of lung tissue upon S-MGB-364 administration during Mtb HN878 infection. in the NIV formulation. This is ﬁrst reported study of S-MGBs against Mtb in vivo. Previous studies have highlighted the ability of intranasally admi- nistered anti-TB compounds to directly target lung macrophages whilst being less toxic relative to oral administration. Furthermore, the intranasal administration of vaccines or drugs against TB is re- ported to be more effective compared with other routes of admin- istration.19,20 Our data indicated that oral S-MGB-364 treatment induced a slight but non-signiﬁcant reduction of cfu in the lungs (1.07-fold) and spleen (1.09-fold) (Figure S5a and b). Additionally, orally administered S-MGB-364 slightly increased li- ver transaminases (AST and ALT) relative to the saline control (Figure S5c and d). However, this increase was statistically non- signiﬁcant. Informed by these, as well as our in vitro data, we elected to directly target the lungs, the site of Mtb infection through the intranasal administration of S-MGB-364 and its NIV encapsulation. Interestingly, intranasal S-MGB-364 administration resulted in decreased serum liver transaminase levels and had a greater reduction on lung Mtb burden, highlighting its superiority to traditional oral administration (Figure 4b, c and f). Intranasal S-MGB-364 has in vivo anti-TB activity encapsulation of S-MGB-364 within NIVs; however, this differ- ence was non-signiﬁcant. encapsulation of S-MGB-364 within NIVs; however, this differ- ence was non-signiﬁcant. Mutagenicity is a signiﬁcant concern for any drug with a DNA-centred mechanism of activity.16–18 In our study, we ob- served a similar low expression of γ-H2AX with S-MGB-364 and the S-MGB-NIV formulation as compared with non-treated macrophages, indicating that S-MGB-364 is non-genotoxic to mammalian cells. These data correlate with our previous studies on cell viability where we showed that macrophages infected with Mtb HN878 remained viable following 5 days of treatment with S-MGB-362 and S-MGB-364, both as a free compound and in the NIV formulation.7 S-MGB-364 alters the recruitment of host lymphoid cells, reduces neutrophils, and recruits interstitial macrophages in the lung during Mtb infection in mice and does not affect mouse lung histopathology However, we noted that NIVs did not have an added antimicrobial beneﬁt that could be attributed to different factors, such as dosing and variable delivery efﬁciency to Mtb-infected areas.21 Despite this, and of note, we demonstrated that 4 weekly treatments with 10 mg/kg S-MGB-364 and S-MGB-364-NIV were less toxic as mea- sured by liver transaminases in comparison with the control saline group (Figure 4f) and reduced the lung burden by ≏1 log in a phy- siological infective dose and high dose Mtb infection model, re- spectively. Additionally, the spleen burdens were unaffected following S-MGB-364 treatment (Figure S6). Daily intranasal ad- ministration of S-MGB-364 may further improve compound efﬁ- cacy. However, due to both technical and physiological limitations of such a regimen (i.e. daily anaesthesia) compounded with the risk of adverse animal welfare outcomes (accidental mor- tality due to repeated intranasal administration),22,23 daily admin- istration was not considered. In the future, nebulized forms of S-MGB-364 could be developed to further assess the effect of daily treatment whilst avoiding the risks detailed above. Despite this lim- itation, these data highlight the anti-TB potential of MGBs. Intranasal S-MGB-364 and S-MGB-364-NIV administration results in decreased lung burden in Mtb-infected mice (d and e) Supernatants from lung homogenates of mice that were infected with 100 cfu HN878 were collected for analysis of cytokine/chemokine concentrations by ELISA. (f) Liver aspartate transaminase (AST), and alanine transa- se (ALT) levels were measured in the sera of Mtb-infected (100 cfu) mice treated intranasally with S-MGB-364, S-MGB-364-NIV, or saline. Data hown as mean+SEM. Students t-test or one-way ANOVA: P,0.05, P,0.01, and *P,0.001. Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 Downloaded from https://academic.oup.com/jac/advance-a Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001 om/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 -article/doi/10.1093/jac/dkac001/6515318 by guest Figure 4. S-MGB-364 and S-MGB-364-NIV treatment is non-toxic and reduced the mycobacterial lung burden in HN878 Mtb-infected mice. (a) C57BL/ 6 mice (n=6 per group) were infected with Mtb HN878 via intranasal challenge. At 1, 2, 3 and 4 weeks post-infection, mice were intranasally treated with 10 mg/kg of S-MGB-364, S-MGB-364-NIV, or saline. Mice were sacriﬁced at 5 weeks post-infection and lungs were isolated and homogenized for cfu enumeration. Bacterial load (cfu) measured in lungs of mice infected with an infective dose of (b) 1000 cfu and (c) 100 cfu Mtb HN878 and in- tranasally treated with S-MGB-364, S-MGB-364-NIV, or saline. (d and e) Supernatants from lung homogenates of mice that were infected with 100 cfu Mtb HN878 were collected for analysis of cytokine/chemokine concentrations by ELISA. (f) Liver aspartate transaminase (AST), and alanine transa- minase (ALT) levels were measured in the sera of Mtb-infected (100 cfu) mice treated intranasally with S-MGB-364, S-MGB-364-NIV, or saline. Data are shown as mean+SEM. Students t-test or one-way ANOVA: P,0.05, P,0.01, and P,0.001. S-MGB-364-NIV, cytokine and chemokine levels were mea- sured in lung homogenates by ELISA. S-MGB-364 and S-MGB-364-NIV signiﬁcantly reduced the levels of IL-1α, IGF-1, TGF-β, IL-5, IL-6, IL-13, IL-17; and the chemokines, CCL2 and CXCL1, relative to the control group (Figure 4d and e). Further, IL-4, IL-12p40, IFN-γ, GM-CSF, TNF, CCL3, CCL5, and CXCL2 were similar to the control group (data not shown). Together, these results indicate a reduction in the lung proin- ﬂammatory response following treatment with S-MGB-364 in Mtb-infected mice. Further, this effect is enhanced by the 6 of 11 6 of 11 Discussion Previously, we reported the intracellular antimycobacterial activ- ity of two S-MGBs; S-MGB-362 and S-MGB-364.7 Further, we showed that NIVs improved compound efﬁcacy whilst being non-toxic in macrophages.7 However, the DNA binding, muta- genicity, as well as the in vivo immunological, and antimycobac- terial properties of these compounds remained unknown. S-MGBs are hypothesized to bind to pathogen DNA in a non- speciﬁc, non-covalent manner.1–3 This association dysregulates various DNA-centric biochemical events by inhibiting DNA–pro- tein interactions. Previous studies have highlighted these DNA-associated mechanisms in antibacterial,4 antiparasitic,3 and antifungal models;2 however, the mechanism of action had not been validated for anti-TB S-MGBs. Herein we show, for the ﬁrst time, that S-MGB-364 binds strongly to and stabilizes DNA, using DNA thermal melting and nMS. Further, we demon- strated that S-MGB-364 binds as a dimer to DNA, similar to the natural product, distamycin. Moreover, we showed that the in- teraction between S-MGB-364 and DNA can lead to inhibition of DNA-centric biochemical events relevant to anti-Mtb activity, such as the action of topoisomerase I. Previous studies have shown that the inhibition of Mtb topo I through the action of small molecules can arrest Mtb growth and can potentially be used as adjunctive anti-TB therapies.14,15 Here, we demon- strated that S-MGB-364 inhibited Mtb topoisomerase relaxation, and not human topoisomerase relaxation or Mtb gyrase super- coiling; however, we do not assert this is the only mechanism of action of S-MGB-364. Instead, we speculate that S-MGB-364 will inhibit a range of different DNA–protein interactions, as has been found for the anti-Gram-positive S-MGB, MGB-BP-3.5 g g p Whilst S-MGB-364 treatment reduced the lung bacterial bur- den, it did not ameliorate lung tissue pathology, nor did it have any effect on the L-arginine metabolic pathway as shown by iNOS or arginase-1 expression in lung tissues. Nevertheless, signif- icant changes in lung cytokine levels were observed after treat- ment with S-MGB-364. The secretion of IL-1α, IGF-1, TGFβ, IL-5, IL-6, IL-13, IL-17; and the chemokines, CCL2 and CXCL1, was sig- niﬁcantly reduced. Furthermore, when incorporated into NIVs, the cytokine responses were either the same as with S-MGB-364 treatment alone or lower in the case of IL-1α and TGFβ. IL-1α, IL-6, and IL-17 are essential cytokines for protection against 7 of 11 b 24 Th f d i i h ki l i i d N hil i d i h i i ure 5. Discussion Increased lung macrophage and B cell recruitment and decreased CD8+ T cells and neutrophil recruitment in S-MGB-364- and MGB-364-NIV-treated mice following Mtb HN878 infection. C57BL/6 mice (n=6 per group) were infected with 100 cfu of Mtb HN878 strain via in- nasal challenge. At 1, 2, 3 and 4 weeks post infection, mice were intranasally treated with 10 mg/kg of S-MGB-364, S-MGB-364-NIV, and saline. ce were sacriﬁced at 5 weeks post-infection and cellular inﬁltration was analysed from lung cell suspensions by ﬂow cytometry. (a) Percentage d (b) total cell numbers of myeloid populations, (c) percentage and (d) total cell numbers of lymphoid populations from lung single cell suspensions mice treated with S-MGB-364, S-MGB-364-NIV or saline. Alveolar macrophages (Alv MΦ)=CD64+SiglecF+CD11c+; interstitial macrophages (Int. Φ)=MERTK+CD64+CD11c−SiglecF−; CD103 dendritic cells (DC)=MHCII+CD11c+CD103+CD11b−, CD11b DC=MHCII+CD11c+CD103−CD11b+; neu- phils (Nw)=LY6G+CD11b+; monocyte-derived dendritic cells (MoDC)=CD64+ CD11b+CD11c+; eosinophils (Eos)=CD64−SiglecF+CD11b+; cells=CD19+CD3−; CD8+ T cells=CD3+CD4−CD8+; CD4+ T cells=CD3+CD4+CD8−; naive T cells=CD62L+CD44−; memory T cells= 62L+CD44+; effector T cells=CD62L−CD44+; TCRγδ Cells=TCRγδ+CD3+. Data shown as mean+SEM. One-way ANOVA: P,0.05, P,0.01; P,0.001, *P,0.0001. eswetter et al. Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 Kieswetter et al. Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001 mic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 Figure 5. Increased lung macrophage and B cell recruitment and decreased CD8+ T cells and neutrophil recruitment in S-MGB-364- and S-MGB-364-NIV-treated mice following Mtb HN878 infection. C57BL/6 mice (n=6 per group) were infected with 100 cfu of Mtb HN878 strain via in- tranasal challenge. At 1, 2, 3 and 4 weeks post infection, mice were intranasally treated with 10 mg/kg of S-MGB-364, S-MGB-364-NIV, and saline. Mice were sacriﬁced at 5 weeks post-infection and cellular inﬁltration was analysed from lung cell suspensions by ﬂow cytometry. (a) Percentage and (b) total cell numbers of myeloid populations, (c) percentage and (d) total cell numbers of lymphoid populations from lung single cell suspensions of mice treated with S-MGB-364, S-MGB-364-NIV or saline. Alveolar macrophages (Alv MΦ)=CD64+SiglecF+CD11c+; interstitial macrophages (Int. MΦ)=MERTK+CD64+CD11c−SiglecF−; CD103 dendritic cells (DC)=MHCII+CD11c+CD103+CD11b−, CD11b DC=MHCII+CD11c+CD103−CD11b+; neu- trophils (Nw)=LY6G+CD11b+; monocyte-derived dendritic cells (MoDC)=CD64+ CD11b+CD11c+; eosinophils (Eos)=CD64−SiglecF+CD11b+; B cells=CD19+CD3−; CD8+ T cells=CD3+CD4−CD8+; CD4+ T cells=CD3+CD4+CD8−; naive T cells=CD62L+CD44−; memory T cells= CD62L+CD44+; effector T cells=CD62L−CD44+; TCRγδ Cells=TCRγδ+CD3+. Data shown as mean+SEM. One-way ANOVA: P,0.05, P,0.01; P,0.001, ***P,0.0001. study. Discussion Neutrophils are associated with excess tissue immuno- pathology and in some cases can provide a permissive environ- ment for Mtb bacterial growth, from which we can infer that the reduction in neutrophils would be a beneﬁcial effect.25 Mtb.24 Therefore, a reduction in these cytokines may result in in- creased susceptibility to Mtb. CCL2 and CXCL1 are known to affect T cell and neutrophil recruitment,24 and a reduction in their levels would explain the reduction of these lung cell populations in this 8 of 11 8 of 11 Intranasal S-MGB-364 has in vivo anti-TB activity 6. S-MGB-364 and NIV encapsulation did not affect pulmonary histopathology. C57BL/6 mice (n=6 per group) were infected with 100 cfu of N878 strain via intranasal challenge. At 1, 2, 3 and 4 weeks post-infection, mice were intranasally treated with 10 mg/kg of S-MGB-364, -364-NIV, or saline. Mice were sacriﬁced at 5 weeks post-infection and lungs were collected in 4% formalin for histopathology analysis. tage of (a) alveolar space, (b) iNOS, (c) CD3, (d) Arg1, and (e) MPO. Data show mean+SEM. Scale bars represent 1000 μm. Magniﬁcation=20×. Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001 Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 Figure 6. S-MGB-364 and NIV encapsulation did not affect pulmonary histopathology. C57BL/6 mice (n=6 per group) were infected with 100 cfu of Figure 6. S-MGB-364 and NIV encapsulation did not affect pulmonary histopathology. C57BL/6 mice (n=6 per group) were infected with 100 cfu of Mtb HN878 strain via intranasal challenge. At 1, 2, 3 and 4 weeks post-infection, mice were intranasally treated with 10 mg/kg of S-MGB-364, S-MGB-364-NIV, or saline. Mice were sacriﬁced at 5 weeks post-infection and lungs were collected in 4% formalin for histopathology analysis. Percentage of (a) alveolar space, (b) iNOS, (c) CD3, (d) Arg1, and (e) MPO. Data show mean+SEM. Scale bars represent 1000 μm. Magniﬁcation=20×. Figure 6. S-MGB-364 and NIV encapsulation did not affect pulmonary histopathology. C57BL/6 mice (n=6 per group) were infected with 100 cfu of Mtb HN878 strain via intranasal challenge. At 1, 2, 3 and 4 weeks post-infection, mice were intranasally treated with 10 mg/kg of S-MGB-364, S-MGB-364-NIV, or saline. Mice were sacriﬁced at 5 weeks post-infection and lungs were collected in 4% formalin for histopathology analysis. Percentage of (a) alveolar space, (b) iNOS, (c) CD3, (d) Arg1, and (e) MPO. Data show mean+SEM. Funding 8 Alsaadi M, Italia JL, Mullen AB et al. The efﬁcacy of aerosol treatment with non-ionic surfactant vesicles containing amphotericin B in rodent models of leishmaniasis and pulmonary aspergillosis infection. J Control Release 2012; 160: 685–91. up.com/jac/a Funding This work was supported by ICGEB, Cape Town Component with Art Falaschi fellowships and EDCTP postdoctoral fellowship to M.O.; gra from the NRF/DSI-South African Research Chair Initiative (SARC South Africa Medical Research Council (SAMRC), and the Internatio Centre for Genetic Engineering & Biotechnology (ICGEB) to F.B.; Knowledge Exchange Development Fund (KEDF) from the University Strathclyde to K.C.C.; the DSI/NRF Collaborative Postgraduate Train Program as well as the BRICS Multilateral Joint Science and Technolo Research Collaboration grant number 110482 to R.G. L.H. was fund by the South African Medical Research Council (SAMRC) through Division of Research Capacity Development under the SAM Internship Scholarship Programme from funding received from South African National Treasury and the L’Oréal-UNESCO For Women Science Sub-Saharan Africa Programme. R.B. is supported by a U Future Leaders Fellowship (Grant Reference MR/T020970/1) and Chancellor’s Fellowship from the University of Strathclyde. I.T.-H funded by an EPSRC Research Excellence Award Studentship. This search was funded in whole, or in part, by the Wellcome Trust a work was done using BSL3 facilities and equipment suppor by Wellcome Trust CIDRI-Africa [203135/Z/16/Z]. Further, the work c ried out under the supervision of F.J.S. was also supported by Wellcome Trust [210103_Z_18_Z]. For the purpose of open access, author has applied a CC BY public copyright licence to any Aut Accepted Manuscript version arising from this submission. The fund had no role in study design, data collection, and analysis, decision to p lish, or preparation of the manuscript. Transparency declarations None to declare. Supplementary data Tables S1 to S5 and Figures S1 to S6 are available as Supplementary d at JAC Online. References 1 Barrett MP, Gemmell CG, Suckling CJ. Minor groove binders as a infective agents. Pharmacol Ther 2013; 139: 12–23. Discussion Scale bars represent 1000 μm. Magniﬁcation=20×. 9 of 11 Kieswetter et al. 2 Scott FJ, Nichol RJO, Khalaf AI et al. An evaluation of minor groove bin- ders as anti-fungal and anti-mycobacterial therapeutics. Eur J Med Chem 2017; 136: 561–72. In conclusion, this study demonstrates that S-MGB-364 binds strongly to DNA as a dimer, inhibits Mtb topoisomerase I relaxation, and is non-genotoxic to mammalian cells. However, further me- chanistic screening to determine the speciﬁc biological mode of action of these compounds will be necessary. Furthermore, S- MGB-364 is active in vivo against Mtb, but future studies of its phar- macokinetics, including different delivery systems, are required to produce more-active formulations. Nonetheless, S-MGB-364 holds promise as an anti-TB compound, and we have provided in vivo proof of concept in mice of the S-MGB class more generally. 3 Giordani F, Khalaf AI, Gillingwater K et al. Novel Minor Groove Binders cure animal African trypanosomiasis in an in vivo mouse model. J Med Chem 2019; 62: 3021–35. 4 Anthony NG, Breen D, Clarke J et al. Antimicrobial lexitropsins containing amide, amidine, and alkene linking groups. J Med Chem 2007; 50: 6116–25. 5 Kerr L, Browning DF, Lemonidis K et al. Novel antibiotic mode of action by repression of promoter isomerisation. bioRxiv 2021: 2020.12.31.424950. 6 Biopharma M. MGB Biopharma Announces Successful End-of-Phase 2 Meeting with FDA for MGB-BP-3, a Potential New Gold Standard, First-Line Treatment for Clostridioides difﬁcile Infection (CDI). https://www.mgb- biopharma.com/mgb-biopharma-successful-end-of-phase-2-meeting-with- fda-for-mgb-bp-3-a-potential-new-gold-standard-ﬁrst-line-treatment-for- clostridioides-difﬁcile-infection-cdi/2021. Funding This work was supported by ICGEB, Cape Town Component with Arturo Falaschi fellowships and EDCTP postdoctoral fellowship to M.O.; grants from the NRF/DSI-South African Research Chair Initiative (SARCHi), South Africa Medical Research Council (SAMRC), and the International Centre for Genetic Engineering & Biotechnology (ICGEB) to F.B.; the Knowledge Exchange Development Fund (KEDF) from the University of Strathclyde to K.C.C.; the DSI/NRF Collaborative Postgraduate Training Program as well as the BRICS Multilateral Joint Science and Technology Research Collaboration grant number 110482 to R.G. L.H. was funded by the South African Medical Research Council (SAMRC) through its Division of Research Capacity Development under the SAMRC Internship Scholarship Programme from funding received from the South African National Treasury and the L’Oréal-UNESCO For Women in Science Sub-Saharan Africa Programme. R.B. is supported by a UKRI Future Leaders Fellowship (Grant Reference MR/T020970/1) and a Chancellor’s Fellowship from the University of Strathclyde. I.T.-H. is funded by an EPSRC Research Excellence Award Studentship. This re- search was funded in whole, or in part, by the Wellcome Trust and work was done using BSL3 facilities and equipment supported by Wellcome Trust CIDRI-Africa [203135/Z/16/Z]. Further, the work car- ried out under the supervision of F.J.S. was also supported by the Wellcome Trust [210103_Z_18_Z]. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. The funders had no role in study design, data collection, and analysis, decision to pub- lish, or preparation of the manuscript. 9 Kieswetter NS, Ozturk M, Jones S-S et al. Deletion of N-acetylmuramyl- L-alanine amidases alters the host immune response to Mycobacterium tuberculosis infection. Virulence 2021; 12: 1227–38. advance-a 10 Roy S, Guler R, Parihar SP et al. Batf2/Irf1 induces inﬂammatory re- sponses in classically activated macrophages, lipopolysaccharides, and mycobacterial infection. J Immunol 2015; 194: 6035–44. article/doi/ 11 Guler R, Mpotje T, Ozturk M et al. Batf2 differentially regulates tissue immunopathology in Type 1 and Type 2 diseases. Mucosal Immunol 2019; 12: 390–402. /10.1093/ 12 Guler R, Parihar SP, Savvi S et al. IL-4Rα-dependent alternative activa- tion of macrophages is not decisive for Mycobacterium tuberculosis pathology and bacterial burden in mice. PLoS One 2015; 10: e0121070. 13 Redon CE, Nakamura AJ, Martin OA et al. Recent developments in the use of γ-H2AX as a quantitative DNA double-strand break biomarker. Aging (Albany NY) 2011; 3: 168–74. Acknowledgements We thank Lizette Fick and Raygaanah Jacobs for their excellent technical assistance with immunohistochemistry staining. We also would like to thank Shandre Pillay, Shelby-Sara Jones, Rudranil Hazra, and Groote Schuur Hospital NHLS diagnostics lab for serum ALT and AST measurements. 7 Hlaka L, Rosslee M-J, Ozturk M et al. Evaluation of minor groove binders (MGBs) as novel anti-mycobacterial agents and the effect of using non- ionic surfactant vesicles as a delivery system to improve their efﬁcacy. J Antimicrob Chemother 2017; 72: 3334–41. Funding 14 Godbole AA, Ahmed W, Bhat RS et al. Inhibition of Mycobacterium tu- berculosis topoisomerase I by m-AMSA, a eukaryotic type II topoisome- rase poison. Biochem Biophys Res Commun 2014; 446: 916–20. 318 by gue 15 Godbole AA, Ahmed W, Bhat RS et al. Targeting Mycobacterium tuber- culosis topoisomerase I by small-molecule inhibitors. Antimicrob Agents Chemother 2015; 59: 1549–57. est on 31 16 Bonin AM, Banks TM, Campbell JJ et al. Mutagenicity of electrophilic N-acyloxy-N-alkoxyamides. Mutat Res 2001; 494: 115–34. Janua Transparency declarations None to declare. Supplementary data Tables S1 to S5 and Figures S1 to S6 are available as Supplementary data at JAC Online. References 1 Barrett MP, Gemmell CG, Suckling CJ. Minor groove binders as anti- infective agents. Pharmacol Ther 2013; 139: 12–23. 17 Snyder RD, Brown JE. Evidence for and role of the dimethylamino group in tamoxifen DNA intercalation in intact Chinese hamster V79 cells. Drug Chem Toxicol 2002; 25: 473–9. ry 2022 effect of instillation volume and form of anesthesia. PLoS One 2012; 7: e31359. 21 Natsheh H, Touitou E. Phospholipid vesicles for dermal/transdermal and nasal administration of active molecules: the effect of surfactants and alcohols on the ﬂuidity of their lipid bilayers and penetration en- hancement properties. Molecules 2020; 25: 2959. 22 Fox JG, Barthold S, Davisson M et al. The mouse in biomedical re- search: normative biology, husbandry, and models. Elsevier, 2006. 23 Miller MA, Stabenow JM, Parvathareddy J et al. Visualization of mur- ine intranasal dosing efﬁciency using luminescent Francisella tularensis: 21 Natsheh H, Touitou E. Phospholipid vesicles for dermal/transdermal and nasal administration of active molecules: the effect of surfactants and alcohols on the ﬂuidity of their lipid bilayers and penetration en- hancement properties. Molecules 2020; 25: 2959. 24 Domingo-Gonzalez R, Prince O, Cooper A et al. Cytokines and chemo- kines in Mycobacterium tuberculosis infection. Microbiol Spectr 2016; 4: doi: 10.1128/microbiolspec.TBTB2-0018-2016. 25 Lovewell RR, Baer CE, Mishra BB et al. Granulocytes act as a niche for Mycobacterium tuberculosis growth. Mucosal Immunol 2021; 14: 229–41. Transparency declarations None to declare. 18 Snyder RD, Hendry LB. Toward a greater appreciation of noncovalent chemical/DNA interactions: application of biological and computational approaches. Environ Mol Mutagen 2005; 45: 100–5. Tables S1 to S5 and Figures S1 to S6 are available as Supplementary data at JAC Online. 19 Grifﬁths G, Nyström B, Sable SB et al. Nanobead-based interventions for the treatment and prevention of tuberculosis. Nat Rev Microbiol 2010; 8: 827–34. 20 Tronde A, Nordén B, Marchner H et al. Pulmonary absorption rate and bioavailability of drugs in vivo in rats: structure–absorption relationships and physicochemical proﬁling of inhaled drugs. J Pharm Sci 2003; 92: 1216–33. 20 Tronde A, Nordén B, Marchner H et al. Pulmonary absorption rate and bioavailability of drugs in vivo in rats: structure–absorption relationships and physicochemical proﬁling of inhaled drugs. J Pharm Sci 2003; 92: 1216–33. 1 Barrett MP, Gemmell CG, Suckling CJ. Minor groove binders as anti- infective agents. Pharmacol Ther 2013; 139: 12–23. 1 Barrett MP, Gemmell CG, Suckling CJ. Minor groove binders as anti- infective agents. Pharmacol Ther 2013; 139: 12–23. 10 of 11 10 of 11 effect of instillation volume and form of anesthesia. PLoS One 2012; 7: e31359. 24 Domingo-Gonzalez R, Prince O, Cooper A et al. Cytokines and chemo- kines in Mycobacterium tuberculosis infection. Microbiol Spectr 2016; 4: doi: 10.1128/microbiolspec.TBTB2-0018-2016. 25 Lovewell RR, Baer CE, Mishra BB et al. Granulocytes act as a niche for Mycobacterium tuberculosis growth. Mucosal Immunol 2021; 14: 229–41. Intranasal S-MGB-364 has in vivo anti-TB activity effect of instillation volume and form of anesthesia. PLoS One 2012; 7: e31359. effect of instillation volume and form of anesthesia. PLoS One 2012; 7: e31359. 24 Domingo-Gonzalez R, Prince O, Cooper A et al. Cytokines and chemo- kines in Mycobacterium tuberculosis infection. Microbiol Spectr 2016; 4: doi: 10.1128/microbiolspec.TBTB2-0018-2016. 25 Lovewell RR, Baer CE, Mishra BB et al. Granulocytes act as a niche for Mycobacterium tuberculosis growth. Mucosal Immunol 2021; 14: 229–41. Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dka Downloaded from https://academic.oup.com/jac/advance-article/doi/10.1093/jac/dkac001/6515318 by guest on 31 January 2022 11 of 11
W2335869149.txt	https://www.ajol.info/index.php/tvl/article/download/133893/124163	en		Resensies: <i>Die potlooddief se bruid en ander stories</i>	Tydskrif vir letterkunde	2,016	cc-by-sa	1,006	Die potlooddief se bruid en ander stories. Keina Swart. Pretoria: Protea Boekhuis, 2015. 139 pp. ISBN: 978�1�4853�0292�6. EBOEK: 978�1�4853�0293�3. EPUB: 978�1�4853�0294�0. DOI: http://dx.doi.org/10.4314/tvl.v53i1.24 Keina Swart is die afgelope veertien jaar redakteur en mede-eienaar van die Waterberg-streekskoerant Die Pos. Uit haar pen het reeds twee kinderboeke verskyn, naamlik Hoekom praat grootmense so snaaks? (2010) en Bleek Bennie se woeste hoes (2011), asook ’n tienerverhaal Bloujaar (2014). Swart is veral bekend as rubriekskrywer en haar eerste boek vir volwassenes, Die potlooddief se bruid en ander stories (2015), is ’n versameling van rubrieke en essays deur hierdie be� kroonde joernalis. Die bundel is verdeel in vier afdelings, naamlik “Mans van my hart”, “Vroue van my hart”, “Kinders van my hart” en “Stories van my hart”, en bevat altesaam 54 stories wat gekenmerk word deur hul eenvoud en Swart se empatie vir haar medemens wat sy verwoord deur die gebruik van humor en nostalgie. Soos die titel van die eerste afdeling te kenne gee, vind die leser hier stories oor verskeie mansfigure. Hierdie afdeling is die sterkste een van die vier, en verhale wat die leser bybly, is “Met woestynsand en duine agter jou�� ”, “�� Woordemans is ge� vaarlik”, “Die potlooddief se bruid”, “Die ou met die overall”, “Dis nog niks, check my skoene!” en “Laatnagversoeke”. Laasgenoemde verhaal kan gelees word as interessante aansluiting by die genre van die Afrikaanse grensverhaal. TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 16_Resensies_02_08 Botha.indd 261 Kenmerkend van hierdie afdeling is die tema van ouderdom wat in menige van die stories voorkom. Hier is dit die onvermydelike aftakelingsproses van die liggaam (en verstand) wat ’n beduidende invloed op die skrywer het. In verhale soos “Wat ’n lieflike geraas”, “Die ou met die overall”, “Lofredes en trompetgeskal” en “Dis nog niks, check my skoene!” is dit die vaderfiguur wat dikwels aangetref word, en in laasgenoemde verhaal ook die plaasvervangende stiefpa. In elkeen van hierdie verhale word die klem ge� plaas op die mans se weerloosheid wat ’n duidelike emosionele uitwerking op die vroulike verteller het. In die tweede afdeling, “Vroue van my hart”, kan verhale gevind word oor uiteenlopende vrouefigure soos ’n ouma, tantes, ’n �� bergvrou, �� ’n �� huiswerker, ’n �� oor� gewig vrou wat agter die parfuumtoon� bak in Edgars werk, en selfs ’n Hongaarse aktrise. Die verhale wat hier beïndruk is “Koms van die bergvrou” en “Jy’s ’n lady”. Weer eens oorheers die tema van ouderdom in verhale soos “Net hier op my skouer” en “Een magtige tagtig jaar”, waarin die fokus in laasgenoemde ook val op die onvermydelike uiteinde van ouderdom, naamlik die dood. Alhoewel die dood ook figureer in die briljante “Jy’s ’n lady”, is dit die tema van die eensame vrou wat so sterk in hierdie uiters visuele vertelling heers, asook in stories soos “Koms van die bergvrou” en “Dagpas vir ’n engel”. “�� Kinders van my hart�� ”�� , die derde afde� ling, bevat meestal stories oor die skrywer se eie kinders, met die fokus op Swart se bewustheid van die veral ouer wordende 261 2016/04/08 08:15:48 AM kind, en haar verwerking van haar eie kinders se verliese en/of teleurstellings. Alhoewel nie een van die stories in hierdie afdeling vir my in die besonder uitstaan nie, slaag Swart om soos �� ’n be� �� kwame rubriekskrywer haar eie emosies en indrukke deur te gee, na gelang van die invloed wat haar kinders op haar as ma, vrou, maar ook as woordmens, skry� wer en boekliefhebber het. ’n Mens kan egter nie help om op te merk hoe die fyn balans tussen humor en patos nie deur� gaans bevredigend gehandhaaf word in hierdie afdeling nie, met van die stories wat by tye sentimenteel aangebied word. In die laaste afdeling van die bun� del, “Stories van my hart”, is dit veral “Laat staan sulke dinge, laat staan”, “Ken Meneer daai huil” en “Dis tyd” wat uitstaan. Eersgenoemde storie is as ’t ware ’n verhaal aanmekaargestik uit enkele reëls uit talle bekende Afri� kaanse volksliedjies om uiteindelik die tragiese liefdesverhaal van oom Kallie en tant Miena te vertel. Hierdie storie beklemtoon Swart se vernuf as skrywer en haar vermoë om kreatief om te gaan met woorde. Omrede dit haar liefde vir die Afrikaanse woord is wat sterk na vore tree, herinner hierdie storie ook aan “Woordemans is gevaarlik” in die eerste afdeling, waar lirieke deur Anton Goosen, Koos Kombuis en David Kramer binne die verhaal verweef is. “Ken Meneer daai huil” dien as ope brief aan die president deur ’n ma wie se seun in ’n plaasaanval vermoor is, en die ontnugtering en pessimisme van hierdie storie kring ook uit na “Wat het hierdie land met ons gedoen?” en “Wat jy nooit moes doen nie”—stories waarin Suid-Afrikaanse realiteite, vernaamlik armoede en geweld, ’n sterk teenwoor� digheid het. Behalwe vir die byna poë� tiese en liriese element wat in “Dis tyd” teenwoordig is, staan hierdie verhaal vir my sentraal binne die bundel vanweë die skrywer se poging om daardie onvaspen� bare emosies weer te gee deur middel van woorde—iets wat sy as rubriekskrywer in elkeen van die stories in die bundel probeer verwoord. Die stories is almal kort vertellinge van ongeveer drie of vier bladsye wat nie baie inspanning tydens die leesproses verg nie, maar met haar persoonlike in� drukke oor ’n verskeidenheid karakters en haar deernis vir mense sal Swart se stories ongetwyfeld menige lesers emo� sioneel stem; een van die belangrikste kenmerke van rubrieke. Dit is egter ook belangrik dat rubrieke die leser aan die dink sit – iets wat Swart nie werklik vir my regkry met al die stories nie. Nietemin is Die potlooddief se bruid en ander stories ’n bewys van Swart se skerp waarnemingsvermoë en ’n bevestiging van haar skryftalent. Op die gebied van die essay en rubriek as genres is hierdie bundel nie ’n literêre mylpaal nie, maar wel goedgeskrewe ontspanningslitera� tuur wat aanklank by ’n wye, populêre lesersmark sal vind. 262 TYDSKRIF VIR LETTERKUNDE • 53 (1) • 2016 16_Resensies_02_08 Botha.indd 262 Frederick J. Botha bothafrederick@gmail.com Noordwes-Universiteit, Potchefstroom 2016/04/08 08:15:48 AM
https://openalex.org/W2593463620	https://www.frontiersin.org/articles/10.3389/frobt.2016.00035/pdf	English	null	Enabling Depth-Driven Visual Attention on the iCub Humanoid Robot: Instructions for Use and New Perspectives	Frontiers in robotics and AI	2,016	cc-by	8,999	1 http://www.cvlibs.net/datasets/kitti/eval_scene_flow.php?benchmark=stereo 2 http://www.cvlibs.net/datasets/kitti/eval_stereo_flow.php?benchmark=stereo Edited by: Edited by: Alexandre Bernardino, Univ. Lisboa, Portugal Reviewed by: Juxi Leitner, Queensland University of Technology (QUT), Australia Aamir Ahmad, Institute for Systems and Robotics, Portugal; Instituto Superior Técnico, Portugal Correspondence: Giulia Pasquale giulia.pasquale@iit.it Keywords: disparity-based segmentation, visual tracking, disparity map, humanoid robotics, iCub E Giulia Pasquale1,2,3, Tanis Mar1,3, Carlo Ciliberto2,4, Lorenzo Rosasco2,3,4 and Lorenzo Natale1 1 iCub Facility, Istituto Italiano di Tecnologia (IIT), Genova, Italy, 2 Laboratory for Computational and Statistical Learning, Istituto Italiano di Tecnologia, (IIT), Genova, Italy, 3 Dipartimento di Informatica, Bioingegneria, Robotica e Ingegneria dei Sistemi, Universitá degli Studi di Genova, Genova, Italy, 4 Poggio Lab, Massachusetts Institute of Technology, Cambridge, MA, USA Reliable depth perception eases and enables a large variety of attentional and interactive behaviors on humanoid robots. However, the use of depth in real-world scenarios is hin- dered by the difficulty of computing real-time and robust binocular disparity maps from moving stereo cameras. On the iCub humanoid robot, we recently adopted the Efficient Large-scale Stereo (ELAS) Matching algorithm (Geiger et al., 2010) for computation of the disparity map. In this technical report, we show that this algorithm allows reliable depth perception and experimental evidence that demonstrates that it can be used to solve challenging visual tasks in real-world indoor settings. As a case study, we consider the common situation where the robot is asked to focus the attention on one object close in the scene, showing how a simple but effective disparity-based segmentation solves the problem in this case. This example paves the way to a variety of other similar applications. Technology Report published: 29 June 2016 doi: 10.3389/frobt.2016.00035 Technology Report 1. INTRODUCTION Specialty section: This article was submitted to Humanoid Robotics, a section of the journal Frontiers in Robotics and AI The main obstacle to stereo vision lies in the process of matching 2D points in the images coming from the cameras on both eyes in order to compute the amount of displacement, or disparity. In this work, we consider the Efficient Large-scale Stereo (ELAS) Matching algorithm (Geiger et al., 2010) and incorporate it in the visual perceptual system of the iCub robot (Metta et al., 2008). Received: 30 December 2015 Accepted: 09 June 2016 Published: 29 June 2016 According to standard KITTI Stereo-Vision Benchmark (Geiger et al., 2012; Menze and Geiger, 2015), ELAS offers a reasonable trade-off between quality of the disparity estimation and computa- tional time, which makes it particularly suited for applications that require real-time performance. Indeed, in the KITTI 2015 and 2012 benchmarks,1,2 ELAS is the first method, among the ones that require less than 1s per frame processing time, which comes with an open-source implementation. This threshold is purely indicative but – given the relatively high resolution of the images constitut- ing the KITTI benchmark – is aimed at excluding those methods that do not provide real-time performance. Those algorithms in the KITTI benchmarks performing better and faster than ELAS Edited by: Alexandre Bernardino, Univ. Lisboa, Portugal 2. RELATED WORK For all these difficulties, active depth sensors have been often preferred when building complex behaviors as, e.g., interactive object learning (Lyubova et al., 2015). The goal of this work is to improve the stereo perception of the iCub robot in order to make dense 3D information usable in action–perception loops. In par- ticular, we show an example object tracking application. Since the main problems affecting the stereo perceptual system of the robot were slowness and robustness, in this work we aim at improving the disparity computation step with respect to these aspects. For camera calibration and image rectification, we adopt the currently implemented technique on the iCub robot, described in Fanello et al. (2014) and briefly resumed in Section 3.1. Depth is a natural cue for object segmentation. For example, consider this common situation for a humanoid robotic platform: a human stands in front of the robot showing to it an object to be recognized or grasped. Both motion- and appearance-based approaches to focus the robot’s attention on the object of interest would impose many constraints even on this simple scenario. Color-based methods work under strict assumptions on the light conditions, kind of background (preferably a table or a wall) and generally fail in cluttered setting. Model-based methods can overcome many of the above limitations, but, more importantly, require the shape type of the object to be known a priori (Greggio et al., 2011). Motion cues are an alternative [see, e.g., Ciliberto et al. (2011) and Kumar et al. (2015)] but clearly require objects to be moving and ego-motion compensation. Perhaps the most distinguishing feature is simply the fact that the object of inter- est is closer to the robot than the background. Indeed, depth information has been exploited in similar robotics settings in the past (Goerick et al., 2005, 2006; Wersing et al., 2006, 2007, 2008; Leitner et al., 2012a; Rudinac et al., 2012). l y We decided to rely on the LIBELAS library because, com- paring to other local dense stereo matching methods, which can be faster [see, e.g., OpenCV’s Block Matching algorithm implementation (Bradski and Kaehler, 2013), for which a GPU accelerated version is also available], LIBELAS provides better matching results in texture-less regions. Indeed, the matching performance of algorithms based on local correspondences is affected by the window size, which is particularly critical in real-world application scenarios, where scenes are characterized by large texture-less elements. Citation: Pasquale G, Mar T, Ciliberto C, Rosasco L and Natale L (2016) Enabling Depth-Driven Visual Attention on the iCub Humanoid Robot: Instructions for Use and New Perspectives. Front. Robot. AI 3:35. doi: 10.3389/frobt.2016.00035 June 2016 \| Volume 3 \| Article 35 Frontiers in Robotics and AI \| www.frontiersin.org 1 Depth-Driven Attention on the iCub Pasquale et al. are either proprietary (Einecke and Eggert, 2010, 2013, 2015) or rely on Convolutional Neural Network architectures and require dedicated GPUs (Mayer et al., 2015).h A different approach proposed the use of machine learn- ing methods as Neural Networks to overcome the calibration problem. In Leitner et al. (2012b), the authors are able to learn a network that maps the 2D projections of a point onto the left and right cameras to its 3D position in the world, being the cameras orientation approximately provided by the robot kinematics. Combining this with an object detection technique that segments an object in both cameras, they are able to localize the object without the need for any calibration. However, by considering only a single point as the object’s centroid, they do not solve the problem of 2-dimensional matching and disparity computation. Moreover, they rely on an appearance-based segmentation algo- rithm that suffers from the limitations of color-based methods mentioned above.fit Therefore, we have integrated the ELAS algorithm, which is available with an open-source license as a self-contained, opti- mized C++ library, LIBELAS,3 into the iCub software system and tested it in different applications, making them readily available for the benefit of the community. In this technical report, we describe the software we have implemented and present a set of quantitative and qualitative experiments to assess the efficacy of the ELAS algorithm in a realistic robotic setting. 2. RELATED WORK In this setting, small windows can be uninformative but too large windows cause border bleeding artifacts and heavier computational time (Geiger et al., 2010). Instead, the ELAS algorithm overcome this problem by propa- gating to non-textured regions disparity information derived on a set of robust correspondences. Moreover, when compared to semi-global methods, including OpenCV’s implementation of Hirschmuller’s Semi-Global Block Matching (SGMB) algorithm (Hirschmuller, 2008) currently in use on the iCub platform, ELAS scales better with respect to the disparity search space, which is generally large in robotics application where the robots must per- ceive both close and far objects. Indeed, in contrast with SGBM, which computes matching costs at each pixel for the full disparity space image, ELAS reduces the search only to plausible values between neighboring support points’ disparities. Finally, accord- ing to recent experimental evidence (Sinha et al., 2014) (and to the KITTI benchmark), ELAS is remarkably fast on large images.h The issues involved by estimation of the disparity map on a humanoid robotic head are related to calibration, speed, and robustness. Indeed, since performing 2-dimensional searches for matching points in the two cameras is computationally expen- sive, a commonly adopted approach for vergent stereo systems (Hartley and Zisserman, 2003) is to first rectify the left and right images, in order to bring corresponding points to lie on the same scanline. Then, disparity can be computed by performing only horizontal searches. However, accurately estimating the recipro- cal position of the stereo pair, needed by the rectification step, can be difficult, particularly on a humanoid robotic head, where the pan and vergence of the robot eyes change continuously and the kinematic information is affected by uncertainty. The fol- lowing horizontal disparity computation step then remains the main computational bottleneck. At present, solutions providing the disparity map with the right trade-off between speed and accuracy are active subject of research. Finally, robustness to lighting conditions, poorly textured regions and inaccuracies in the rectification is another key requirement for an algorithm to be usable on a robotic platform. These are also the main reasons why LIBELAS has been the library of choice for many previous robotic applications. LIBELAS is adopted in Tombari et al. 3 http://www.cvlibs.net/software/libelas/ Frontiers in Robotics and AI \| www.frontiersin.org 3.1. Rectificationi Image rectification consists in the process of transforming a set of multiple images onto the same plane and is a fundamental step to most depth estimation algorithms. Rectification requires knowledge of both the intrinsic (camera specific) parameters of the two (or more) cameras and extrinsic parameters, i.e., the position and orientation of the cameras with respect to the world reference frame. More formally, any 3D point with coordinates X = (x, y, z, 1)⟙ with respect to the world reference frame is mapped on the camera image plane x = (u, v, 1)⟙ via the transformation Based on these motivations, we decided to integrate LIBELAS into the iCub’s depth perception system. The spirit of our work is close to Leitner et al. (2008), where the authors adapt the algorithm for disparity computation presented in Bernardino and Santos-Victor (2002) to work on a humanoid robotic head and benchmark its performance and computational efficiency. In this work, we validate the integration of LIBELAS in the iCub’s stereo perceptual system. We focus in particular to situations where 3D information is used in visuo-motor tasks, specifically object detection and tracking in indoor settings. The contribution of the paper is twofold: we first improve the iCub stereo percep- tual system, by providing the possibility of employing LIBELAS, beyond OpenCV’s SGBM, for disparity computation; by building on faster and more robust 3D information, we are then able to implement a disparity-based tracker that allows for detection of never-seen objects on cluttered and dynamic background; this, finally, allows us to realize an interactive object learning applica- tion that we use also for semi-automatic ground-truth collection. All the code of the applications that we show in this work is made publicly available to the iCub community.l sx = PX (1) (1) where s ∈ R is a scaling factor, P ∈ R3×4 is the Projection Matrix that can be factorized as P = K[R\|t], with K ∈ R3×3 and [R\|t] ∈ R3×4 the matrices of intrinsic and extrinsic parameters, respectively.f Intrinsic and extrinsic parameters can be estimated off-line during a calibration phase. However, while intrinsic parameters are camera specific and do not change over time, on the iCub the relative pose of the cameras changes when the robot fixates objects at different distance. To circumvent this issue, Fanello et al. (2014) pre-compute the intrinsic parameters matrices KL and KR using standard calibration procedure (Hartley and Zisserman, 2003). 3.1. Rectificationi Extrinsic parameters are then re-estimated at runtime. This calibration employs SIFT matching to estimate the Fundamental Matrix between the two camera planes [details are in Fanello et al. (2014)]. To achieve real-time performance, Fanello et al. (2014) exploit the robot’s kinematics to approximate the camera transformation between subsequent frames and perform re-calibration using SIFT matching at a lower frame rate. This procedure is implemented in the SfM (Structure from Motion) module included in the iCub stereo-vision repository.4 Once the projection matrices PL and PR associated with the left and right cameras are known, the corresponding images can be mapped onto the same plane, i.e., they are recti- fied. They are, therefore, ready for the subsequent stage: disparity estimation. In Section 3 we briefly review the processing steps currently adopted on the iCub for depth estimation; in Section 4 we describe the application that we devised to focus the robot’s atten- tion on the closest object in its workspace. Finally, in Section 5 we experimentally demonstrate the effectiveness of this approach. 4 https://github.com/robotology/stereo-vision 2. RELATED WORK (2011) for an object recog- nition task, and in Van den Bergh and Van Gool (2012) it is used in conjunction with color and optical flow to provide a real-time June 2016 \| Volume 3 \| Article 35 Frontiers in Robotics and AI \| www.frontiersin.org 2 Depth-Driven Attention on the iCub Pasquale et al. super-pixel segmentation of the scene. In Mitzel and Leibe (2012) and Baumgartner et al. (2013) LIBELAS is at the basis of an algorithm for people detection and inference engine that learns interactions between people and objects. Moreover, (Lin et al., 2012) show that LIBELAS can be implemented on an embedded ARM-based processor with real-time on mid-resolution images. Frontiers in Robotics and AI \| www.frontiersin.org 3. DEPTH ESTIMATION In this section we briefly describe the depth estimation pipeline adopted in this work. Following the standard approach from multi-view geometry (Hartley and Zisserman, 2003), the process is organized into two main phases: image rectification and dispar- ity computation.hi 3.2.2. Bayesian Inferenceh The second phase relies on the two-view geometry parameters estimated in Section 1 and the support points S to predict the most likely disparity values for the remaining image pixels. In particular, the authors adopt a Bayesian framework to model the likelihood: p d x x x L R n R ( ... ) ( ) ( ) ( ) \| , , , , 1 S of observing a disparity d for a given point x(L) on the left image and a set of candidate corresponding points x x R n R 1 ( ) ( ) ... , , on the right image. The most likely disparity value is, therefore, esti- mated by factorizing such likelihood and performing a Maximum A Posteriori (MAP) procedure. 1. Filtering: a 5 × 5 Gaussian filter (σx = σy = 1.5) is applied to the disparity map in order to smooth the surfaces. Then, as we are interested in the foremost object, background is suppressed by putting to black/zero all pixels whose grayscale value is under a threshold, set to 30 ÷ 50, so that farther (darker) pixels are removed. A sequence of 4 dilation and 2 erosion operations with a 3 × 3 kernel matrix, interleaved by another 5 × 5 Gaussian filtering (σx = σy = 2) follows, in order to suppress small blobs and fill holes in large blobs. As pointed out in Geiger et al. (2010), this procedure can be carried out independently for each image point and it is fast and parallelizable. Clearly, this is a critical feature for the robotic setting, where the disparity estimation process must be com- puted at frame rate. LIBELAS offers two presets of parameters, MIDDLEBURY and ROBOTICS, the latter being specifically tuned for higher robustness to dynamic lighting conditions and real-world scenes. We integrated the OpenMP parallelization of LIBELAS, available at the same website of the library, in the stereo-vision repository. 2. i 2. Blob selection: a simple routine to localize the closest blob of “reasonable” size (i.e., larger than a predefined threshold to avoid spurious detections) is devised. Iteratively:i 3.2. Disparity Computation with ELAS 3.2. Disparity Computation with ELAS Disparity estimation consists in the process of evaluating the displacement of pixels from one (rectified) image to the other. Disparity is usually computed after rectification since at this stage the corresponding image points from the left and right cameras lie on the same scanline and, therefore, matching can be restricted to horizontal lines. A variety of disparity estimation methods have been proposed in the literature. The Efficient Large-Scale Stereo (ELAS) Matching algorithm proposed in Geiger et al. (2010) consists in the following two phases: The rectification step estimates the geometrical transforma- tion matrix relating left and right image planes in order to align the epipolar lines with the image scanlines. After this operation, horizontal disparity is computed for each pixel in the left (right) rectified image, by searching its correspondent point in the right (left) rectified image along its scanline. The resulting disparity map provides an estimation of the 3D structure of the scene as a cloud of points (whose projections end up on the image pixels) with respect to the observer. To recover the 3D position of the point corresponding to a specific pixel, the camera’s extrinsic parameters can be used, in combination with its disparity, to re-project it. 1. A set of robust support points is detected and matched across the two images. Regarding the estimation of the camera parameters, we follow the procedure described in Fanello et al. (2014), which involves online calibration starting from the initial off-line calibration (online re-calibration is required when the reciprocal position of the eyes change). As mentioned already, for disparity estima- tion, we adopt the Efficient Large-scale Stereo (ELAS) Matching algorithm proposed in Geiger et al. (2010). 2. Dense disparity on a uniform group of points is obtained from these support points in a Bayesian framework. June 2016 \| Volume 3 \| Article 35 3 Depth-Driven Attention on the iCub Pasquale et al. In this section we offer a very brief overview of the ideas underlying ELAS, while referring the reader to the original paper for a more detailed description of the algorithm (Geiger et al., 2010). object recognition (Section 6.1). We are exploiting this applica- tion to acquire a dataset of images depicting multiple objects held in the hand of a human teacher who is showing them to the iCub, which is going to be released soon. 5 https://github.com/robotology/segmentation/tree/master/dispBlobber 3.2. Disparity Computation with ELAS In fact, a similar strategy, based however on independent motion detection, was previously explored on the iCub robot (Fanello et al., 2013a,b) for previous releases of the iCubWorld dataset. We show that in such an application disparity information results in a more reliable and stable cue. 3.2.1. Support Pointshi The first phase of ELAS is performed on a predetermined grid on the image plane, where candidate points are selected depending on their local appearance. To do so, the authors used a vector of local orientations (response to oriented Sobel filters) and performed robust matching between such feature vectors to eliminate unstable pairs of points. The outcome of this stage is a set S of points s = (u, v, d)⟙ that encode the position (u,v) of a support point on the left (rectified) image and the disparity d with respect to the matched point on the right (rectified) image. In the following, we describe the algorithm we devised to seg- ment the object closest to the iCub cameras and focus the robot’s gaze on it. 4.1. Foremost Object Segmentation To achieve real-time performance, we reduced the post-processing operations on the disparity map to the minimum. Therefore, we implemented a proof-of-concept segmentation algorithm that can provide a reasonably stable and accurate blob around the closest proto-object in the scene. We are aware of the existence of more sophisticated algorithms, which may provide more precise segmentations [e.g., Li et al. (2013)]. These algorithms could be easily plugged in the present pipeline to realize other behaviors that require more accurate segmentation. Below we report the algorithm together with the parameters currently used on the robot. The code, implemented by using standard OpenCV func- tions, has been made available in the dispBlobber module, part of the iCub segmentation repository.5 Frontiers in Robotics and AI \| www.frontiersin.org 4.2. Foremost Object Fixation j At this stage, the foremost object’s centroid on the reference (left) image is available for further usage. In this application, the dis- pBlobber module asks the SfM module to re-project the com- puted centroid to its corresponding 3D position in the Cartesian space. This 3D point is finally fed to the module in charge of controlling the robot’s gaze [iKinGazeCtrl (Pattacini, 2011)], which moves the iCub’s eyes accordingly. This solution is more accurate than performing the triangulation using the robot kin- ematics, because the SfM module performs a visual estimation of the relative orientation of the cameras [using the algorithm in Fanello et al. (2014)]. It can be noticed that the LIBELAS implementation is fast (achieving a 15 fps rate with respect to the 5 fps provided by SGBM) and robust enough to allow for further applications of the computed disparity, such as the disparity-driven attention behavior described in the following. As a consequence, the head and eyes positions of the robot are continuously updated to keep the focus of attention fixed on the required 3D target, i.e., the closest object in the visual field, while the human moves it in front of the cameras. This pipeline is looped in real time so that the robot is able to follow the closest object with the gaze. It is then clear that relying on a fast and robust disparity map (eventually at the expenses of some sub- pixel precision) in this kind of application is critical, and the reported results confirm that LIBELAS is suited to this task.f For these experiments, the disparity range was set to [0,127] for both ELAS and SGBM. In Table 2, we report the parameters of the SGBM algorithm, which have been tuned to the specific iCub’s indoor setting. Those not reported were left to their default value [see OpenCV’s documentation (Bradski and Kaehler, 2013)]. In the case of LIBELAS implementation, we chose the ROBOTICS preset of parameters offered by the library. In order to speed up computations we set the post_process_only_ left and the subsampling parameters to true and employed the OpenMP accelerated version of the library. All other parameters were left to their default value. 4. OUR BENCHMARK: DISPARITY-DRIVEN ATTENTION (a) find the 2D image location of the brightest (closest) pixel as a vector (u,v), (b) generate a candidate blob from the seed pixel by aggrega- ting all neighboring pixels with disparity value between the brightest one (d) and a lower threshold defined by 0.9*d, For validation, we consider the following benchmark. We designed a segmentation procedure based on the disparity map. This procedure identifies distinct 3-dimensional entities in the scene and it focuses the robot’s gaze toward the object that is closest to the cameras. By following this strategy, we were able to implement a simple but effective tracking algorithm that continuously focuses the robot’s attention and gaze toward the closest object in the scene, while at the same time providing also an approximate visual segmentation.i (c) suppress (putting to black/zero) the aggregated region if its size is lower than a threshold (in our experiment set to 300 pixels for 320 × 240 images and to 1400 pixels for 640 × 480 images), (d) start again from a until one region satisfying the size re- quirement is found, and We first employed this basic tracking system to perform a qualitative and quantitative analysis of the disparity map produced by ELAS in a real-world indoor robotic setting (Sections 5.1 and 5.2). Then, within this general scenario we defined a reliable protocol to acquire ground-truth for visual (e) check whether the returned region is composed by a single blob or by multiple connected blobs, by selecting only the largest one satisfying the size threshold fixed June 2016 \| Volume 3 \| Article 35 4 Pasquale et al. Depth-Driven Attention on the iCub 4.2. Foremost Object Fixation We note also that this is a very basic (yet effective) implemen- tation for a disparity-driven attention system and that further improvements as, e.g., applying a Kalman filter to the trajectory of the 3D centroid, could be introduced to smooth and stabilize the resulting tracking system. Frontiers in Robotics and AI \| www.frontiersin.org 5.1. Real-Time Disparity-Driven Segmentation above, in the latter case. This is achieved by first compu- ting the contours of the region and then iterating on the connected components, discarding those whose area is under the threshold. We collected a sequence of 200 frames at the resolution of 640 × 480, acquired across 2 minutes and recorded from the iCub cameras while a human subject was moving his hand in front of the robot. We computed the performance of the disparity- based segmentation protocol described in Section 1 when using ELAS or SGBM. In Table 1, we report the computational time required on our platform [Intel(R) Core(TM) i7 3770QM CPU at 3.40 GHz with 16-GB RAM] to perform the disparity estimation and segmentation, averaged over the whole acquisition sequence. We also report the ratio of “missed” blobs: the ratio of frames for which the segmentation algorithm failed to detect any blob. Our experiments show that ELAS significantly outperforms the baseline and moreover leads to better segmentation. In Figure 1, we report three exemplar images from this sequence: the first row depicts the rectified images acquired from the left camera (those from the right camera are not reported); the second and third rows report, respectively, the disparity maps and the resulting segmentation, obtained with ELAS (odd columns) or SGBM (even columns). We collected a sequence of 200 frames at the resolution of 640 × 480, acquired across 2 minutes and recorded from the iCub cameras while a human subject was moving his hand in front of the robot. We computed the performance of the disparity- based segmentation protocol described in Section 1 when using ELAS or SGBM. In Table 1, we report the computational time required on our platform [Intel(R) Core(TM) i7 3770QM CPU at 3.40 GHz with 16-GB RAM] to perform the disparity estimation and segmentation, averaged over the whole acquisition sequence. 3. Computing the blob’s centroid and ROI: if any blob is found, its center of mass and its smallest enclosing rectangular bound- ing box (ROI), with an arbitrary margin, are computed.fi 4. Averaging over a temporal buffer: finally, the centroid and the ROI are averaged over a buffer of n frames (with, e.g., n = 3) in order to mitigate isolated mis-detections. June 2016 \| Volume 3 \| Article 35 5.2. Disparity-Driven Visual Attention Bottom: segmentation of the closest blob in the scene. TABLE 2 \| SGBM parameter setting. TABLE 2 \| SGBM parameter setting. we recorded the coordinates (umodel, vmodel) of the center of the red ball in the same image plane, provided by the red-ball detector. TABLE 2 \| SGBM parameter setting. OpenCV’s parameter name Value preFilterCap 63 SADWindowSize 7 P1 8 ⋅ 7 ⋅ 7 P2 32 ⋅ 7 ⋅ 7 uniquenessRatio 15 speckleWindowSize 50 speckleRange 16 disp12MaxDiff 0 The left column reports the parameter name in the OpenCV implementation and the right column its value in our application. Figure 3 reports the image plane coordinates (top rows) with red and blue colors, respectively, for disparity and model-based tracker, and their difference (bottom rows). Notice that, in some frames, ELAS estimates the wrong position for the blob (sudden jumps in the red curves); instead, the red ball tracker fails to detect its target within a 2 s interval around t = 18 s in the plot. In Figure 2, we provide a short sequence showing the ELAS failure around t = 2.6 s. Indeed, it does happen that, since the robot is moving and we are in an uncontrolled setting, the disparity map is affected by noise that cause false blob detections. Notice, however, that these errors occur on isolated frames and can be removed by filtering the 2D image position detected by raw segmentation. In Figure 4, we report instead a short sequence extracted from the interval in which the red ball detector fails: in this case, the error is due to a constant mis-detection caused by the slightly changed lighting condition. This unexpected behavior by the way offers the occasion to highlight that the proposed approach based on the disparity cue for tracking and segmentation is not only a viable solution but can also be even more robust than appearance-based information when the 3D position of the target is a more stable signal than its color. The left column reports the parameter name in the OpenCV implementation and the right column its value in our application. order to provide the gaze controller with a more frequent feed- back and the disparity range was reduced to [0, 95]. For SGBM, we used the same parameter set of the previous experiment. 5.2. Disparity-Driven Visual Attention For LIBELAS, there was no need to enable the subsampling since the lower resolution already allowed to achieve frame-rate performance (30 fps) on our platform. Experiments with SGBM were performed off-line because the lower efficiency (~10 fps) did not allow for a smooth tracking. As a reference ground-truth to compare with the result of the disparity-based segmentation, we used the output of a model-based object tracker (Taiana et al., 2010). In particular, we used a red ball for which a well-established particle filter tracker is implemented in the pf3dTracker module, part of the icub-basic-demos repository.6 As the operator moved the red ball in front of the robot, the gaze was focused toward it (since it was the closest object in the scene). The information about its estimated position was acquired independently using the disparity-based segmentation procedure described above and the color/shape-based particle filter tracker (see Figure 2). We recorded the coordinates (udisp, vdisp) of the closest blob’s centroid on the left image plane as provided at each frame by the segmen- tation module on top of ELAS disparity map. At the same time, Figure 5 shows the same quantities of Figure 3 but computed on the disparity map provided by SGBM. We computed the dis- parity map off-line on the same set of rectified images acquired when tracking with the ELAS algorithm. As can be clearly noticed, the unstable behavior of the disparity produced by SGBM is not sufficient to provide a fast and reliable signal to track the ball. 5.2. Disparity-Driven Visual Attention p y In this section we test the presented simple attention system driven by disparity information, whose underlying principle is to keep the robots’ gaze focused on the closest object in the scene. In particular, we consider the following setting: a human moves an object in front of the robot camera, and we evaluate the stabil- ity of the resulting “tracking” application. In this section, we present a qualitative as well as quantitative analysis of the depth estimation process described in Section 3, with particular focus on the improvements provided by the ELAS algorithm, which represents the novel element of the pipeline for disparity computation. As we are mainly concerned in assessing the possibility to employ this algorithm in practical, real-time, robotics applications, we first evaluate the disparity- based segmentation protocol introduced in Section 1 and then we evaluate this approach for disparity-driven visual attention behavior. We employed the pipeline described in Section 4. In the cur- rent experiment, we used low-resolution images (320 × 240) in TABLE 1 \| Average computational time (splitted for disparity computation and segmentation) and percentage of blobs missed by LIBELAS and SGBM over the sequence represented in Figure 1. SGBM ELAS Time disparity [ms] 190 60 Time segmentation [ms] 20 5 Time total [ms] 210 65 Missed blobs [%] 11.2 2 June 2016 \| Volume 3 \| Article 35 TABLE 1 \| Average computational time (splitted for disparity computation and segmentation) and percentage of blobs missed by LIBELAS and SGBM over the sequence represented in Figure 1. For our experiments, we employed the OpenCV (Bradski and Kaehler, 2013) implementation of the Semi-Global Block Matching algorithm (SGBM) (Hirschmuller, 2008) as a base- line to compare the performance of ELAS. Indeed, SGBM was considered the “off-the-shelf” disparity estimation algorithm for the iCub robot used in the SfM module in the iCub stereo- vision repository [see Fanello et al. (2014)]. 5 Depth-Driven Attention on the iCub Pasquale et al. FIGURE 1 \| Examples from a sequence recorded on the iCub’s cameras, with fixed eyes and head. Top: left rectified images. Middle: disparity maps computed by LIBELAS and OpenCV’s SGBM methods. Bottom: segmentation of the closest blob in the scene. FIGURE 1 \| Examples from a sequence recorded on the iCub’s cameras, with fixed eyes and head. Top: left rectified images. Middle: disparity maps computed by LIBELAS and OpenCV’s SGBM methods. 6 https://www.github.com/robotology/icub-basic-demos Frontiers in Robotics and AI \| www.frontiersin.org 6. APPLICATIONS OF DISPARITY ON HUMANOID ROBOTS In this section we show how the disparity-driven attention system described in Section 5 can be employed to improve the robot perception of the surrounding environment. In particular, we consider a basic interaction between the robot June 2016 \| Volume 3 \| Article 35 Frontiers in Robotics and AI \| www.frontiersin.org 6 Depth-Driven Attention on the iCub Pasquale et al. FIGURE 2 \| Frames extracted from the sequence represented in Figure 3 around t = 2.6 s, when LIBELAS fails to detect the closest object. Top: output of the red ball tracker. Middle: disparity map. Bottom: disparity segmentation. FIGURE 2 \| Frames extracted from the sequence represented in Figure 3 around t = 2.6 s, when LIBELAS fails to detect the closest object. Top: output of the red ball tracker. Middle: disparity map. Bottom: disparity segmentation. FIGURE 3 \| Coordinates of the red ball target, recorded while the human operator was moving it in front of the robot. Top: u and v coordinates of the closest blob’s centroid on the left image plane (red trace), provided at each frame by the disparity segmentation module, and of the center of the red ball in the same image plane (blue trace), provided by the red ball detector. Bottom: difference between the two. LIBELAS is used to provide the disparity map. FIGURE 3 \| Coordinates of the red ball target, recorded while the human operator was moving it in front of the robot. Top: u and v coordinates of the closest blob’s centroid on the left image plane (red trace), provided at each frame by the disparity segmentation module, and of the center of the red ball in the same image plane (blue trace), provided by the red ball detector. Bottom: difference between the two. LIBELAS is used to provide the disparity map. 6.1. On the Fly Object Recognition 6.1. On the Fly Object Recognition 6.1. On the Fly Object Recognition and a human teacher or the situation in which the robot needs to visually select objects on a table. In this section, our observations are mainly qualitative. and a human teacher or the situation in which the robot needs to visually select objects on a table. In this section, our observations are mainly qualitative. We focus on the setting used in Fanello et al. (2013), where a human teacher shows new objects to the iCub in order for the June 2016 \| Volume 3 \| Article 35 Frontiers in Robotics and AI \| www.frontiersin.org 7 Depth-Driven Attention on the iCub Pasquale et al. FIGURE 4 \| Frames extracted from the sequence represented in Figure 3 in the period from t = 18 s to t = 20 s, when the red ball tracker fails to detect its target. Top: output of the tracker. Middle: disparity map. Bottom: disparity segmentation. FIGURE 5 \| Similar to Figure 3, but in this case SGBM provides the disparity map. The blue trace is the (u,v) coordinate compute from the color tracker, while the red trace is obtained using the blob detector on the disparity map. Because of the low efficiency, the blob detector is computed off-line. For comparison in this experiment, we use the same sequence of Figure 3. FIGURE 4 \| Frames extracted from the sequence represented in Figure 3 in the period from t = 18 s to t = 20 s, when the red ball tracker fails to detect its target. Top: output of the tracker. Middle: disparity map. Bottom: disparity segmentation. robot to focus its attention toward them and learn their visual appearance. Communication between the human and the robot occurs through speech, i.e., commands and object labels are ver- bally provided by the human teacher [see Fanello et al (2013b) for more details and a throughout overview of the system]. We show how, by replacing the motion-based segmentation and tracking, used in Fanello et al. (2013b), with the disparity-based approach described in Section 4 we are able to remarkably improve the FIGURE 4 \| Frames extracted from the sequence represented in Figure 3 in the period from t = 18 s to t = 20 s, when the red ball tracker fails to detect its target. Top: output of the tracker. Middle: disparity map. Bottom: disparity segmentation. 6.1. On the Fly Object Recognition FIGURE 5 \| Similar to Figure 3, but in this case SGBM provides the disparity map. The blue trace is the (u,v) coordinate compute from the color tracker, while the red trace is obtained using the blob detector on the disparity map. Because of the low efficiency, the blob detector is computed off-line. For comparison in this experiment, we use the same sequence of Figure 3. FIGURE 5 \| Similar to Figure 3, but in this case SGBM provides the disparity map. The blue trace is the (u,v) coordinate compute from the color tracker, while the red trace is obtained using the blob detector on the disparity map. Because of the low efficiency, the blob detector is computed off-line. For comparison in this experiment, we use the same sequence of Figure 3. robot to focus its attention toward them and learn their visual appearance. Communication between the human and the robot occurs through speech, i.e., commands and object labels are ver- bally provided by the human teacher [see Fanello et al. (2013b) for more details and a throughout overview of the system]. We show how, by replacing the motion-based segmentation and tracking, used in Fanello et al. (2013b), with the disparity-based approach described in Section 4, we are able to remarkably improve the June 2016 \| Volume 3 \| Article 35 Frontiers in Robotics and AI \| www.frontiersin.org 8 Depth-Driven Attention on the iCub Pasquale et al. FIGURE 6 \| Three frames (extracted from the attached video) showing the effectiveness of the proposed segmentation system. Top: resulting crop in the left rectified frame, labeled by the operator using speech. Middle: disparity map. Bottom: segmented disparity blob, its centroid, and the enclosing ROI. FIGURE 6 \| Three frames (extracted from the attached video) showing the effectiveness of the proposed segmentation system. Top: resulting crop in the left rectified frame, labeled by the operator using speech. Middle: disparity map. Bottom: segmented disparity blob, its centroid, and the enclosing ROI. FIGURE 6 \| Three frames (extracted from the attached video) showing the effectiveness of the proposed segmen the left rectified frame, labeled by the operator using speech. Middle: disparity map. Bottom: segmented disparity blob, its c (extracted from the attached video) showing the effectiveness of the proposed segmentation system. Top: resulting crop ed by the operator using speech. Middle: disparity map. Bottom: segmented disparity blob, its centroid, and the enclosing ROI. 6.1. On the Fly Object Recognition usability of the resulting application and the naturalness of the human-robot interaction. train/benchmark off-line visual recognition systems on the robot’s visual experience. This is particularly useful because it allows to collect object recognition ground-truth by consistently reducing the effort of the manual annotation phase: indeed not only the label is provided verbally by the teacher but also the ROI around the object is automatically provided by the disparity segmentation. The acquisition application through which we are currently build- ing a large-scale visual recognition dataset of objects part of the iCub’s world is available online at the iCubWorld8 repository. In Figure 6, we report three frames extracted from three corresponding sequences, recorded while tracking three dif- ferent objects following the disparity-based strategy previously described. The top row shows the output of the pipeline: the object (in this case a cup, a toy octopus, and a lemon squeezer) is localized in the scene using the ROI provided by the disparity segmentation module, the label being provided verbally by the human teacher. The middle row reports the associated dispar- ity map by ELAS and the bottom row reports its segmentation, together with the centroid, that is used for the tracking (red dot, average over three frames; green dot, current frame) and the ROI, used for the segmentation (averaged over three frames to account for spurious mis-segmentations). The ROI is computed as the smallest rectangular region enclosing the segmented blob, with a margin of 20 pixels.h We conclude that the strategy proposed in this report is a viable alternative to the motion-based tracker employed in Ciliberto et al. (2013), Fanello et al. (2013a,b) and Pasquale et al. (2015). Another advantage is that this strategy is more effective because it does not require the human teacher to continuously shake the object in front of the robot (as it is the case when motion is used instead). This results in a more natural interaction, where the user is free to move the object slowly or keep it still at all. The segmentation is more accurate, especially because it allows to improve the detection using low-pass filtering, and the quality of the acquired images is higher. The full video, recorded from the iCub cameras while the robot was focusing on the objects to be learned and showing the differ- ent stages of the tracking pipeline, is available as Supplementary Material to this paper. 7 https://github.com/robotology/onthefly-recognition Frontiers in Robotics and AI \| www.frontiersin.org 8 https://github.com/GiuliaP/icubworld 6.1. On the Fly Object Recognition Notice that the disparity-driven control of attention results in stable object tracking.hi 6.2. Object Exploration and Manipulation Finally, we consider a setting in which the robot is standing in front of a table and uses disparity to distinguish separate objects. In this setting, by relying on LIBELAS disparity map, we could reconstruct the scene in front of the robot and the system could determine the optimal hand pose for a reliable grasp (Gori et al., 2014). The uses of this tracking application can be multiple: first of all, we plugged it into our object recognition pipeline to teach new objects to the robot (code available in the iCub onthefly-rec- ognition repository7). Then, we are currently employing it as a fast and natural method to collect large-scale annotated datasets of images containing objects “as seen by the robot,” to be used to June 2016 \| Volume 3 \| Article 35 9 Pasquale et al. Depth-Driven Attention on the iCub FIGURE 7 \| Top left: rectified frame recorded from the iCub’s left camera while the robot was looking at a table in front of it. Top right: segmentation of the three closest objects on the table obtained from the disparity map of the scene (bottom). FIGURE 7 \| Top left: rectified frame recorded from the iCub’s left camera while the robot was looking at a table in front of it. Top right: segmentation of the three closest objects on the table obtained from the disparity map of the scene (bottom). In Figure 7, we report the left rectified image (top left) and the corresponding segmentation (top right), obtained by putting a threshold on the disparity map (bottom). For the purpose of demonstration, such threshold was chosen manually; however, in a real application more sophisticated processing of the disparity map could applied to cluster 3D point clouds and better detect separate objects. behaviors of the humanoid robotic system, such as interaction with the human or with the surrounding environment. The sys- tem described in this paper for depth estimation is made publicly available: it can be used as an off-the-shelf solution for the benefit of the whole iCub community and, with minor adaptations, on other robots. 7. CONCLUSION All authors have contributed to the conceptual design or devel- opment of the work described in this paper and participated in drafting and revising its content. They also approve publication of the paper and agree to be accountable for all aspects of the work described therein. In this work, we have described the current system implemented on the iCub robot to perform depth estimation and how it ben- efits from the recent incorporation of the state-of-the-art dispar- ity computation algorithm ELAS (Geiger et al., 2010). We have evaluated a few real applications of the information provided by the disparity map produced by ELAS to typical robotics settings. We experimentally demonstrated that this approach is computa- tionally efficient and robust for the real-world scenario. The work presented in this report may be at the basis of more complex SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/frobt.2016.00035 The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/frobt.2016.00035 REFERENCES Ciliberto, C., Fanello, S., Santoro, M., Natale, L., Metta, G., and Rosasco, L. (2013). “On the impact of learning hierarchical representations for visual recognition in robotics,” in IEEE/RSJ International Conference on Intelligent Robots and Systems (IROS). Tokyo. Baumgartner, T., Mitzel, D., and Leibe, B. (2013). “Tracking people and their objects,” in IEEE Conference on Computer Vision and Pattern Recognition (CVPR) (Portland, OR: IEEE Computer Society), 3658–3665. 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Syst. 9. doi:10.5772/54657 Conflict of Interest Statement: The work described in this paper was conducted in the absence of any commercial or financial relationship that can be construed as a potential conflict of interest. Conflict of Interest Statement: The work described in this paper was conducted in the absence of any commercial or financial relationship that can be construed as a potential conflict of interest. Li, C., Lu, L., Hager, G. D., Tang, J., and Wang, H. (2013). “Robust object tracking in crowd dynamic scenes using explicit stereo depth,” in Asian Conference on Computer Vision (ACCV) (Daejeon: Springer), 71–85. Copyright © 2016 Pasquale, Mar, Ciliberto, Rosasco and Natale. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Lin, K. W., Lau, T. K., Cheuk, C. M., and Liu, Y. (2012). “A wearable stereo vision system for visually impaired,” in IEEE International Conference on Mechatronics and Automation (ICMA), 1423–1428. Lyubova, N., Ivaldi, S., and Filliat, D. (2015). From passive to interactive object learning and recognition through self-identification on a humanoid robot. Auton. Robots 40, 33–57. doi:10.1007/s10514-015-9445-0 June 2016 \| Volume 3 \| Article 35 Frontiers in Robotics and AI \| www.frontiersin.org 11
https://openalex.org/W4230369854	https://hal.inrae.fr/hal-02544162/file/2020_mesmin_PeerJ.pdf	English	null	<i>Ooctonus vulgatus</i> (Hymenoptera, Mymaridae), a potential biocontrol agent to reduce populations<i> </i>of <i>Philaenus spumarius</i> (Hemiptera, Aphrophoridae) the main vector of <i>Xylella fastidiosa</i> in Europe	null	2,019	cc-by	7,777	Ooctonus vulgatus (Hymenoptera, Mymaridae), a potential biocontrol agent to reduce populations of Philaenus spumarius (Hemiptera, Aphrophoridae) the main vector of Xylella fastidiosa in Europe Xavier Mesmin, Marguerite Chartois, Guénaëlle Genson, Jean-Pierre Rossi, Astrid Cruaud, Jean-Yves Rasplus To cite this version: Xavier Mesmin, Marguerite Chartois, Guénaëlle Genson, Jean-Pierre Rossi, Astrid Cruaud, et al.. Ooctonus vulgatus (Hymenoptera, Mymaridae), a potential biocontrol agent to reduce populations of Philaenus spumarius (Hemiptera, Aphrophoridae) the main vector of Xylella fastidiosa in Europe. PeerJ, 2020, 8, pp.e8591. 10.7717/peerj.8591. hal-02544162 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02544162 https://hal.inrae.fr/hal-02544162v1 Submitted on 16 Apr 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Ooctonus vulgatus (Hymenoptera, Mymaridae), a potential biocontrol agent to reduce populations of Philaenus spumarius (Hemiptera, Aphrophoridae) the main vector of Xylella fastidiosa in Europe Xavier Mesmin1,2, Marguerite Chartois2, Guénaëlle Genson2, Jean-Pierre Rossi2, Astrid Cruaud2 and Jean-Yves Rasplus2 Xavier Mesmin1,2, Marguerite Chartois2, Guénaëlle Genson2, Jean-Pierre Ross Astrid Cruaud2 and Jean-Yves Rasplus2 1 AGAP, INRAE, CIRAD, Montpellier SupAgro, Univ Montpellier, San Giuliano, France 2 CBGP, INRAE, CIRAD, IRD, Montpellier SupAgro, Univ Montpellier, Montpellier, France Subjects Agricultural Science, Biodiversity, Entomology, Spatial and Geographic Information Science Keywords Insect vector, Oophagous, Meadow spittlebug, Parasitoid, Biological control, Natural regulation How to cite this article Mesmin X, Chartois M, Genson Géë, Rossi J-P, Cruaud A, Rasplus J-Y. 2020. Ooctonus vulgatus (Hymenoptera, Mymaridae), a potential biocontrol agent to reduce populations of Philaenus spumarius (Hemiptera, Aphrophoridae) the main vector of Xylella fastidiosa in Europe. PeerJ 8:e8591 http://doi.org/10.7717/peerj.8591 INTRODUCTION Xylella fastidiosa (Wells, 1987) is a xylem-dwelling insect-borne bacterium that originates from the Americas, infects more than 500 species of plants (EFSA, 2015) and causes a variety of scorch-like diseases in many cultivated species (Almeida & Nunney, 2015; EFSA, 2018; Sicard et al., 2018). Studies on the economic impact of X. fastidiosa have primarily focused on the wine and grape industries. Yield reduction and management costs to the California grape industry are estimated at more than US$100 million per year (Tumber, Alston & Fuller, 2014) and a potential introduction of the bacterium in Australia is estimated to cost up to AUD 7.9 billion over 50 years (Hafi et al., 2017). X. fastidiosa has been recently detected in Europe and is present in Italy (Saponari et al., 2013), France (Denancé et al., 2017), Spain (Olmo et al., 2017), and Portugal (DGAV, 2019). Furthermore, niche modelling has shown that a large part of Europe is climatically suitable for the bacterium (Godefroid et al., 2018; Godefroid et al., 2019). Hence, X. fastidiosa represents a serious threat to European agriculture and natural ecosystems. The spread of X. fastidiosa depends on several interacting factors, mainly insect vectors and plant communities as well as landscape, climate features and population dynamics of the bacterium itself (Krugner et al., 2019). As a consequence, disease management is complex. Reducing bacterium spread requires acting on a set of different biotic and abiotic factors (Almeida et al., 2005) and modelling approaches may help setting up effective strategies (Fierro, Liccardo & Porcelli, 2019). Here we focus on a possible management strategy to control populations of the most common vector of X. fastidiosa reported in Europe so far: the meadow spittlebug Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Aphrophoridae) (Saponari et al., 2014; Cornara et al., 2016). P. spumarius is highly polyphagous (Cornara, Bosco & Fereres, 2018), widely distributed in the Palearctic from sea level to high elevation (about 2,000 m; e.g., Halkka, Raatikainen & Vilbaste, 1975; Lees, Dent & Gait, 1983; Drosopoulos & Asche, 1991; Loukas & Drosopoulos, 1992; Quartau, Borges & André, 1992; Stewart & Lees, 1996; Drosopoulos & Remane, 2000), and was probably introduced to the New World (Whittaker, 1973). Its ability to acquire and transmit X. fastidiosa was previously demonstrated (Severin, 1950; Saponari et al., 2014; Cornara et al., 2016). So far, a few studies have assessed the impact of different insecticides to reduce juvenile populations of P. ABSTRACT As a vector of Xylella fastidiosa (Wells, 1987) in Europe, the meadow spittlebug Phi- laenus spumarius (Linnaeus, 1758) (Hemiptera, Aphrophoridae) is a species of major concern. Therefore, tools and agents to control this ubiquitous insect that develops and feeds on hundreds of plant species are wanted. We conducted a field survey of P. spumarius eggs in Corsica and provide a first report of Ooctonus vulgatus Haliday, 1833 (Hymenoptera, Mymaridae) as a potential biocontrol agent of P. spumarius in Europe. To allow species identification, we summarized the main characters distinguishing O. vulgatus from other European species of Ooctonus and generated COI DNA barcodes. Parasitism rates were variable in the four localities included in the survey but could reach 69% (for an average number of eggs that hatched per locality of 109). Based on the geographic occurrences of O. vulgatus obtained from the literature, we calibrated an ecological niche model to assess its potential distribution in the Holarctic. Obviously, several questions need to be addressed to determine whether O. vulgatus could become an effective biocontrol agent of P. spumarius in Europe. So far, O. vulgatus has been reared only from P. spumarius eggs, but its exact host-range should be evaluated to ensure efficiency and avoid non-target effect. The top-down impact of the parasitoid on vector populations should also be assessed on large data sets. Finally, the feasibility of mass rearing should be tested. We hope this report serves as a starting point to initiate research on this parasitoid wasp to assess whether it could contribute to reduce the spread and impact of X. fastidiosa in Europe. Submitted 6 September 2019 Accepted 17 January 2020 Published 24 March 2020 Corresponding author Jean-Yves Rasplus, jean-yves.rasplus@inrae.fr Academic editor Ilaria Negri Additional Information and Declarations can be found on page 12 DOI 10.7717/peerj.8591 Copyright 2020 Mesmin et al. Distributed under Creative Commons CC-BY 4.0 Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 INTRODUCTION spumarius in Europe (Dongiovanni et al., 2018; Dader et al., 2019). However, there is a growing awareness of the need to encourage management practices that safeguard harvests, human health, biodiversity and the environment. Thus, the development of effective biological control programs is desirable. Among biocontrol strategies, augmentative biological control consists in enhancing the effectiveness of naturally occurring natural enemies by the periodic release of specimens (Eilenberg, Hajek & Lomer, 2001; Aubertot & Savary, 2005). Compared to classical biological control it eliminates unintended effects of the introduction of new, non-native, parasitoids or predaceous arthropods (Hoy, 2008). However, as for all biological control programs, augmentative biocontrol requires field investigations to identify potential natural enemies of the target pest. 2/18 Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 Currently, information about the natural enemies of the meadow spittlebug are scattered (Cornara, Bosco & Fereres, 2018). Species of birds, frogs, arachnids, and insects (Hymenoptera, Diptera, and Coleoptera) occasionally feed on P. spumarius (Phillipson, 1960; Halkka & Kohila, 1976; Harper & Whittaker, 1976; Henderson, Hoffman & Jeanne, 1990; Pagliano & Alma, 1997) but predation by native natural enemies does not appear to be an important source of mortality. Studies are in progress to test whether the invasive assassin bug Zelus renardii Kolenati, 1857 (Hemiptera, Reduviidae) could be used to control populations of P. spumarius in olive orchards (Salerno et al., 2017). However, mass release of this species may be risky for local biodiversity, especially for beneficial arthropods (Ables, 1978). Indeed, it is considered as a generalist predator (Ables, 1978; Cisneros & Rosenheim, 1998; Weirauch, Alvarez & Zhang, 2012; Salerno et al., 2017, but see Cohen & Tang, 1997 who suggest a strong effect of prey body size). So far, only few parasitoids of P. spumarius have been recorded. Adults are attacked by Verralia aucta (Fallen, 1817) (Diptera, Pipunculidae) in Europe with relatively high parasitism rates in England: in average 31% in females and 46% in males over four years (Whittaker, 1969; Whittaker, 1973). Parasitism by V. aucta has a direct effect on P. spumarius population dynamics because it renders the host sterile (Whittaker, 1973). However, this parasitoid does not have an immediate effect on bacterium transmission because adults are only killed after 10–11 weeks of parasitism (Whittaker, 1969), a period during which they are probably still able to spread the bacterium. INTRODUCTION Contrastingly, an interesting feature of egg parasitoids is that they kill the host in the egg stage, that is, before it can inflict damage to its host plants (Mills, 2010). In the case of P. spumarius, the insect is killed before it acquires the bacterium from an infected host plant and becomes able to transmit it. A few egg parasitoids have been recorded in the US: Ooctonus vulgatus Haliday, 1833 (Hymenoptera, Mymaridae) and at least two unnamed species of Centrodora (Hymenoptera, Aphelinidae) (Weaver & King, 1954). Indeed, the genus Tumidiscapus, which is cited as parasitoid of P. spumarius in the US (Weaver & King, 1954), is in fact a synonym of Centrodora (Hayat, 1983). However, little is known about the biology and efficacy of egg parasitoids in natura. In this study, a field survey was conducted to identify major egg parasitoids of P. spumarius in Corsica. We provide a first report of Ooctonus vulgatus in this area. We summarized the main characters separating O. vulgatus from other Palearctic species to facilitate identification and generate COI DNA barcodes to accurately identify the species. Finally, we reviewed the literature and gathered all available occurrence data (i.e., geographical coordinates) of previously detected populations of O. vulgatus. This allowed us to calibrate an ecological niche model linking different climate descriptors to species occurrence data and estimate the potential distribution of the parasitoid in the Holarctic region for comparison with the distribution of P. spumarius. Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 MATERIALS AND METHODS Sampling and calculation of parasitism rate Five to ten handfuls of about eight top branches of Cistus monspeliensis L. 1753 (cut at 50 cm below the end of the branch) were sampled in four localities (Fig. 1). These localities 3/18 3/18 Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 8.5 9.0 9.5 10.0 41.5 42.0 42.5 43.0 G G G G n = 135 n = 122 n = 80 n = 100 100 % 80 % 20 % 51 % 49 % 31 % 69 % Longitude Latitude N Philaenus spumarius Ooctonus vulgatus Figure 1 Parasitism rate of Philaenus spumarius eggs in the four sites sampled in Corsica. Size of the pie chart is proportional to the total number of eggs that hatched from each locality (n). Slices indicate the relative proportion of O. vulgatus (dark grey) and nymphs of P. spumarius (light grey) that emerged from the pool of eggs. GPS coordinates of sampling localities: 42.984205◦N, 9.395287◦E (Ersa); 42.338849◦N, 9.180636◦E (Tralonca); 42.274756◦N, 9.487185◦E (Canale-di-Verde); 41.931726◦N, 9.343731◦E (Ventiseri). The map was built with the R package maps, using data from UNESCO (1987) through UNEP/GRID-Geneva. Full-size DOI: 10.7717/peerj.8591/fig-1 Figure 1 Parasitism rate of Philaenus spumarius eggs in the four sites sampled in Corsica. Size of the pie chart is proportional to the total number of eggs that hatched from each locality (n). Slices indicate the relative proportion of O. vulgatus (dark grey) and nymphs of P. spumarius (light grey) that emerged from the pool of eggs. GPS coordinates of sampling localities: 42.984205◦N, 9.395287◦E (Ersa); 42.338849◦N, 9.180636◦E (Tralonca); 42.274756◦N, 9.487185◦E (Canale-di-Verde); 41.931726◦N, 9.343731◦E (Ventiseri). The map was built with the R package maps, using data from UNESCO (1987) through UNEP/GRID-Geneva. Full-size DOI: 10.7717/peerj.8591/fig-1 4/18 Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 4/18 were part of a larger field survey of population dynamics of P. spumarius in Corsica. We targeted C. monspeliensis to maximize our chances to find eggs of P. spumarius. Indeed, we demonstrated in a previous study that, in Corsica, adults of P. spumarius seemed to be mainly associated with this species (Cruaud et al., 2018). Sampling was performed between the 12th and the 15th of February 2019. The back of each leaf (about 900 leaves per handful of branches) was inspected in the laboratory for whitish clusters, which were retained and inspected under a binocular microscope to confirm the presence of eggs of P. MATERIALS AND METHODS spumarius (Appendix S1; 109, 148, 167, 187 eggs obtained per site, hence a total of 611 eggs monitored). The morphological identification of P. spumarius eggs and first stage nymphs was performed using the descriptions of Weaver & King (1954) (Appendix S1). The pieces of leaf containing the eggs were placed on filter papers in unaerated Petri dishes (i.e., without spur) at room temperature (20.2 ± 1.5 ◦C), with natural light. Filter papers were kept moist by adding drops of water when necessary. Hatching was monitored every morning from the 18th of February to the 15th of March 2019. Emerging nymphs and parasitoids were killed and stored in 70% Ethanol at 4 ◦C. Parasitism rates were computed in each locality with the following formula: Parasitism rate = Number of parasitized eggs Number of parasitized eggs+Number of unparasitized eggs (Costello & Altieri, 1995). Morphological identiﬁcation of the parasitoids Identification to species was performed using the Ooctonus keys by Triapitsyn (2010) and Huber (2012). Specimens were desiccated using HMDS (Heraty & Hawks, 1998) and glued on grey cards. Imaging was performed with a Keyence digital microscope (VHX- 5000 Camera color CMOS and the VH-Z100UT lens). Images were then edited in Adobe Photoshop CS6 c⃝software. Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 Species distribution modelling framework Occurrences of O. vulgatus were retrieved from the literature and the GBIF database (GBIF.org, 2019) (Tables S1 and S2). Two hundred and five occurrences were obtained from the literature (Table S2), eight of which were not included in the analysis as no geographic coordinates were available. Forty occurrences were obtained from GBIF (last access: 22 August 2019; Table S2), but were all discarded due to dubious identification or lack of information on sample origin. Therefore, no occurrence obtained from GBIF could be included in the analysis. We fitted a correlative model linking different climate descriptors to species occurrences. The Maxent algorithm was chosen to conduct analyses because it does not require absence data (i.e., locations in which we can presume that a species is truly absent) (Phillips, Anderson & Schapire, 2006). We summarized below the main step of our analysis and details are provided in Appendix S2. The mean temperature and precipitation of the wettest, driest, warmest, and coldest quarters as well as precipitation seasonality were extracted from the Worldclim 2.0 database (Fick & Hijmans, 2017) and used as bioclimatic descriptors (Hijmans et al., 2005). In absence of formal knowledge about climatic factors constraining O. vulgatus distribution, we constituted three sets of bioclimatic variables and performed modelling with each of them (Qiao, Soberón & Peterson, 2015; Godefroid et al., 2019). The first set (CLIM1) comprised the mean temperature of the wettest, driest, warmest, and coldest quarters to reflect the impact of temperature constraints on distribution. To highlight the precipitation constraint, we added the precipitation seasonality to CLIM1 and constituted the second set (CLIM2). Finally, we built a third set (CLIM3) by assembling CLIM1 and the precipitation of the wettest, driest, warmest, and coldest quarters to fully account for both extreme temperatures and precipitations in the species distribution models (SDMs). The Maxent algorithm requires a set of locations where the species has been found (here, a random 70% of the available occurrences, the other 30% being used for model validation) and a set of locations where no information about the presence of the species are available (referred to as background points). A total of 10,000 background points were randomly generated in North America and Europe. Molecular identiﬁcation of the parasitoids Six individuals were used for molecular identification. Three of them were handled individually (sample codes = XMES00042_0101, XMES00077_0101, XMES00091_0101) and the remaining three were pooled to increase DNA yield (sample code = XMES00041_0189). Total genomic DNA was isolated using the Qiagen DNeasy Blood & Tissue kit without destruction of the specimens. We followed manufacturer’s protocol with the following modifications. Samples (whole insects, without dissection or crushing) were incubated overnight in an Eppendorf thermomixer (temperature = 56 ◦C, mixing frequency = 300 rpm). To increase DNA yield, two successive elutions (50 µL each) were performed with heated buffer AE (56 ◦C) and an incubation step of 15 min followed by centrifugation (6,000 g for 1 min at room temperature; see Cruaud et al. (2019) for a detailed description of the protocol). Eppendorf microtubes LoBind 1.5 ml were used for elution and to store DNA at minus 20 ◦C until PCR amplification. Vouchers were deposited at Centre de Biologie pour la Gestion des Populations (CBGP), Montferrier-sur- Lez, France. The mitochondrial Cytochrome c oxidase I standard barcode fragment (COI) was amplified with a cocktail of M13-tailed primers as detailed in Germain et al. (2013). Unpurified PCR products were sent to Eurofins MWG Operon (Ebersberg, Germany) for sequencing using the M13F and M13R primers (Germain et al., 2013; Ivanova et al., Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 5/18 2007). Both strands for each overlapping fragment were assembled in Geneious v11.1.4 (https://www.geneious.com). Geneious was also used to translate consensus sequences to amino acids to detect premature codon stops. All COI sequences available on BOLD (Ratnasingham & Hebert, 2007) for Ooctonus species were downloaded (last access July 12, 2019) and aligned with the newly generated sequences using MAFFT v7.245 (Katoh & Standley, 2013). A maximum likelihood tree was inferred with raxmlHPC-PTHREADS- AVX version 8.2.4 (Stamatakis, 2014). A rapid bootstrap search (100 replicates) followed by a thorough ML search (-m GTRGAMMA) was conducted. Tree visualization and annotation was performed with TreeGraph 2.13 (Stöver & Müller, 2010). Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 Parasitism rates Out of the 611 eggs monitored, 437 (i.e., 71.5%) hatched. 277 (63.4%) gave rise to P. spumarius nymphs and parasitoids emerged from 160 eggs (36.6%). All parasitoids were identified as O. vulgatus (Fig. 2). No parasitoid emerged from eggs collected in one of the four localities. We observed parasitism rates of 20.5, 48.9 and 69.0% in the three other localities (Fig. 1). Species distribution modelling framework To render complex response to environmental constraints while reducing model overfitting we first fitted 48 Maxent models using six regularization multiplier (RM) combinations (L, LQ, H, LQH, LQHP, LQHPT with L = linear, Q = quadratic, H = hinge, P = product and T = threshold) and feature class (FC) values (eight values ranging from 0.5 to 4 with Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 6/18 increments of 0.5) (Radosavljevic & Anderson, 2014). Optimal FC and RM combinations were determined for each of the three bioclimatic datasets (CLIM1–CLIM3) using the R language (R Core Team, 2019) and the package ENMeval (Muscarella et al., 2014). Optimal parameters were then used to fit a set of 10 replicate Maxent models using 70% of the dataset. The performance of each model was evaluated using the remaining 30% of occurrences using the area under the receiver–operator curve (AUC, Fielding & Bell, 1997) and the true skill statistics (TSS, Allouche, Tsoar & Kadmon, 2006). Models with AUC <0.8 were excluded from further analyses (Vicente et al., 2013). Habitat suitability maps (logistic output ranging from 0 to 1) were transformed into binary projections using the threshold that optimized the TSS statistics on the testing data (Guisan, Thuiller & Zimmermann, 2017). Maxent replicate models were fitted and evaluated using the R package biomod2 (Thuiller et al., 2009). Two different outputs were generated using the set of model prediction. (i) Binary predictions were averaged to produce the committee (consensus) averaging (Araújo & New, 2007; Marmion et al., 2009) showing the likelihood of the presence of O. vulgatus. This consensus model ranges from 0 (all the models predict absence) to 100% (all the models predict presence) and (ii) the median of the logistic outputs (Guisan, Thuiller & Zimmermann, 2017) of the models that depicts the climate suitability across the different models. Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 Guidelines for the identiﬁcation of O. vulgatus To help identification, we list below the main features that differentiate O. vulgatus from its closest relatives. The genus Ooctonus has been recently revised in the Palearctic and Nearctic regions respectively by Triapitsyn (2010) and Huber (2012). Ooctonus can be distinguished from other genera of Mymaridae by the following set of characters: tarsi 5-segmented, propodeum with diamond-shaped pattern of carinae (Fig. 2F), fore wing venation about one-third the wing length (Fig. 2C), with short marginal and stigmal vein, parastigma with hypochaeta next to proximal macrochaeta (Huber, 2012). In the Holarctic region, O. vulgatus can be distinguished from other species of Ooctonus by the following unique combination of features (Fig. 2): vertex without stemmaticum; mesoscutum without median groove; posterior part of scutellum and frenum smooth with weak sculpture laterally; metanotum and propodeum without reticulate sculpture; propodeum without median carina, but with a pentagonal areole formed by dorsolateral carinae; short petiole, 0.9–1.2x as long as metacoxa; forewing at least slightly truncate apically; females funicle with multiporous placoid sensilla (mps) on F7 and F8 only, F5 and F6 without mps; single Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 7/18 Figure 2 Morphology of Ooctonus vulgatus Haliday, 1833. (A) Male antenna (B) Female antenna (C) Habitus. (D) Head front view. (E) Mesosoma lateral view. (F) Male propodeum. (G) Mesosoma dorsal view. All scales = 100 µm except habitus. Photo credit: Jean-Yves Rasplus INRA. Full-size DOI: 10.7717/peerj.8591/fig-2 10 7717/peerj 8591 8/1 Figure 2 Morphology of Ooctonus vulgatus Haliday, 1833. (A) Male antenna (B) Female antenna (C) Habitus. (D) Head front view. (E) Mesosoma lateral view. (F) Male propodeum. (G) Mesosoma dorsal view. All scales = 100 µm except habitus. Photo credit: Jean-Yves Rasplus INRA. Full-size DOI: 10.7717/peerj.8591/fig-2 Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 8/18 Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 row of six bullae inside the female clava; ovipositor at most 1. 4× as long as metatibia and only slightly exerted beyond apex of gaster. Molecular identiﬁcation of the parasitoid Barcode sequences were successfully generated from all samples. All sequences were identical. Phylogenetic analysis confirmed that the most likely identification was O. vulgatus (Fig. S1). Species distribution modelling A total of 200 occurrences (197 obtained from the literature plus the three localities where we sampled O. vulgatus) (Fig. 3A) were used to model the distribution of O. vulgatus in Europe. The optimal Maxent parameters were RM = 4 and FC = hinge; RM = 4 and FC = hinge and RM = 2.5 and FC = hinge for CLIM1, CLIM2, and CLIM3, respectively. With the exception of one model of CLIM2, all models based on these optimal values yielded AUC values >0.8, which indicated that the different bioclimatic data subsets performed well. The consensus model was therefore computed from a set of 29 estimates of climate suitability. Figure 3B shows the median of the climate suitability values for the 29 models considered. Figure 3C depicts the proportion of the 29 models indicating that the climate is suitable for O. vulgatus. Both Figs. 3B and 3C show that the climate is favorable in very large areas covering most of Western Europe and around the Black Sea. These areas are overlapping with the geographical range of P. spumarius (Cruaud et al., 2018). Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 DISCUSSION Ooctonus Haliday, 1833 is a medium-sized genus of Mymaridae containing 37 described species that occur in all zoogeographical regions of the world (Holt et al., 2013) excepted Australasia (Noyes, 2019). O. vulgatus has been reared from the eggs of P. spumarius and studied only once in North America (Weaver & King, 1954). This species is thus poorly known as confirmed by the limited barcoding record. Indeed, only four barcodes are available in BOLD (two from Virginia United States, one from Ontario Canada, and one from British Columbia Canada). As a likely component of aerial plankton, O. vulgatus is expected to be a widespread species distributed in the Holarctic region (ranging from Ireland to the Sakhalin peninsula and from eastern to western coasts of North America, as south as California (Huber, 2012)). The species has been also reported from China (Bai, Jin & Li, 2015) but authors’ illustration casts some doubts about specimen identification. There are only a few unquestionable occurrences in the literature for this species (n = 197). Here, we provide a first report of O. vulgatus in Corsica and assess, for the first time in Europe, its biology as parasitoid of P. spumarius. We also confirm its potential large distribution throughout Europe with modelling approaches. More importantly we show that O. vulgatus potential distribution in Europe (Fig. 3) overlaps that of its host P. spumarius (Cruaud et al., 2018), which is not surprising from a biological point of view but is an interesting result in the framework of biological control. This study is preliminary and predictions, especially because they are based on a limited number of occurrences, are indicative only. This study Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 9/18 20 30 40 50 60 −100 −50 0 50 longitude latitude A 0km 250km 500km N 30 40 50 60 70 −20 0 20 40 longitude latitude 0.2 0.3 0.4 0.5 0.6 median of predicted habitat suitability values B 0km 250km 500km N 30 40 50 60 70 −20 0 20 40 longitude latitude 0 25 50 75 100 proportion of models predicting presence (%) C Figure 3 Geographical distribution of O. vulgatus. (A) Distribution of O. vulgatus occur lected from the literature. (B) Consensus model of climate suitability estimated by Maxent: model outputs. (C) Consensus model of climate suitability estimated by Maxent: proportio predicting O. vulgatus presence in Europe. Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 DISCUSSION Full-size DOI: 10.7717/p 20 30 40 50 60 −100 −50 0 50 longitude latitude A A B 0km 250km 500km N 30 40 50 60 70 −20 0 20 40 longitude latitude 0.2 0.3 0.4 0.5 0.6 median of predicted habitat suitability values B C B C 0km 250km 500km N 30 40 50 60 70 −20 0 20 40 longitude latitude 0 25 50 75 100 proportion of models predicting presence (%) C Figure 3 Geographical distribution of O. vulgatus. (A) Distribution of O. vulgatus occurrences col- lected from the literature. (B) Consensus model of climate suitability estimated by Maxent: median of model outputs. (C) Consensus model of climate suitability estimated by Maxent: proportion of models predicting O. vulgatus presence in Europe. Full-size DOI: 10.7717/peerj.8591/fig-3 10/18 Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 is a starting point to encourage investigations in other parts of Europe. Sampling efforts should more specifically target areas predicted as suitable for P. spumarius but non-suitable for O. vulgatus such as eastern areas of Europe. When studied in North America, observed parasitism rates did not exceed 10% of the sampled eggs of P. spumarius (Weaver & King, 1954). Here, we obtained parasitism rates of up to 69%, but absence of parasitism in one sampled site. While we acknowledge sampling four sites is not enough to have a representative view of P. spumarius egg parasitism in Corsica, our results show that parasitism rate can be high though very variable. Further surveys are obviously necessary to better assess the spatial and temporal variability of parasitism rate and understand what is(are) the cause(s) of such variations in Corsica and throughout Europe. Identifying such drivers could open new avenues for conservation biological control against P. spumarius, through the implementation of environments favorable to O. vulgatus in the vicinity of crops susceptible to X. fastidiosa. The use of mymarids in biological control program has a long history. The most notable instance being the use of Anaphes nitens (Girault, 1928) in several countries to successfully control the eucalyptus weevil, Gonipterus scutellatus Gyllenhal, 1833 (Coleoptera, Curculionidae), which feeds and reproduces on Eucalyptus trees (Doull, 1955). More recently, Cleruchoides noackae Lin and Huber, 2007 has been used in South America to control an invasive sap-feeding pest of Eucalyptus, Thaumastocoris peregrinus Carpintero and Dellapé, 2006 (Hemiptera, Thaumastocoridae) (Martinez, González & Dicke, 2018). Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 Competing Interests Competing Interests The authors declare there are no competing interests. DISCUSSION Mymarid species were used to control leafhopper vectors of plant pathogens (Hemiptera, Cicadellidae). Anagrus armatus (Ashmead, 1887) regulated Edwardsiana froggatti (Baker, 1925) (Hemiptera, Cicadellidae), a pest of apple in New Zealand, with parasitism rates of the eggs reaching 80% (Dumbleton, 1937). More recently, Cosmocomoidea species were used to target Homalodisca vitripennis (Germar, 1821) (Hemiptera, Cicadellidae) a vector of X. fastidiosa in California (Irvin & Hoddle, 2010). In all these cases, mymarids helped regulate pest population growth. However, before any attempts to regulate populations of P. spumarius are made, we need to enrich our knowledge on O. vulgatus. In particular, the degree of specificity of the P. spumarius – O. vulgatus interaction needs to be determined to avoid non-target effect of augmentative biocontrol (Van Driesche & Hoddle, 2016). We also need to evaluate our ability to consistently rear O. vulgatus in controlled conditions, one of the key obstacles to the use of mymarids in biological control programs (but see Martinez, González & Dicke, 2018). Finally, parasitoids can have complex effects on vector-borne disease by either increasing (Jeger et al., 2011) or decreasing (Martini, Pelz-Stelinski & Stelinski, 2014) pathogen spread. Further research is still needed to better understand the impact of such tri-trophic interactions on plant disease dynamics. While O. vulgatus does not directly impact transmission capacity of P. spumarius, by killing its host at an early stage of development, it reduces the number of vectors that may acquire the bacterium from an infected host-plant and become able to transmit it. Again, we consider this study as a starting point to encourage research on this parasitoid wasp to assess whether it could contribute to reduce the spread and impact of X. fastidiosa in Europe. Increasing egg parasitism of P. spumarius in the fall might significantly reduce Mesmin et al. (2020), PeerJ, DOI 10.7717/peerj.8591 11/18 population size in the next year and possibly the transmission of the bacterium, without resorting to chemical treatments. population size in the next year and possibly the transmission of the bacterium, without resorting to chemical treatments. Funding This work was funded by the Collectivité Territoriale de Corse and the European Union Horizon 2020 research and innovation program under Grant Agreement No. 727987 XF-ACTORS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Grant Disclosures Grant Disclosures The following grant information was disclosed by the authors: Collectivité Territoriale de Corse and the European Union Horizon 2020 research and innovation program: 727987 XF-ACTORS. The following grant information was disclosed by the authors: Collectivité Territoriale de Corse and the European Union Horizon 2020 research and innovation program: 727987 XF-ACTORS. Author Contributions • Xavier Mesmin conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. • Marguerite Chartois analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. • Guénaëlle Genson performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft. • Jean-Pierre Rossi, Astrid Cruaud and Jean-Yves Rasplus conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. 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https://openalex.org/W2802999206	https://zenodo.org/record/3384912/files/RESEARCH%20OF%20MICROBIOLOGICAL%20INDICATORS%20OF%20QUALITY%20OF%20SURFACE%20WATERS%20OF%20NATURAL%20ENVIRONMENTAL%20TERRITORIES%20OF%20THE%20DANUBE%20BASIN.pdf	English	null	RESEARCH OF MICROBIOLOGICAL INDICATORS OF QUALITY OF SURFACE WATERS OF NATURAL ENVIRONMENTAL TERRITORIES OF THE DANUBE BASIN	Eureka, Physics and Engineering./Eureka, Physics and Engineering	2,018	cc-by	4,966	Original Research Article: full paper Original Research Article: full paper Original Research Article: full paper (2018), «EUREKA: Physics and Engineering» Number 2 Abstract A comparative analysis of the sanitary and ecological state of surface watercourses in the upper part of the Danube basin (on the territory of Ukraine) was carried out according to microbiological indicators. Similar hygienic studies were previously conducted in the middle and lower Danube in Austria, Slovakia, Hungary and Romania. In Ukraine, the river network of the Danube River basin was not studied by microbiological indicators. 3 3 Chemical Engineering (2018), «EUREKA: Physics and Engineering» Number 2 (2018), «EUREKA: Physics and Engineering» Number 2 Original Research Article: full paper The object of research is the watercourses on the territory of various zones of the nature protection object, which are different in function. This approach makes it possible to use hygienic indicators of water in protected areas of nature conservation areas as a reference for conducting background monitoring.i The original design of the treatment plant based on the use of “Viya” fibrous carrier and “paste” technical structure is proposed. It was established that as the transition from the reserve to the economic zone occurs, the nitrate content in the water increases, the BOD increases in water, and the dissolved oxygen in the water decreases for all the watercourses studied. Significant differences in microbiological indices of watercourses of various functional zones of the protected object have been revealed. The possibility of using the sanitary-microbiological indicators of the river network as a reliable rapid test for assessing the state of environmental safety of nature conservation areas is shown. Keywords: nature protection areas, functional zones, surface waters, Danube basin, sanitary-microbiological indicators. © Andrew Masikevych, Mikhail Kolotylo, Valery Yaremchuk, I: 10.21303/2461-4262.2018.00590 Yurij Masikevych, Valentyn Myslytsky, Ivan Burdeniuk, Konstantin Dombrovskyi © Andrew Masikevych, Mikhail Kolotylo, Valery Yaremchuk, 1. Introduction Sanitary-microbiological indicators are successfully used as indicators of environmental pollution: water, air, soil [1–3], as well as to monitor the status of ecosystems, forecasting and modeling of development. In connection with the rapid reaction to environmental changes, bac- teria proved to be ideal markers of microbial contamination of surface waters [4]. This approach is especially valuable for protected areas. Protected areas serve as background sites for assessing the ecological status and changes in the environment. Studying their condition makes it possible to predict changes in the environment to a distant future. The object of research is the sanitary and epidemiological indicators of the water network of the Siret River. This river enters the upper part of the Danube basin and forms the basis of the hydrological network of the protected nature reserve “Vyzhnytsia National Nature Park”. The Dan- ube is one of the most important rivers of Europe and the world and, simultaneously, according to a number of authors [4], is one of the most vulnerable aquatic ecosystems. For monitoring the water quality of the Danube River, the coastal countries currently use different approaches and methods of sanitary and ecological analysis, which does not allow the formation of a holistic picture of the state of the ecosystem of an important waterway in Europe. That is why the use of microbiological analysis as a universal approach for water quality monitoring by the coastal countries of the inter- national river Danube is relevant at this time. DOI: 10.21303/2461-4262.2018.00590 Yurij Masikevych, Valentyn Myslytsky, Ivan Burdeniuk, Konstantin Dombrovskyi Yurij Masikevych, Valentyn Myslytsky, Ivan Burdeniuk, Konstantin Dombrovskyi 2. Literature review and problem statement To date, there is a strong demand for monitoring water quality. Researches of scientists of the Institute of Hydrometeorology from Bratislava [5] suggest the use of autoregressive models for studying the impact of natural and technogenic changes on the quality of the Danube. In terms of monitoring the water network, the assessment of the presence of pathogenic bacteria in water is of particular interest, according to the American School of Researchers at the University of California, Davis and Iowa State University. In particular, this concerns the problems associated with the use of indicator organisms for this purpose. This approach is the main problem of protecting human and animal health [6, 7]. The results obtained by Hungarian researchers [8] indicate that the bacteriological proper- ties of water can be a specific indicator of fecal pollution and organic contamination. That is why, according to the authors, microbiological contamination by fecal (pathogenic) bacteria is consid- ered to be the most important issues of surface water quality, and especially in the Danube basin. The accumulation index of E. coli is one of the specific indicators of fecal contamination of the aquatic environment, and can be used for express diagnostics of the aquatic environment. An interesting experience is the assessment of the quality of the surrounding aquatic en- vironment of the Kingdom of the Netherlands [11]. The national program for monitoring surface water in this country, in addition to hydrochemical indicators, including also hydrobiological mon- itoring (including epidemiological). In a number of EU countries (most of which have their own 4 4 Chemical Engineering (2018), «EUREKA: Physics and Engineering» Number 2 Original Research Article: full paper (2018), «EUREKA: Physics and Engineering» Number 2 y Number 2 biological assessment of surface waters), bacteria, algae, invertebrate hydrobionts, phytoplankton are used as bioindicators [12, 13]. Researchers [9] have summarized existing methods of microbiological bioindication of the aquatic environment. It should be noted that the standards of the USSR in the field of protection of the hydrosphere operating in Ukraine do not take into account bacteriological indicators of water quality. This was, in all probability, the reason that data on the microbi- ological state of the upper part of the Danube basin (in the Ukrainian part: Tisza, Prut, Siret rivers) are practically absent. Since water quality assessment is expensive [14], the development of rapid assessment meth- ods remains an urgent problem. 3. The aim and objectives of research The aim of research is studying the epidemiological state of the surface waters of the river network of the Siret River (the upper part of the Danube basin, the Ukrainian territory) of various functional zones of the nature reserve object. To achieve the aim, the following tasks are set: To achieve the aim, the following tasks are set: – to determine the main sanitary and microbiological indicators (nitrite content, biological oxygen consumption, dissolved oxygen, coli-index, total microbial number), determine the quality of surface waters; – to conduct a comparative analysis of sanitary and microbiological indicators, various in terms of the functional purpose of the zones, a nature reserve site;i – to study the possibility of using the proposed “biofilter”, created on the basis of “Viya” artificial fibrous carrier and “paste” technical design, for assessing the quality of surface waters of various functional zones of a nature reserve site. 2. Literature review and problem statement The introduction of this approach in many developed countries can reduce the cost of monitoring. It should also be noted that the use of sanitary and microbiological indicators of the river net- work for assessing the status of protected sites, to date, is episodic [4, 10]. The question of assessing the sanitary and hygienic quality of surface waters of different purpose and status of functional zones of nature protection territories remains to be fully investigated.i The analysis of literature data confirms the need to use methods of sanitary-microbiological analysis of surface waters for assessing the ecological status of nature reserves and monitoring of aquatic ecosystems as a whole. 4. Materials and methods for research of surface water quality indicators The watercourses have been studied: rivers Stebnyk, Sukhyi Strumok, Solonets entering the Ukrainian part of the Danube Basin. To assess the quality of the surface waters of the waterways of the Vyzhnytsia national nature park (NNP), Viya fibrous carrier (TU (995990)) was used. It made from a textured filament yarn (TU 6-06-C116-87, text 350) is described in [15].i Earlier [15, 16] it was established that a fibrous carrier of this type can be successfully used to construct “biofilters” for cleaning surface water. In particular, it was shown that the effectiveness of the “bioreactor” depends on the temperature, the concentration of microorganisms and that the accumulation of phytoplankton and heterotrophic microorganisms on carriers is most effective in the summer period (July-August). Using the approach described by the authors, a “biofilter” was mounted on the basis of special wooden structures – “paste”, have long been used by local residents to saturate the streams with oxygen (Fig. 1). “Biofilter” is intended for adsorption on its surface of bacteria on hydrobionts. The studies were carried out during the summer season 2016 and 2017. Sampling of water (23–25 °C) was carried out immediately before and after the “biofilters” from different functional zones of the waterways of the NNPs indicated in the scheme (Fig. 2), in accordance with the exist- ing standard techniques. The choice of water sampling points was determined by their belonging to the functional zones of the protected object. The linking of the research region to the map of Europe is shown in Fig. 3. 5 5 Chemical Engineering (2018), «EUREKA: Physics and Engineering» Number 2 (2018), «EUREKA: Physics and Engineering» Number 2 Original Research Article: full paper Original Research Article: full paper Fig. 1. When installing a cleaning structure using “Viya” fibrous carrier: A – a «paste» wooden structure; B – “Viya” fibrous carrier Fig. 1. When installing a cleaning structure using “Viya” fibrous carrier: A – a «paste» wooden structure; B – “Viya” fibrous carrier Fig. 2. 4. Materials and methods for research of surface water quality indicators Map-scheme of functional zoning of Vyzhnytsia national nature park: sampling points: 1 – the upper part of the Stebnik riverbed, 2 – the upper part of the Sukhyi Strumok canal, 3 – the upper part of the Slavets riverbed, 4 – “OIKOS” recreational zone, 5 – “Myslyvskyi budynok” recreational zone, 6 – the lower part of the Stebnik riverbed, 7 – the lower part of the Sukhyi Strumok canal, 8 – the middle part of the Solonets riverbed Fig. 2. Map-scheme of functional zoning of Vyzhnytsia national nature park: sampling points: 1 – the upper part of the Stebnik riverbed, 2 – the upper part of the Sukhyi Strumok canal, 3 – the upper part of the Slavets riverbed, 4 – “OIKOS” recreational zone, 5 – “Myslyvskyi budynok” recreational zone, 6 – the lower part of the Stebnik riverbed, 7 – the lower part of the Sukhyi Strumok canal, 8 – the middle part of the Solonets riverbed Fig. 2. Map-scheme of functional zoning of Vyzhnytsia national nature park: sampling points: 1 – the upper part of the Stebnik riverbed, 2 – the upper part of the Sukhyi Strumok canal, 3 – the upper part of the Slavets riverbed, 4 – “OIKOS” recreational zone, 5 – “Myslyvskyi budynok” recreational zone, 6 – the lower part of the Stebnik riverbed, 7 – the lower part of the Sukhyi Strumok canal, 8 – the middle part of the Solonets riverbed 6 Chemical Engineering Original Research Article: ull paper (2018), «EUREKA: Physics and Engineering» Number 2 Fig. 3. Geographical location of the research area on the map of Europe (2018), «EUREKA: Physics and Engineering» Number 2 (2018), «EUREKA: Physics and Engineering» Number 2 Original Research Article: full paper (2018), «EUREKA: Physics and Engine Number 2 Fig. 3. Geographical location of the research area on the map of Europe Fig. 3. Geographical location of the research area on the map of Europe The investigated watercourses are a rather convenient model for studying the quality of surface waters of various functional zones of the nature reserve object, because they include three zones: a reserved zone in the upper part of the riverbed, a recreational zone in the middle of the riverbed and a recreational zone in the lower part. 4. Materials and methods for research of surface water quality indicators The content of nitrates was determined in accordance with DSTU 40780-2001, biological oxygen consumption, dissolved oxygen, coli-index, total microbial number was determined by con- ventional methods in accordance with methodological guidelines [17].i g g The hydrobiological material (biocenosis of the periphyton overgrowth of the Viya” fi- brous carrier) was selected in the second decade of August. The material was delivered to the lab- oratory in an open vessel. The infusoria, rotifers and turbolarium of the fouling biocenosis were studied in a living state under the Biola R-14 microscope, with an increase of 150–600 times. Other organisms (larvae of amphibiotic insects and nematodes) were fixed with 70¤ ethanol and determined their species affiliation. The species were determined according to determinants and scientific works [18, 19]. The statistical processing of the results was carried out using the computer program Excel. 5. Research results of surface water quality of various functional areas of the Vyzhnytsia NNP 5. Research results of surface water quality of various functional areas of the Vyzhnytsia NNP The carried out researches have shown that there is an increase in the value of sanitary and microbiological indicators downstream along all these watercourses. Especially it concerned the increase in the amount of lactose-positive E. coli (E. coli) per 1 liter of water (coli-index). It should be noted that the E. coli is a sanitary indicator and indicates fecal contamination in this case of water bodies of the environment. Comparing the indices of coli-index in samples of river water of the protected zone and selected water samples in the economic zone, an increase in the coliform index was found to be 2 times on average (Table 1). At the same time, the total microbial number (CFU/ml) exceeded by 2–4 times the normative indicators adopted in the EU countries (Surface Water Directive: 75/440 EU) and amounted to 1500–1700 (for the protected zone), 2300–3500 (for the stationary zone recreation) and more than 5000 (for the economic zone). Downstream of the watercourses, and the transition from the reserve to the economic zone, there is a growth in the water content of nitrates, an increase in the BOD index and a decrease in dissolved oxygen in the water. The obtained results indicate an increase in pollution of the river net- work by organic residues, in particular, they can be fecal connections of the economic zone where there are no existing treatment facilities. The study of the qualitative species composition of organisms (periphyton) that inhabited the fibrous substrate showed bacteria, protozoa, fungi, algae, worms, crustaceans, bivalves and others to form fouling. 7 Chemical Engineering (2018), «EUREKA: Physics and Engineering» Number 2 Original Research Article: full paper Original Research Article: full paper The data are presented in Fig. 4 indicate that the “Viya” fibrous material is able to accumu- late in considerable quantities the bacteria of the E. coli group (Fig. 4). 5. Research results of surface water quality of various functional areas of the Vyzhnytsia NNP Table 1 The values of the main sanitary-microbiological indices of various functional zones of the Vyzhnytsia NNP* Sampling points BODtot (mg O2/dm3) Dissolved oxygen (mg О2/dm3) Coli-index (dm3) Microbial number (CFU/ml) Nitrogen (mg N/dm3) protected area 1 1,8±0,10 7,5±0, 55 85±10,5 1500±85,4 0,01±0,001 2 2,3±0,12 7,1±0,50 80±8,0 1700±90,2 0,02±0,001 3 2,0±0,08 8,0±0, 70 82±12,2 1600±65,5 0,02±0,002 zone of stationary recreation 4 5,1±0,30 5,2±0, 60 90±11,5 2300±92,5 0,02±0,03 5 4,5±0,25 6,0±0,55 100±9,4 3500±150,4 0,05±0,004 economic zone 6 8,8±0,50 4,2±0, 20 120±15,3 5800±250,5 0,15±0,02 7 9,2±0,45 4,0±0,25 105±12,5 5200±280,3 0,10±0,01 8 9,5±0,40 3,8±0,20 110±14,6 5500±255,2 0,14±0,02 Note: * – the data are reliable for p<0.05; the name of the sampling points corresponds to the notation of Fig. 2 Table 1 The values of the main sanitary-microbiological indices of various functional zones of the Vyzhnytsia NNP alues of the main sanitary-microbiological indices of various functional zones of the Vyzhnytsia NNP* Note: * – the data are reliable for p<0.05; the name of the sampling points corresponds to the notation of Fig. 2 Note: * – the data are reliable for p<0.05; the name of the sampling points corresponds to the notation of Fig. 2 Fig. 4. The coefficient of accumulation of bacteria on fibrous carriers “Viya”: the name of the sampling points corresponds to the notation in Fig. 2; coefficient of accumulation – the multiplicity of excess of content in comparison with the surrounding water environment. 0 4 8 12 16 1 2 3 4 5 6 7 8 0 4 8 12 16 1 2 3 4 5 6 7 8 Fig. 4. The coefficient of accumulation of bacteria on fibrous carriers “Viya”: the name of the sampling points corresponds to the notation in Fig. 2; coefficient of accumulation – the multiplicity of excess of content in comparison with the surrounding water environment. As the data in Fig. 4 for sampling points 1, 2, 3, included in the NNP protected area is characterized by a high coefficient (from 11 to 14 times) of the accumulation of bacteria on fibrous carriers. 6. Discussion of the research results of surface water quality of various functional zones of the Vyzhnytsia NNP Comparison of the obtained results (Table 1) with the normative indicators adopted in the EU countries (Surface Water Directive: 75/440 EU) give grounds to assert that the surface waters of the river network of the NNP reserve and recreational zone refer to the permissible level of pollution. At the same time, the river water of the economic zone does not meet the sanitary and hygienic requirements and refers to a moderate and high pollution degree. The use of water of this pollution degree, without proper cleaning, can lead to the appearance of a number of diseases in the population. The conducted studies showed (Fig. 4) that in the protected zone and the zone of NNP stationary recreation, the maximum values of the accumulation coefficient of bacteria on fibrous carriers take place. This coefficient characterizes the multiplicity of exceeding the amount of E. coli group on a synthetic carrier in comparison with the surrounding aqueous medium. It is known that the presence of E. coli group in water indicates fecal contamination and, according- ly, the possible contamination of water by pathogenic microorganisms of the intestinal group (typhoid, paratyphoid, dysentery, etc.) [8]. However, the detection and isolation of pathogens is complicated by the low concentration of bacteria in surface waters, the high financial costs and the length of detection technology [23]. The use of natural adsorbents is well-established in this regard [24]. In order to overcome this methodological obstacle, “biofilter” based on a synthetic material such as “Viya” was used. It was mounted on special «paste» wooden structures. The data in Fig. 3 indicate that the use of the “biofilter” makes it possible to concentrate the E. coli group for their identification and possible destruction. It is also shown that as the transition to the economic zone and the increase in the amount of organic pollution of waterways, there is a sharp increase in the magnitude of the coli-index and the microbial number (Table 1). At the same time, the accumulation coefficient of E. coli group on the “biofilter” is characterized by minimal indicators. This can be explained by the saturation of the absorption surface by microorganisms, and also by their partial incorporation into nutrient chains. One of the proofs of this statement is an increase in the species composition of hydrobionts on the “biofilters” of the watercourses of the economic zone. (2018), «EUREKA: Physics and Engineering» Number 2 (2018), «EUREKA: Physics and Engineering» Number 2 (2018), «EUREKA: Physics and Engineering» Number 2 6. Discussion of the research results of surface water quality of various functional zones of the Vyzhnytsia NNP Thus, representatives of rotifers, copepods (and other lithophilic species) prevailed in water samples collected in the protected zone (upper part of the current). At the same time, in the water of the economic zone, with the growth of organic detritus, the species composition of the fouling is replenished at the expense of detritophages, incl. nematodes, oligochaetes, dreissena and others.iii Thus, a specific “biofilter” in the form of an artificially created microecosystem is formed downstream on fibrous carriers. In this microecosystem fibrous carrier serves as a kind of “home” for microorganisms, plant and invertebrate animal organisms, they are able to accumulate, which is the basis for cleaning reservoirs. In addition, some bacteria, in all probability, become an ele- ment of the feeding chain and serves as food for invertebrate hydrobionts. Thus, water reservoirs are cleaned in two stages: due to adsorption on synthetic carriers in the first stage and trophic chains on the second stage.i In general, in the estuary part of the tributaries of the Siret basin there is a significant con- tamination of surface waters (III-IV quality class), which is consistent with the results of studies [25], which showed that it is the Siret and Prut rivers that are significant factors in the pollution of the lower part of the Danube riverbed. 5. Research results of surface water quality of various functional areas of the Vyzhnytsia NNP In water samples taken in the zone of stationary recreation (points 4–5), this coefficient also has a sufficiently high index in the range of 9–10 units, that is, the carrier accumulates on its surface an order of more bacteria than the river water of the sampling points.i In the periphyton of the “Viya” fibrous carrier, 12 species and subspecies of hydrobionts were found. Most of these hydrobionts belong to amphibiotic insects (8 taxa). Rotifers and in- fusoria are represented by 2 taxa, respectively. Other systematic groups (ciliated worms, nem- atodes) were represented by one species, respectively. The species composition of the hydrobi- onts was supplemented with new species as the transition from the protected zone – 5 species (points 1, 2, 3) to the zone of stationary recreation – 7 species (points 4, 5) and the economic zone – 12 species (points 6, 7, 8). 8 8 8 Chemical Engineering Original Research Article: full paper Original Research Article: full paper Original Research Article: full paper (2018), «EUREKA: Physics and Engineering» Number 2 References [1] Sumampouw, O. J., Risjani, Y. (2014). Bacteria as Indicators of Environmental Pollution: Review. International Journal of Ecosystem, 4 (6), 251–258. [2] Ashbolt, N. J. (2015). Microbial Contamination of Drinking Water and Human Health from Community Water Systems. Current Environmental Health Reports, 2 (1), 95–106. doi: 10.1007/s40572- 014-0037-5 [2] Ashbolt, N. J. (2015). Microbial Contamination of Drinking Water and Human Health from Community Water Systems. Current Environmental Health Reports, 2 (1), 95–106. doi: 10.1007/s40572- 014-0037-5 [3] Hermans, S. M., Buckley, H. L., Case, B. S., Curran-Cournane, F., Taylor, M., Lear, G. (2016). Bacteria as emerging indicators of soil condition. Applied and Environmental Microbiology, 83 (1). doi: 10.1128/aem.02826-16 [3] Hermans, S. M., Buckley, H. L., Case, B. S., Curran-Cournane, F., Taylor, M., Lear, G. (2016). Bacteria as emerging indicators of soil condition. Applied and Environmental Microbiology, 83 (1). doi: 10.1128/aem.02826-16 [4] Pall, E., Niculae, M., Kiss, T., Sandru, C. D., Spinu, M. (2013). Human impact on the mi- crobiological water quality of the rivers. Journal of Medical Microbiology, 62, 1635–1640. doi: 10.1099/ jmm.0.055749-0 [4] Pall, E., Niculae, M., Kiss, T., Sandru, C. D., Spinu, M. (2013). Human impact on the mi- crobiological water quality of the rivers. Journal of Medical Microbiology, 62, 1635–1640. doi: 10.1099/ jmm.0.055749-0 [5] Pekarova, P., Onderka, M., Pekar, J., Roncak, P., Miklanek, P. (2009). Prediction of Water Quality in the Danube River Under extreme Hydrological and Temperature Conditions. Journal of Hy- drology and Hydromechanics, 57 (1), 3–15. doi: 10.2478/v10098-009-0001-5 [6] Pandey, P. K., Kass, P. H., Soupir, M. L., Biswas, S., Singh, V. P. (2014). Contamination of water resources by pathogenic bacteria. AMB Express, 4 (1), 51. doi: 10.1186/s13568-014-0051-x [7] Pandey, P. K., Soupir, M. L. (2013). Assessing the Impacts ofE. coliLaden Streambed Sedi- ment onE. coliLoads over a Range of Flows and Sediment Characteristics. JAWRA Journal of the Amer- ican Water Resources Association, 49 (6), 1261–1269. doi: 10.1111/jawr.12079 [8] Bayoumi Hamuda, H. E. A. F., Patko, I. (2012). Ecological monitoring of Danube water quality in Budapest region. American Journal of Environmental Sciences, 8 (3), 202–211. doi: 10.3844/ ajessp.2012.202.211 [9] Ryl’s’kyi, O. F., Masikevych, Yu. G. (2012). Mikrobiolohichna bioindykatsiyi dovkillya zabrudnenoho vazhkymy metalamy ta inshymy ksenobiotykamy. Visnyk Zaporiz’koho natsional’noho universytetu, 3, 139–147. [10] Mudrak, O. V. (2012). Zbalansovanyy rozvytok ekomerezhi Podillya: stan, problemy, pers- pektyvy. Vinnytsya, 914. [11] Schets, F. M., van Wijnen, J. H., Schijven, J. F., Schoon, H., de Roda Husman, A. M. (2008). 7. Conclusions 1. It is shown that as the transition from the protected to the economic zone occurs, the ni- trate content in the water increases, the BOD index increases and the dissolved oxygen in the water decreases for all the investigated watercourses.i 1. It is shown that as the transition from the protected to the economic zone occurs, the ni- trate content in the water increases, the BOD index increases and the dissolved oxygen in the water decreases for all the investigated watercourses.i 2. “Viya” fibrous carrier is capable of effective accumulation on the adsorbing surface of microorganisms of bacteria of the group of Escherichia coli and invertebrate hydrobionts for water- courses with insignificant content of organic pollutants. 2. “Viya” fibrous carrier is capable of effective accumulation on the adsorbing surface of microorganisms of bacteria of the group of Escherichia coli and invertebrate hydrobionts for water- courses with insignificant content of organic pollutants. 9 9 Chemical Engineering (2018), «EUREKA: Physics and Engineering» Number 2 (2018), «EUREKA: Physics and Engineering» Number 2 Original Research Article: full paper Original Research Article: full paper Number 2 3. Significant differences in microbiological indices of watercourses of various functional zones of the protected object have been established. The total microbial number (CFU/ml) was 1500–1700 (for the protected zone), 2300–3500 (for the zone of stationary recreation) and more than 5000 (for the economic zone). 3. It is shown that during the growth of organic pollution of water bodies, especially in the economic zone of nature protection territories, a kind of “biofilters” – artificial microecosystems – are formed on fibrous carriers. 3. It is shown that during the growth of organic pollution of water bodies, especially in the economic zone of nature protection territories, a kind of “biofilters” – artificial microecosystems – are formed on fibrous carriers. 4. The obtained results supplement the idea of the sanitary and ecological state of the Danube basin in its upper part on the territory of Ukraine. 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Mykolaiv: PMBSNU, Bristol: UWE, 308. [17] Sanitarno-virusolohichnyy kontrol’ vodnykh ob»yektiv: metod. vkazivky MV 10.2.1-145- 2007. Pro zatverdzhennya metodychnykh vkazivok «Sanitarno-virusolohichnyy kontrol’ vodnykh ob»yektiv» (2007). Ministerstvo okhorony zdorovia Ukrayiny, No. 284. Dodatok 1. Available at: http:// www.moz.gov.ua/docfiles/8203_dodatok.rar Last accessed: 18. 01.2018 [18] Berger, H. (2008). Monograph of the Amphisiellidae and Trachelostylidae (Ciliophora, Hy- potricha). Salzburg, 736. doi: 10.1007/978-1-4020-8917-6 [19] Kutikova, L. A., Starobogatov, Ya. I. (1977). Opredelitel presnovodnykh bespozvonochnykh Evropeyskoy chasti SSSR (plankton i bentos). Leningrad, 511. [19] Kutikova, L. A., Starobogatov, Ya. I. (1977). Opredelitel presnovodnykh bespozvonochnykh E k h ti SSSR ( l kt i b t ) L i d 511 [19] Kutikova, L. A., Starobogatov, Ya. I. (1977). Opredelitel presnovodnykh bespozvono Evropeyskoy chasti SSSR (plankton i bentos). Leningrad, 511. i Evropeyskoy chasti SSSR (plankton i bentos). Leningrad, 511. i [20] Barinova, S. (2017). On the Classification of Water Quality from an Ecological Point of View. International Journal of Environmental Sciences & Natural Resources, 2 (2). doi: 10.19080/ ijesnr.2017.02.555581 [21] Odume, O. N.; Tutu, H. (Ed.) (2017). Ecosystem Approach to Managing Water Quality. Water Quality. doi: 10.5772/65707 [22] Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy (2000). Official Journal of the European Communities, 43, 72. [23] Thompson, D. E., Rajal, V. B., De Batz, S., Wuertz, S. (2006). Detection of Salmonella spp. in water using magnetic capture hybridization combined with PCR or real-time PCR. Journal of Water and Health, 4, 67–75. [24] Malyovanyi, M., Sakalova, G., Chornomaz, N., Nahurskyi, O. (2013). Some kinetic regular- ities of intracellular substance extracting. 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https://openalex.org/W1978452806	https://link.springer.com/content/pdf/10.1007/s10750-013-1567-9.pdf	English	null	A simple solution enabling quantitative sampling of freshwater and marine sediments covered by dense submerged vegetation	Hydrobiologia	2,013	cc-by	2,193	Hydrobiologia (2013) 716:1–4 DOI 10.1007/s10750-013-1567-9 Hydrobiologia (2013) 716:1–4 DOI 10.1007/s10750-013-1567-9 OPINION PAPER Keywords Corer-type samplers Sediments Submerged vegetation Abstract The biological and physico-chemical structure of near bottom habitats located under densely growing submerged vegetation, and their signiﬁcance in the functioning of whole aquatic ecosystems, are very little known due to difﬁculties in sampling. Corer- type samplers, believed to be the best in littoral studies, do not work properly in such places, because their tube opening is easily clogged by plant shoots, acting as a stopper. In order to overcome this problem, an alteration to the shape of the tube ending is proposed. This can be done by its trimming at an accurate angle, or by ﬁtting (permanently or interchangeably) a metal collar made of stainless steel to its end, so that the ending would resemble the shape of a needle. The modiﬁcation can be applied to virtually all corer samplers. It was found to be efﬁcient while sampling sediment cores for the purposes of both hydrobiolog- ical and paleolimnological studies on heavily over- grown macrophyte-dominated water ecosystems. A simple solution enabling quantitative sampling of freshwater and marine sediments covered by dense submerged vegetation Ryszard Kornijo´w • Timo Kairesalo Received: 14 February 2013 / Revised: 5 April 2013 / Accepted: 4 May 2013 / Published online: 15 June 2013 The Author(s) 2013. This article is published with open access at Springerlink.com Keywords Corer-type samplers Sediments Submerged vegetation Introduction sediment into the tube. In such a case, the sample collected contains turbid water instead of the actual sediment material. This particularly concerns bottoms overgrown with thick beds of creeping charales or mosses, and to a lesser extent those covered by protruding plants such as pondweeds Potamogeton spp. or water milfoils Myriophyllum spp. In such a situation, the researcher usually attempts sampling sediments in other places. This might ﬁnally be successful, but in places without or with sparse vegetation. The material obtained this way is not representative of either weedy or open water habitats. A solution might be the application of a tube of a very small diameter. This, however, has negative consequences, for example the necessity to collect a high number of samples to obtain sufﬁcient quantity of material. In the case of paleolimnological studies, additional disadvantages of this approach are the compression of sediment and smearing (Glew et al., 2001; IAEA, 2003). Therefore, a core obtained this way does not meet the criteria of an intact core. While using the metal collar, the possibility of chemical contamination of the sediment collected with metals contained in the collar should be considered. This is particularly important should the sediments be analyzed for heavy metals. In such a case, it is safer to alter the shape of the acrylic tube ending by its trimming at an accurate angle. The issue is very rarely addressed in the literature (Stoner et al., 1983), and no effective solution has been developed so far permitting the collection of samples of bottom sediments in habitats heavily overgrown by submerged vegetation. In consequence, the chemical and physical properties of the sediments, as well as benthic biotic communities in such habitats are very little known. The modiﬁcation described can be used in virtually any coring equipment. In its simplest and easiest-to-use version, it can be an open-barrel corer—a tube of up to approximately2 minlength,andopenatboththetopand bottom, driven vertically into the sediment (Fig. 1B). At the end of the drive, the top of the tube is closed, and the tube together with the sediment is retrieved. This kind of device is very easy to operate, and is sufﬁcient for effective sampling of sediments in many shallow hab- itats. The asymmetric ending of the tube can also be applied in more sophisticated gravity corers, like Kajak or UWITEC corers, to sample at greater depths (Fig.1B). Introduction Sediment sampling methods have undergone a long evolution (Elliott & Tullett, 1978; Downing, 1984; IAEA, 2003). In the past, box-type samplers were used for both sediment sampling and sectioning. Currently, corer-type samplers, including both open-barrel and gravity corers, are believed to be the most suitable. They are particularly useful for sampling in the littoral zone, where various hard objects, like stones, sticks, or shoots and roots of aquatic vegetation occur. More- over, they permit the collection of so-called undis- turbed sediment cores (Blomqvist, 1991; Wright, 1991; Glew et al., 2001; Schumacher, 2005). The diameter of a tube sampler should be large enough to provide sufﬁcient material for analysis, minimize the possibility of sediment compression, and avoid the impact of smearing along the core edge. An internal tube diameter of 4–12 cm is considered optimal for most hydrobiological and paleolimnolog- ical investigations (Downing, 1984; IAEA, 2003). However, in situations where the bottom of a water ecosystem is covered with a dense carpet of sub- merged vegetation, sampling by means of such a core sampler becomes problematic or even impossible. The plant material clogs the tube opening, acting as a stopper. This does not allow the penetration of Handling editor: Jasmine Saros Handling editor: Jasmine Saros R. Kornijo´w (&) Department of Fisheries Oceanography and Marine Ecology, National Marine Fisheries Research Institute, Kołła˛taja 1, 81-332 Gdynia, Poland e-mail: ryszard.kornijow@mir.gdynia.pl T. Kairesalo Department of Ecological and Environmental Sciences, University of Helsinki, Niemenkatu 73, 15140 Lahti, Finland 12 3 3 2 Hydrobiologia (2013) 716:1–4 Changing the shape of the tube ending can be obtained by its trimming at an angle of about 30, or by ﬁtting (permanently or interchangeably) a metal collar made of stainless steel at the end of the tube (Fig. 1A). The advantage of the latter solution is that the sharp metal edges facilitate the penetration by the apparatus in hard sediment. They also easily intersect plant shoots, if they stop on the edge of the tube. The internal diameter of the metal collar should be the same as the internal diameter of the corer tube, but the external one can be larger by about 2 mm. The external diameter of the collar slightly larger than that of the tube, like the oblique cutting edge, additionally decreases friction forces on the outer tube wall when it is driven, and increases the ability of the device to penetrate with greater efﬁciency. Description of the improvement Restricted access to the sediment due to the presence of densely growing vegetation can be overcome by modifying the shape of the ending of the sampler tube in a way that it resembles a needle. Owing to this solution, penetration through macrophyte carpets progresses smoothly. This permits taking quantitative samples of sediments, and even intact cores of any length. Introduction This paperpresents a simple proposal that may permit overcoming the problem of clogging of tube samplers with macrophytic shoots. It involves changing the shape of the sampler tube ending. It has been successfully applied in both hydrobiological and paleolimnological studies on heavily overgrown macrophyte-dominated lowland lakes, with submerged plant biomass during summer varying from about 3 to 5 kg wet weight m-2 (e.g., Tarkowska-Kukuryk & Kornijo´w, 2008; Kornijo´w et al., 2013; Kowalewski et al., 2013). After collecting a sample, it is possible to take a water sample (using a vacuum pump) from above the sediment for chemical analysis, or to measure the water properties in situ by means of appropriate probes inserted to the tube, if it can be detached from the head sampler (Fig. 1C). The sediment core can be analyzed as a whole or sectioned at a given resolution using special devices for slicing (e.g., Wright, 1991; Winterhalter, 2001; Kornijo´w, 2013). Concluding remarks An advantage of a needle-shaped asymmetrical cut- ting edge, applied in our solution, is that it can 12 123 3 Hydrobiologia (2013) 716:1–4 m Fig. 1 The needle-shaped ending of a tube, with a metal collar at the bottom (A) left applied to a simple open-barrel corer, right—to UWITEC gravity corer (B) after disconnection from the sampler head, with an extruder inside and sediments ready for near- bottom water measurements or sediment sectioning (C) Fig. 1 The needle-shaped ending of a tube, with a metal collar at the bottom (A) left applied to a simple open-barrel corer, right—to UWITEC gravity corer (B) after disconnection from the sampler head, with an extruder inside and sediments ready for near- bottom water measurements or sediment sectioning (C) open-barrel corers. They may be expected in the case of some gravity corers where the water ﬂow may be disrupted by the ﬂow-actuated seal. Therefore, we recommend that the initial phase of driving a tube into the sediment be proceeded very gently. open-barrel corers. They may be expected in the case of some gravity corers where the water ﬂow may be disrupted by the ﬂow-actuated seal. Therefore, we recommend that the initial phase of driving a tube into the sediment be proceeded very gently. concentrate the cutting force to a small part of the advancing cutting edge. This decreases the initial resistance of penetration. The asymmetry of the cutting edge, however, may raise certain doubts concerning the dynamics of sediment collection. As a result of the uneven distribution of the corer mass, the sampler might have a tendency for tipping over when it begins penetrating the sediment. This is not the case here, however, due to the frequently occurring softness of highly hydrated surface sediment, and the considerable weight of a sampler lowered on a rope. In places with more compact sediments the problem may be overcome by pushing the sampler by hand into the sediment using a drive rod, connected to the top of the core tube. Acknowledgments The work was partly supported by the State Committee for Scientiﬁc Research, Project No. P06S00127. Our thanks are due to Małgorzata Kornijo´w for the linguistic veriﬁcation of the paper, and to Leszek Błe˛dzki for valuable comments on the manuscript. Concluding remarks Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Another potential problem is related to uneven entering the sediment by an asymmetrical tube edge. The surface sediment is unsupported on the side of the sample opposite the cutting edge. Under these condi- tions, the surface material may be deﬂected away from the core tube, and not be incorporated into the sample (the issue discussed in details, e.g., by Glew et al., 2001, p. 82, Fig. 5). In our opinion, such forces should not be produced if the oblique core ending is applied to References Blomqvist, S., 1991. Quantitative sampling of soft-bottom sediments. Problems and solutions. Marine Ecology Pro- gress Series 72: 295–304. Downing, J. A., 1984. Sampling the benthos of standing waters. In Downing, J. A. & F. H. Rigler (eds), A Manual on Methods for the Assessment of Secondary Productivity in Fresh Waters. Blackwell Scientiﬁc Publications, Oxford: 87–130. 12 3 3 3 Hydrobiologia (2013) 716:1–4 4 Elliott, J. M. & P. A. Tullett, 1978. A Supplement to a Bibli- ography of Samplers for Benthic Invertebrates. Freshwater Biological Association, Occasional Publication No. 20, Ambleside. Wasiłowska. & T. Namiotko, 2013. Disentangling natural and anthropogenic drivers of changes in a shallow, mac- rophyte dominated lake Rotcze (Eastern Poland). Aquatic Botany 106: 1–13. Wasiłowska. & T. Namiotko, 2013. Disentangling natural and anthropogenic drivers of changes in a shallow, mac- rophyte dominated lake Rotcze (Eastern Poland). Aquatic Botany 106: 1–13. Glew, J. R., J. P. Smol & W. M. Last, 2001. Sediment core collection and extrusion. In Last, W. M. & J. P. Smol (eds), Tracking Environmental Change Using Lake Sediments, Vol. 1., Basin analysis, coring, and chronological tech- niques. Kluwer Academic Publishers, Dordrecht: 73–105. Schumacher, B., 2005. Collection of Undisturbed Surface Sediments: Sampler Design and Initial Evaluation Testing. U.S. Environmental Protection Agency Ofﬁce of Research and Development, Washington, DC [available on internet at http://www.epa.gov/esd/cmb/pdf/USS_Sampler_FINAL_ REPORT.pdf]. Accessed 13 May 2013. IAEA, 2003. Collection and Preparation of Bottom Sediment Samples for Analysis of Radionuclides and Trace Ele- ments. IAEA-TECDOC-1360. IAEA, Vienna [available on internet at http://www-pub.iaea.org/MTCD/publications/ PDF/te_1360_web.pdf]. Accessed 13 May 2013. EPORT.pdf]. Accessed 13 May 2013. Stoner, A. W., H. S. Greening, J. D. Ryan & R. J. Livingston, 1983. Comparison of macrobenthos collected with cores and suction sampler in vegetated and unvegetated marine habitats. Estuaries 6: 76–82. Kornijo´w, R., 2013. A new sediment slicer for rapid sectioning of the uppermost sediment cores from marine and fresh- water habitats. Journal of Paleolimnology 49: 301–304. doi:10.1007/s10933-012-9655-9. Tarkowska-Kukuryk, M. & R. Kornijo´w, 2008. Inﬂuence of spatial distribution of submerged macrophytes on Chiro- nomidae assemblages in shallow lakes. Polish Journal of Ecology 56: 747–758. Kornijo´w, R., G. A. Kowalewski., S. McGowan., A. Kaczorowska, M. Ga˛siorowski., M. Wasiłowska., M. Obremska. & M. Suchora, 2013. Over 200 years intensive drainage practices induce ecosystem state change in a shallow lake from Poland. Ecosystems (submitted). Winterhalter, B., 2001. GEMAX – The Ultimate Corer for Soft Sediments. Oy Kart Ab, Helsinki [available on internet at www.kolumbus.ﬁ/boris.winterhalter/GEMAX.pdf]. Wright, H. E. Jr., 1991. Coring tips. Journal of Paleolimnology 6: 37–49. References Accessed 13 May 2013. Wright, H. E. Jr., 1991. Coring tips. Journal of Paleolimnology 6: 37–49. Kowalewski, G. A., R. Kornijo´w., S. McGowan., M. Suchora., K. Bałaga., A. Kaczorowska., M. Ga˛siorowski., A. 123 12
https://openalex.org/W3089235223	https://judikatif-upiyptk.org/ojs/index.php/judikatif/article/download/2/2	Indonesian	null	Branding Lasi Dairy Farm	Judikatif	2,021	cc-by-sa	1,629	Abstrak Di Sumatera Barat perkembangan populasi ternak sapi perah selama beberapa tahun menunjukan adanya peningkatan pertahun. Walaupun terjadi penurunan populasi sapi perah di provinsi Sumatera Barat pada tahun 2014 namun pada tahun berikutnya kembali mengalami peningkatan, hal ini menggambarkan bahwa minat peternak terhadap usaha peternakan sapi perah ini cukup besar. Satu - satunya usaha sapi perah yang saat ini sedang dikembangkan di Kenagarian Lasi, Kecamatan Candung, Kabupaten Agam adalah usaha mandiri yang dikelola oleh Bapak Suhatril, berdiri pada pertengahan tahun 2016 dengan nama peternakan sapi perah ”Lassy Dairy Farm”. Konsep awal pengerjaan logo Lassy Dairy Farm ini adalah bagaimana menciptakan image yang berdasarkan konsep S.W.O.T (Strength, Weakness, Opprtunity, Threat), hasil kesimpulan wawancara dengan owner dari Lassy Dairy Farm yaitu Healty, Pure, Family dan sentra. Kata kunci: Branding, Logo, Lassy Dairy Farm. © 2021 Judikatif identitas usaha Lassy Dairy Farm membuat usaha tersebut tidak mengalami peningkatan dan belum dikenal oleh orang banyak. Untuk meningkatkan sebuah perusaahan supaya citranya lebih dikenal oleh masyarakat luas maka di butuhkan sebuah Branding. Branding secara sederhana dapat diartikan sebagai proses pengelolaan merek sebuah organisasi yang bertujuan untuk meningkatkan ekuitas merek (brand equity) dalam jangka panjang. Manajemen juga dapat berarti bagian tertentu dalam perusahaan yang memiliki tanggung jawab diantaranya merancang identitas merek dan mengelolanya untuk mendapatkan efektivitas maksimal dan memastikan bahwa merek tidak terganggu oleh tindakan taktis pesaing. Dalam hal ini penulis menemukan kendala dalam usaha peternakan ini, yaitu kurangnya branding yang membuat citra dari usaha tersebut tidak terlalu di kenal oleh orang lain dan identitas logo yang di pakai perusahaan saat sekarang ini belum menampilkan citra perusahaan dan logo tersebut hanya di ambil dari internet. sesuai dengan wawancara dengan owner tersebut, Lassy Dairy Farm mempunyai keinginan untuk menjadikan daerah Nagari Lasi menjadi sentra sapi perah di Sumatera Barat, sedangkan dari nama Lassy tersebut penulis menemukan nama Lassy tersebut terletak di daerah Spanyol Untuk mengefektif Jurnal Desain Komunikasi Kreatif https://judikatif-upiyptk.org/ojs 2021 Vol. 3 No. 1 Hal: 6-8 e-ISSN: 2745-9454 Jurnal Desain Komunikasi Kreatif https://judikatif-upiyptk.org/ojs e-ISSN: 2745-9454 e-ISSN: 2745-9454 Branding Lasi Dairy Farm Aditya Pisma1, Riki Iskandar2 1,2Universitas Putra Indonesia YPTK Padang Abstract In West Sumatra, the population of dairy cattle for several years has shown an increase every year. Although there was a decline in the dairy cattle population in the province of West Sumatra in 2014 but the following year it increased again, this illustrates that the interest of farmers in dairy farming is quite large. The only dairy business currently being developed in Kenagarian Lasi, Candung District, Agam Regency is an independent business managed by Mr. Suhatri, established in mid- 2016 under the name "Lassy Dairy Farm". The initial concept of working on the Lassy Dairy Farm logo is how to create an image based on the S.W.O.T (Strength, Weakness, Opportunity, Threat) concept, the conclusion of an interview with the owner of Lassy Dairy Farm, namely Health, Pure, Family and Sentra. Keywords: Branding, Logo, Lassy Dairy Farm. Gambar 2. Final logo Gambar 2. Final logo 1. Pendahuluan Di Sumatera Barat perkembangan populasi ternak sapi perah selama beberapa tahun menunjukan adanya peningkatan pertahun. Walaupun terjadi penurunan populasi sapi perah di provinsi Sumatera Barat pada tahun 2014 namun pada tahun berikutnya kembali mengalami peningkatan, hal ini menggambarkan bahwa minat peternak terhadap usaha peternakan sapi perah ini cukup besar. Satu - satunya usaha sapi perah yang saat ini sedang dikembangkan di Kenagarian Lasi, Kecamatan Candung, Kabupaten Agam adalah usaha mandiri yang dikelola oleh Bapak Suhatril, berdiri pada pertengahan tahun 2016 dengan nama peternakan sapi perah ”Lassy Dairy Farm”. Bapak Suhatril merupakan alumni ITB, ia mendirikan usaha ini didorong oleh hobi yang beliau miliki. Lassy Dairy Farm telah mendapatkan surat izin usaha mikro dan kecil (IUMK) pada tanggal 6 September 2018 oleh pemerintahan kabupaten Agam, dan untuk lisensi BPOM (badan pengawasan obat dan makanan) masih dalam proses. tujuan beliau mendirikan usaha ini untuk meningkatkan ekonomi keluarga dan masyarakat sekitar Kenagarian Lasi Kecamatan Candung, Kabupaten Agam serta membantu penyediaan susu di Sumatera Barat. Tidak ada 1 \| Revisi: 10-05-2021 \| Diterbitkan: 30-06-2021 \| DOI: 10.35134/judikatif.v131.1 Diterima: 01-04-2021 \| Revisi: 10-05-2021 \| Diterbitkan: 30-06-2021 \| DOI: 10.35134/judikatif.v131.1 6 Aditya Pisma, dkk promosi Lassy Dairy Farm, maka dibutuhkan sebuah Branding dari segi Identitas Visual untuk meningkatkan citra perusahaan dan pembeda dengan peternakan sapi perah lainnya, serta dibutuhkan promosi dalam media cetak. Karena media cetak dapat memberikan informasi yang lebih rinci. Dengan demikian promosi tersebut akan diterima dengan baik oleh masyarakat. Maka dari itu penulis akan memuat sebuah karya akhir yang berjudul “Branding Lasi Dairy Farm”. promosi Lassy Dairy Farm, maka dibutuhkan sebuah Branding dari segi Identitas Visual untuk meningkatkan citra perusahaan dan pembeda dengan peternakan sapi perah lainnya, serta dibutuhkan promosi dalam media cetak. Karena media cetak dapat memberikan informasi yang lebih rinci. Dengan demikian promosi tersebut akan diterima dengan baik oleh masyarakat. Maka dari itu penulis akan memuat sebuah karya akhir yang berjudul “Branding Lasi Dairy Farm”. dengan karakter yang sama dengan visi dan misi yang dijalankan peternakan. Branding yang jelas dalam pemilihan nama, logo, warna, media, image, tipografi dan penerapan identitas, sehingga menimbulkan brand image yang dapat di ingat di lingkungan masyarakat. 3. Hasil dan Pembahasan Pada Branding Lasi Dairy Farm, media utama adalah logo. Logo menjelaskan wajah suatu perusahan yang menggabarkan identitas, karakter dan image perusahaan tersebut. Visual pada logo menampilkan stilisasi dari data inventaris visual. Dharsono Sony Kartika (2007:71) menjelaskan, “Stilisasi merupakan cara penggambaran untuk mencapai bentuk keindahan dengan cara meng-gayakan objek atau benda yang digambar, yaitu dengan cara meng-gayakan setiap kontur pada objek atau benda tersebut.” Stilisasi dilakukan terhadap objek yang berhubungan dengan konsep logo yang didapat dari S.W.O.T dan USP (Unique Selling Proposition), bertujuan untuk mendapatkan keywork design brief yang merepresentasikan identitas dan karakteristik dari Lasi Dairy Farm. Setelah melalui beberapa tahap menghasilkan beberapa logo yang akan diseleksi kembali menjadi 3 alternatif seperti pada gambar 1 sampai dengan gambar 5. 2.2. Pesan Verbal Bahasa dalam penerapannya merupakan unsur yang sangat penting dalam menunjang proses komunikasi, Bahasa berperan sebagai penghubung seseorang dengan orang lain dalam mencapai keinginan yang dimaksud, agar orang lain bisa mengetahui dan mengerti maksud yang diinginkannya, maka dapat dikatakan Bahasa sebagai fungsi utama dalam komunikasi. Dalam perancangan ini sedapat mungkin menimbulkan citra bahwa perancangan Branding Lassy dairy Farm, sangat membantuk peternakan itu sendiri dan memberikan informasi yang baik kepada masyarakat. Maka dari itu dalam perancangan Branding Lassy Dairy Farm ini penulis menggunakan Bahasa Indonesia yang sesuai dengan ejaan yang disempurnakan (EYD), agar pesan yang disampaikan dapat dengan mudah dimengerti dan dipahami oleh masyarakat yang menyaksikannya. 2.1. Metode Analisis Data Dalam Perancangan Branding Lassy Dairy Farm ini, adanya faktor faktor yang mempengaruhi dan menghambat, maka perlu suatu analisis internal dan eksternal sehingga sejauh mana factor-faktor kunci yang menjadi sasaran perancangan objek studi penelitian ini. Setelah melakukan identifikasi data, didapat bermacam jenis data. Maka selanjutnya adalah menganalisis data yang kemudian dapat diberi arti dan makna yang berguna dalam memecahkan masalah perancangan ulang. Analisis merupakan upaya untuk mensintetis data untuk menghasilkan simpulan data. Tujuannya menemukan pokok-pokok pikiran untuk pemecahan masalah yaitu membangun citra Lassy Dairy Farm yang berfungsi sebagai konsep perancangan Branding Analisis masalah yang digunakan dalam Branding Lassy Dairy Farm ini adalah analisis S.W.O.T (Strength, Weakness, Opprtunity, Threat) dan USP (Unique Selling Proposition). Gambar 1. Alternatif logo Gambar 2. Final logo Gambar 1. Alternatif logo Gambar 2. Final logo Daftar Rujukan Gambar 4. Pengaplikasian Logo Gambar 4. Pengaplikasian Logo [1] Kusrianto, Adi. 2007. Pengantar Desain Komunikasi Visual. Yogyakarta: Andi. Gambar 5. Pengaplikasian logo [2] Rusman. 2012. Pembelajaran Berbasis Teknologi dan Informasi Mengembangkan Professionalitas Guru. Jakarta: Raja Grafindo Persada [3] Rustan, Surianto, 2017. Mendesain Logo. Edisi 2017. Jakarta : Gramedia Pustaka Utama. [4] Layout: Dasar & penerapannya. Edisi 2017. Jakarta: PT Gramedia Pustaka Utama [5] Sachari, Agus. 2005. Pengantar Metode Penelitian Budaya dan Desain (Arsitektur, Seni Rupa, dan Kriya). Jakarta : Erlangga [6] Safanayong, yongki. 2013. Desain Komunikasi Visual Terpadu. Jakarta: Arte Intermedia [7] Tinarbuko, Sumbo 2008. Semiotika Komunikasi Visual. Yogyakarta: Jalasutra Gambar 5. Pengaplikasian logo 2.3. Pesan Visual Di dalam pesan visual, strategi secara keseluruhan menggunakan visual yang menarik dan berbeda, sesuai dengan karakter yang di tampilkan perusahan yaitu sapi dan perbukitan, dan peternakan tersebut telah menciptakan sebuah Branding yang sesuai Jurnal Desain Komunikasi Kreatif Vol. 3 No. 1 (2021) 6-8 Jurnal Desain Komunikasi Kreatif Vol. 3 No. 1 (2021) 6-8 7 Aditya Pisma, dkk identitas tempat usaha, selain itu, Branding Lasi Dairy Farm ini bertujuan untuk membangun citra yang lebih baik dari sebelumnya melalui media komunikasi visual kepada khalayak umum. Lewat perancangan ini diharapkan makin banyaknya masyarakat yang lebih mengenal Lasi Dairy Farm dan cangkupan produksipun bisa lebih meluas dari pada sebelumnya. Branding Lasi Dairy Farm dalam berbagai jenis media aplikasi sebagai media promosi kepada masyarakat menggunakan prinsip dan unsur desain untuk menghasilkan desain yang sempurna. Disamping itu juga melakukan observasi, beberapa analisis, dan proses berkarya untuk menghasilkan desain yang baik. Dalam Proses pencarian data dan penganalisaan masalah, penulis mendapat kemudahan dan kelancaran dalam mendapatkan data tersebut. Owner terbuka dalam memberikan informasi, baik data visual maupun verbal. Keseluruhan data yang didapatkan selanjutnya diolah untuk mendapatkan elemen-elemen desain yang akan di gunakan dalam perancangan ini. Secara umum, Branding perusahaan sekaligus mempromosikan perusahaan kepada masayarakat untuk mendapatkan citra yang lebih baik lagi agar menambah minat masyarakat luas tidak hanya di golongan distributor saja tapi bisa merambah lebih luas lagi. Selain itu, brand adalah identitas untuk membedakannya dengan yang lain dan tanda kepemilikan untuk membedakan milik tempat usaha walaupun bergerak di bidang yang sama, kemudian aplikasi perancangan sebagai alat memvisualkan citra kepada khalayak umum bahwa perusahaan tersebut konsekuen dan professional sekaligus untuk mencegah pembajakan dari sebuah image perusahaan Gambar 3. Pattern Illustrasi Gambar 3. Pattern Illustrasi Gambar 4. Pengaplikasian Logo 4. Kesimpulan Melalui proses berkarya Tugas Akhir ini, telah diciptakan sebuah Branding Lasi Dairy Farm sebagai Jurnal Desain Komunikasi Kreatif Vol. 3 No. 1 (2021) 6-8 Jurnal Desain Komunikasi Kreatif Vol. 3 No. 1 (2021) 6-8 8
https://openalex.org/W2017821949	https://boris.unibe.ch/16884/1/Huber%20%26%20Huber%20-%20CRS%20%282012%29.pdf	English	null	The Centrality of Religiosity Scale (CRS)	Religions	2,012	cc-by	8,336	Religions 2012, 3, 710–724; doi:10.3390/rel3030710 Religions 2012, 3, 710–724; doi:10.3390/rel3030710 Keywords: centrality of religiosity; measurement; dimensions; interreligious; religious construct system Stefan Huber 1,* and Odilo W. Huber 2 1 Faculty of Theology, Interreligious Studies, University of Berne, Unitobler, Länggassstrasse 51, CH-3012 Bern, Switzerland 1 Faculty of Theology, Interreligious Studies, University of Berne, Unitobler, Länggassstrasse 51, CH-3012 Bern, Switzerland 1 Faculty of Theology, Interreligious Studies, University of Berne, Unitobler, Länggassstrasse 51, CH-3012 Bern, Switzerland 2 Department of Psychology, University of Fribourg, Rue de Faucigny 2, CH-1700 Fribourg, Switzerland; E-Mail: odilo.huber@unifr.ch; Tel.: +41-26-300-76-34 2 Department of Psychology, University of Fribourg, Rue de Faucigny 2, CH-1700 Fribourg, Switzerland; E-Mail: odilo.huber@unifr.ch; Tel.: +41-26-300-76-34 2 Department of Psychology, University of Fribourg, Rue de Faucigny 2, CH-1700 Fribourg, Switzerland; E-Mail: odilo.huber@unifr.ch; Tel.: +41-26-300-76-34 * Author to whom correspondence should be addressed; E-Mail: stefan.huber@theol.unibe.ch; Tel.: +41-31-631-48-63; Fax: +41-31-631-82-24. * Author to whom correspondence should be addressed; E-Mail: stefan.huber@theol.unibe.ch; Tel.: +41-31-631-48-63; Fax: +41-31-631-82-24. Received: 9 July 2012; in revised form: 30 July 2012 / Accepted: 8 August 2012 / Published: 20 August 2012 Received: 9 July 2012; in revised form: 30 July 2012 / Accepted: 8 August 2012 / Published: 20 August 2012 Abstract: The Centrality of Religiosity Scale (CRS) is a measure of the centrality, importance or salience of religious meanings in personality that has been applied yet in more than 100 studies in sociology of religion, psychology of religion and religious studies in 25 countries with in total more than 100,000 participants. It measures the general intensities of five theoretical defined core dimensions of religiosity. The dimensions of public practice, private practice, religious experience, ideology and the intellectual dimensions can together be considered as representative for the total of religious live. From a psychological perspective, the five core-dimensions can be seen as channels or modes in which personal religious constructs are shaped and activated. The activation of religious constructs in personality can be regarded as a valid measure of the degree of religiosity of an individual. The CRS thus derives from the five dimensional measures a combined measure of the centrality of religiosity which is suitable also for interreligious studies. The paper presents the theoretical basis and rationale of its construction with different versions of the CRS in 20 languages with norm values for 21 countries. Furthermore, the paper presents versions of different extension and describes specific modifications that were developed for studies with Buddhists, Hindus and Muslims. Religions 2012, 3 711 2. Basic Ideas and Construction Principles General measures of religiosity refer to its intensity, salience, importance or centrality in the individual. Most common are single item scales asking for a self report on the subjective importance of religion or the salience of religious identity, e.g., ‗How important is religion for you‘ or ‗How religious do you consider yourself‘. These allow the most economical assessment of the general intensity of religiosity. However, there are at least two fundamental problems with this approach. First, the reliability of one item measures is undefined. Second, also the validity of such measures is debatable, because it remains unclear which criteria a respondent assesses in order to produce the response. The answer may have been generated based on belief, private religious practice, interest in religious questions, or the affiliation to a religious community. Thus, different respondents may generate their assessment based on different criteria. The centrality scale takes an inverse approach: It asks for the general intensities of theoretical defined core dimensions of religiosity which can be considered as representative for the total of religious live and derives from them a combined measure of the centrality of religiosity. This measurement strategy is based on two prerequisites. First is the problem of representativeness. A theoretically founded decision has to be made of which expressions of religiosity are representative for the whole of religious life. Second is the problem of generalizability of the religious content targeted by the indicators. Contents have to be identified that are meaningful and acceptable in most religious traditions allowing for transreligious generalization of the measure. 1. Introduction The Centrality of Religiosity Scale (CRS) is a measure of the centrality, importance or salience of religious meanings in personality. It has been developed by Huber [1–5] and has yet been applied in more than 100 studies in sociology of religion, psychology of religion and religious studies in 25 countries with in total more than 100,000 participants. The largest single application is in the global Religion Monitor with representative samples in 21 countries [6]. However, no comprehensive overview on the scale comprising a base for its practical application is yet available in English. The present paper aims to close this desideratum. It consists of four parts: first we introduce the basic ideas and construction principles of the CRS, second we sketch the model of religiosity on which the CRS is based on. Third, we provide a taxonomy of the different versions of the CRS. Finally, norm values from 21 nations are provided. Religions 2012, 3 712 Religions 2012, 3 social expectations. For instance, ―The intellectual dimension has to do with the expectation that the religious person will be informed and knowledgeable about the basic tenets of his faith and its sacred scriptures.‖ ([8], p. 11) or ―The experiential dimension gives recognition to the fact that all religions have certain expectations, however imprecisely they may be stated, that the religious person will at one time or another achieve direct knowledge of ultimate reality or will experience religious emotion.‖ ([8], p. 10). There are two major achievements of Glocks model with respect to earlier conceptualizations: first is the theoretical discussion of the problem of the universal frame of reference for empirical research on religion and religiosity. Second is the identification of a limited set of core dimensions which cover the general scope of religious life. The five core dimensions, while being developed from a sociological perspective, do also cover religiosity from a psychological perspective as they denote distinguishable psychological modes of the representation of religious contents. The intellectual and ideological dimensions refer to thought, the dimensions of public and private practice refer to action, and the experiential dimension refers to experience, emotion and perception. Thus they can be considered as representative for religiosity from both perspectives, the sociological perspective reflecting social expectations as well as for the psychological representation of religious contents. 2.1. Identification of Dimensions of Religiosity With respect to the first problem of the identification of representative dimensions of religiosity, the CRS refers to the multidimensional model of religion by Charles Glock [7,8]. Glock‘s approach is originated in sociology of religion. He defined five core dimensions of religion constituting a general frame of reference for empirical research: the intellectual, the ideological, the ritualistic, the experiential, and the consequential dimension. In 1968, Stark and Glock eliminated the consequential dimension from the model and split the ritualistic dimension into public and private practice, thus maintaining five dimensions [9]. The approach of Glock was centered on religious institutions and Religions 2012, 3 Religions 2012, 3 may not be predicted with sufficient accuracy from each other [1]. These findings strongly support Glock‘s claim that the five core dimensions allow representative measurement of a broad scope of religious life. Thus, in order to assess the religiosity of a person, it is necessary to measure all 5 dimensions. 2.3. The Problem of the General Importance of Religiosity Glock‘s model, however, does not address the general importance of religion for the individual as conceptualized by one-item scales. Hence, the relation between the postulated multidimensional structure of religiosity and the general importance of religion remains unclear. A first attempt to solve this problem was undertaken by Wimberly [14] referring to identity theory of Stryker [15] and exchange theory of Homans [16]. Wimberley assumes that the pattern of the five dimensions in the individual depends on two factors: first on the salience of the religious identity and second on the ratio of costs and rewards of religious life in the realms of the five dimensions. In the case of high identity salience he expects a strong intrinsic religious motivation which determines all or nearly all facets of the religious life of the individual. As a result of this strong and unifying cause, the five dimensions of religiosity loose their relative autonomy. They should be similarly high developed regardless of their costs and rewards. In the contrasting case of low salience of religious identity the unifying causation of religious life by an intrinsic motivation is missing. As a result the pattern of five dimensions depends mainly on the different ratios of the costs and rewards, which are connected with these dimensions causing a high degree of relative autonomy. On the basis of these considerations Wimberley hypothesized that in empirical studies the relative autonomy of the five dimensions depends on the share of respondents with a low or medium salience of the religious identity. The higher this share is the greater the measured relative autonomy of the five dimensions should be. In the border case of a sample in which most of the respondents have a very salient religious identity, no autonomy of the dimensions should be detectable. Unfortunately, Wimberly never tested this hypothesis empirically. 2.2. Generalizability of Religious Contents The second problem is the generalizability of the religious contents used for the operationalization of the five core dimensions. In his empirical investigations, Glock focused on North American Christianity (e.g., [9]), hence the indicators with which the multidimensional model was investigated have a Christian bias and contradict his theoretical claim of universality. This threatens the generalizability of the measure as well as of the results. A more practical problem of some operationalizations of the Glock model is the lack of distinction between the five core dimensions. Weigert and Thomas [10] showed this paradigmatically in their critics of the operationalization of the Glock model by Faulkner and DeJong [11]. In this questionnaire most items measuring other dimensions were confounded with the dimension of religious ideology (see [1], pp 136–144 for an in-depth discussion). To overcome these restrictions, two principles for an universal operationalization of the five core dimensions should be considered [1]. First, items should be strongly related to typical expressions of the respective dimensions For instance, the intellectual dimension should only refer to processes or results of intellectual activities but not to the belief in these results. The experiential dimension, e.g., should refer to situations in which direct contact with an ultimate reality is perceived but not to attitudes towards such experiences. Second, to provide generalizability, the religious contents measured should be as general as possible and should be relevant and meaningful in the context of different religious traditions. Within the realm of Christian religiosity, a multitude of studies investigated Glock‘s model and confirmed the factors. Additional confirmation has been found also in studies not referring to Glock‘s model. In a meta-analysis of sociological studies, most of the factors found could be re-categorized in Glock‘s dimensions [1,12]. Additionally, it has been shown that the religious indicators applied in the international survey programme ISSP refer to one of the Glock dimensions each [13]. Furthermore, all five dimensions are necessary to describe religiosity because they are relatively autonomous, i.e., they 713 Religions 2012, 3 714 Religions 2012, 3 Religions 2012, 3  From a sociological perspective, the intellectual dimension refers to the social expectation that religious people have some knowledge of religion, and that they can explain their views on transcendence, religion and religiosity. In the personal religious construct system this dimension is represented as themes of interest, hermeneutical skills, styles of thought and interpretation, and as bodies of knowledge. A general indicator for the intellectual dimension is the frequency of thinking about religious issues. It indicates how often religious contents are ―updated‖ through the medium of thinking, which leads into the heart of the intellectual dimension. Furthermore, the content of this indicator is independent of any confessional bias or religious affiliation. It can therefore be applied across religions.  From a sociological perspective, the intellectual dimension refers to the social expectation that religious people have some knowledge of religion, and that they can explain their views on transcendence, religion and religiosity. In the personal religious construct system this dimension is represented as themes of interest, hermeneutical skills, styles of thought and interpretation, and as bodies of knowledge. A general indicator for the intellectual dimension is the frequency of thinking about religious issues. It indicates how often religious contents are ―updated‖ through the medium of thinking, which leads into the heart of the intellectual dimension. Furthermore, the content of this indicator is independent of any confessional bias or religious affiliation. It can therefore be applied across religions.  The dimension of ideology refers to the social expectation that religious individuals have beliefs regarding the existence and the essence of a transcendent reality and the relation between the transcendence and human. In the personal religious construct system this dimension is represented as beliefs, unquestioned convictions and patterns of plausibility. General indicators of this dimension should focus only on the aspect of the plausibility of the existence of a transcendent reality, e.g., ―To what extend do you believe in the existence of God or something divine‖. This ―basic-belief‖ is common to most religious traditions, because it is a prerequisite for all further concepts and dogmas concerning the essence of this reality. Once a respondent considers transcendent reality as plausible, specific constructions of transcendence as prevalent in different traditions can become psychologically relevant. 3. Revision of the Five-Dimensional Model of Religiosity A second proposal for the clarification of the relation between the five core-dimensions of religiosity and the general importance of religion was made by Huber [1]. He approaches the problem from the perspective of personality psychology inspired by ideas of Allport and Ross [17] and Kelly [18], suggesting the concept of the personal religious construct-system as the unifying psychological entity in which the core-dimensions merge. Referring to Kelly‘s personality theory a personal construct is a pattern for the anticipation of events. Accordingly the personal system of religious constructs can be defined as a superstructure in personality which consists of all personal constructs which are related to the individually defined realm of religion and religiosity. A personal religious construct is activated when the individual anticipates something with a religious meaning. In relation to this approach, the five core-dimensions can be seen as channels or modes in which personal religious constructs are activated. The interplay between the sociologically defined core-dimensions and the psychologically defined personal religious construct-system can be described as follows: Religions 2012, 3 Religions 2012, 3 of religious perceptions and as a body of religious experiences and feelings. Analogously to private practice, two basic forms of experiencing the transcendence can be distinguished, ―one-to-one experiences‖ which correspond to a dialogical spirituality pattern and ―experiences of being at one‖ corresponding to a participative one. Hence, we recommend the use of both expressions of religious experience for the measurement of its general intensity. As discussed above, the five core-dimensions can be seen as representative for the whole of religious life. This is the key for the construction of the CRS. It rests on two assumptions: 1. The measurement of the general intensity of the five core-dimensions allows a representative estimation of the frequency and intensity of the activation of the personal religious construct system. estimation of the frequency and intensity of the activation of the personal religious const 2. The probability of a central position of the religious construct-system in personality increases with the overall intensity and frequency of its activation. Religions 2012, 3  The dimension of public practice refers to the social expectation that religious individuals belong to religious communities which is manifested in the public participation in religious rituals and in communal activities. In the personal religious construct system this dimension is represented as patterns of action and as a sense of belonging with respect to a certain social body as well as to a certain ritualized imagination of the transcendence. The general intensity of this dimension can be measured easily by inquiring about the frequency with which somebody takes part in religious services. In interreligious studies it is recommendable to vary the label for religious service according to the religious affiliation of the respondents—e.g. ―church attendance‖ for Christians, and ―Friday prayer‖ for Muslims.  The dimension of private practice refers to the social expectation that religious individuals devote themselves to the transcendence in individualized activities and rituals in private space. In the personal religious construct system this dimension is represented as patterns of action and a personal style of devotion to the transcendence. It makes sense to consider both prayer and meditation when measuring the general intensity of private practices, because they express basic and irreducible forms of addressing oneself to transcendence. Inherent to the structure of prayer is the act of addressing a ―counterpart.‖ This dynamic implies a dialogical pattern of spirituality. In contrast, meditation is structured more fundamentally with reference to the self and/or an all-pervasive principle, and is therefore more in line with a participative pattern of spirituality. Considering both forms of private religious practice means that both basic patterns of spirituality are covered.  The dimension of religious experience refers to the social expectation that religious individuals have ―some kind of direct contact to an ultimate reality‖ ([9], p. 126) which affects them emotionally. In the personal religious construct system this dimension is represented as patterns  The dimension of religious experience refers to the social expectation that religious individuals have ―some kind of direct contact to an ultimate reality‖ ([9], p. 126) which affects them emotionally. In the personal religious construct system this dimension is represented as patterns 715 4. Versions of the Centrality Scale The centrality scale is available in different versions (see Table 1 with the latest revised items in English, Table 2 for the German original versions; versions in 19 languages are available online http://www.theol.unibe.ch/ipt/huber.html). They differ first in economics of measurement, and second in range of inter-religious applicability. All versions operationalize the before mentioned five core dimensions on a most general level with items that measure either the objective or subjective frequency, or the intensity of the activation of personal religious constructs specific to the modi of the dimensions. Wherever possible, objective frequencies were asked (items 03, 04, and 04b in Tables 1 and 2). These items consider religious practice which in most religions traditions are undertaken regularly and are easily accessible in frequency format. For events that may occur less regularly, subjective frequencies were asked in five levels (never, rarely, occasionally, often, and very often). The different frequency formats require the recoding of the objective frequencies into the five levels of the subjective frequencies (see Table 2 for the recoding procedure). For items where frequencies have little meaning as e.g., the belief in something divine, its intensity or importance is assessed in five levels (not at all, not very much, moderately, quite a bit, and very much so). 3.1. Construct Validity of the CRS It is important to keep in mind that the construction of the CRS follows a probabilistic logic. This means, that in general individuals with higher scores on the CRS have a more central religious construct system. The validity of this measurement strategy was confirmed empirically. There are very high correlations between the CRS and self reports of the salience of the religious identity, which are traditionally applied as one item scales for religiosity. They amount to 0.83 in a students‘ sample [1,2] and 0.73 in the international Religion Monitor [19]. Furthermore, there are also high correlations between CRS values and self-reports of the importance of religion for daily life, with coefficients of 0.78 in a students‘ sample [1,2] and 0.67 in the international Religion Monitor [19]. An alternative way to validate the CRS consists in the test of differential predictions for categorical groups of respondents based on their CRS-score. Huber [1–5] distinguishes between the groups of the ―highly-religious‖ with a central position of the religious construct system in the individual, the ―religious‖ with a subordinated position of the personal religious construct system and the ―non-religious‖ with hardly any religious construct system (see [3], pp. 36–38 and [4], pp. 44–48, for an in-depth discussion of the categorization in three groups and the resulting strategies for empirical research). Theoretically it can be expected that the group of the ―highly religious‖ differ at least in two constitutive features form the two other groups. First, in the group of the ―highly-religious‖ the system of personal religious constructs should be much more differentiated than in the groups of the ―religious‖ and ―non-religious‖ (thesis of differentiation). Second, religious contents, e.g., the experience of forgiveness by God, which are salient in the religious construct system of the ―highly-religious‖, should have a much stronger relevance for general psychological dispositions, e.g., the willingness to forgive others in social situations, than in the groups of the ―religious‖ and ―non-religious‖ (thesis of differentiation). Both predictions were already tested empirically. The thesis of differentiation was confirmed in relation to the theological complexity of positive and negative religious emotions [4]. The thesis of relevance was confirmed in relation to the political relevance of religious concepts [4,5], and in relation to social relevance of the experience of forgiveness by God [3,20]. Religions 2012, 3 Religions 2012, 3 716 4.1. Length of the CRS Versions Experience 10: How often do you experience situations in which you have the feeling that God or something divine wants to communicate or to reveal something to you? 10b: How often do you experience situations in which you have the feeling that you are touched by a divine power? Intellect 11: How often do you keep yourself informed about religious questions through radio, television, internet, newspapers, or books? Ideology 12: In your opinion, how probable is it that a higher power really exists Public practice 13: How important is it for you to be connected to a religious community? Private practice 14: How often do you pray spontaneously when inspired by daily situations? 14b: How often do you try to connect to the divine spontaneously when inspired by daily situations? Experience 15: How often do you experience situations in which you have the feeling that God or something divine is present? 1. Items and versions of the Centrality of Religiosity Scale (CRS)—English versions. of the Centrality of Religiosity Scale (CRS)—English versions. versions Basic CRS versions Additional Items for the interreligious versions only Interreligious CRSi versions ssues? CRS-5 CRS-10 CRS-15 CRSi-7 CRSi-14 CRSi-20 God or ervices? 04b: How often do you meditate? in which ng divine 05b: How often do you experience situations in which you have the feeling that you are in one with all? e about religious n afterlife—e.g. f the dead or services? u? 09b: How important is meditation for you? ns in which you divine wants to 10b: How often do you experience situations in which you have the feeling that you are touched by a divine power? ormed about religious newspapers, or books? a higher power really nnected to a religious when inspired by daily 14b: How often do you try to connect to the divine spontaneously when inspired by daily situations? in which you have the nt? Table 1. Items and versions of the Centrality of Religiosity Scale (CR Dimension Items for both the basic and interreligious versions Basic CRS versions Additional It only Intellect 01: How often do you think about religious issues? CRS-5 CRS-10 CRS-15 Ideology 02: To what extent do you believe that God or something divine exists? Public practice 03: How often do you take part in religious services? Private practice 04: How often do you pray? 4.1. Length of the CRS Versions The basic scale is provided in three lengths with 15 (CRS-15), with 10 (CRS-10) and with 5 items (CRS-5). These versions suitable at least for Abrahamitic religions (Judaism, Christianity, Islam) are nested in each other and grow more economical (see Tables 1 and 2). The original versions with 10 or 15 items with a slightly different wording in some items is documented in several publications [1–3]. The modifications are explained below. The CRS-15 has three items per dimension. It is the version with the highest dimensional discriminance, i.e., it allows the measurement of the core dimensions with the highest reliability and accuracy and thus is best being applied if the differential influence of the dimensions on other phenomena is of interest. In three studies reliabilities of the individual dimensions ranged from 0.80 to 0.93, and from 0.92 to 0.96 for the whole CRS-15 [3]. The CRS-10 is a reduced and more economical version containing only two questions per dimension (reliability in eight studies from 0.89 to 0.94; [2]). Selected were the items which had the respectively highest theoretical relevance to the respective dimensions. The CRS-5 is the most economical version. Religions 2012, 3 Religions 2012, 3 717 Table 1. Items and versions of the Centrality of Religiosity Scale (CRS)—English versions. Dimension Items for both the basic and interreligious versions Basic CRS versions Additional Items for the interreligious versions only Interreligious CRSi versions Intellect 01: How often do you think about religious issues? CRS-5 CRS-10 CRS-15 CRSi-7 CRSi-14 CRSi-20 Ideology 02: To what extent do you believe that God or something divine exists? Public practice 03: How often do you take part in religious services? Private practice 04: How often do you pray? 04b: How often do you meditate? Experience 05: How often do you experience situations in which you have the feeling that God or something divine intervenes in your life? 05b: How often do you experience situations in which you have the feeling that you are in one with all? Intellect 06: How interested are you in learning more about religious topics? Ideology 07: To what extend do you believe in an afterlife—e.g. immortality of the soul, resurrection of the dead or reincarnation? Public practice 08: How important is to take part in religious services? Private practice 09: How important is personal prayer for you? 09b: How important is meditation for you? 4.1. Length of the CRS Versions Intellect 06: Wie stark interessieren Sie sich dafür, mehr über religiöse Themen zu erfahren? Ideology 07: Wie stark glauben Sie daran, dass es ein Leben nach dem Tod gibt?—z.B. Unsterblichkeit der Seele, Auferstehung von den Toten oder Reinkarnation? Public practice 08: Wie wichtig ist Ihnen die Teilnahme an Gottesdiensten? Private practice 09: Wie wichtig ist für Sie das persönliche Gebet? 09b: Wie wichtig ist für Sie Meditation? Experience 10: Wie oft erleben Sie Situationen, in denen Sie das Gefühl haben, dass Gott oder etwas Göttliches Ihnen etwas sagen oder zeigen will? 10b: Wie oft erleben Sie Situationen, in denen Sie das Gefühl haben, dass Sie von einer göttlichen Kraft berührt werden? Intellect 11: Wie oft informieren Sie sich durch Radio, Fernsehen, Internet, Zeitschriften oder Bücher über religiöse Fragen? Ideology 12: Wie hoch ist Ihrer Ansicht nach die Wahrscheinlichkeit, dass es eine höhere Macht gibt? Public practice 13: Wie wichtig ist Ihnen die Verbindung zu einer religiösen Gemeinschaft? Private practice 14: Wie oft richten Sie mitten in Ihrem Alltag ein kurzes Gebet an Gott? 14b: Wie oft suchen Sie mitten in Ihrem Alltag Kontakt zu einer göttlichen Kraft? Experience 15: Wie oft erleben Sie Situationen, in denen Sie das Gefühl haben, dass Gott oder etwas Göttliches anwesend ist? Table 2. Items and versions of the Centrality of Religiosity Scale (CRS)—Original German versions. b th th b i d i t li i i B i Additi l It f th i t li i i I t li Table 2. Items and versions of the Centrality of Religiosity Scale (CRS)— Dimension Items for both the basic and interreligious versions Basic CRS versions Additional It only Intellect 01: Wie oft denken Sie über religiöse Fragen nach? CRS-5 CRS-10 CRS-15 Ideology 02: Wie stark glauben Sie daran, dass Gott oder etwas Göttliches existiert? Public practice 03: Wie häufig nehmen Sie an Gottesdiensten teil? Private practice 04: Wie häufig beten Sie? 04b: Wie häu Experience 05: Wie oft erleben Sie Situationen, in denen Sie das Gefühl haben, dass Gott oder etwas Göttliches in Ihr Leben eingreift? 05b: Wie oft das Gefühl ha Intellect 06: Wie stark interessieren Sie sich dafür, mehr über religiöse Themen zu erfahren? Ideology 07: Wie stark glauben Sie daran, dass es ein Leben nach dem Tod gibt?—z.B. Unsterblichkeit der Seele, Auferstehung von den Toten oder Reinkarnation? Public practice 08: Wie wichtig ist Ihnen die Teilnahme an Gottesdiensten? 4.1. Length of the CRS Versions 04b: How ofte Experience 05: How often do you experience situations in which you have the feeling that God or something divine intervenes in your life? 05b: How of which you ha all? Intellect 06: How interested are you in learning more about religious topics? Ideology 07: To what extend do you believe in an afterlife—e.g. immortality of the soul, resurrection of the dead or reincarnation? Public practice 08: How important is to take part in religious services? Private practice 09: How important is personal prayer for you? 09b: How imp Experience 10: How often do you experience situations in which you have the feeling that God or something divine wants to communicate or to reveal something to you? 10b: How oft have the feelin Intellect 11: How often do you keep yourself informed about religious questions through radio, television, internet, newspapers, or books? Ideology 12: In your opinion, how probable is it that a higher power really exists Public practice 13: How important is it for you to be connected to a religious community? Private practice 14: How often do you pray spontaneously when inspired by daily situations? 14b: How ofte when inspired Experience 15: How often do you experience situations in which you have the feeling that God or something divine is present? 09b: How important is meditation for you? 10b: How often do you experience situations in which you have the feeling that you are touched by a divine power? 14b: How often do you try to connect to the divine spontaneously when inspired by daily situations? Religions 2012, 3 Religions 2012, 3 718 Table 2. Items and versions of the Centrality of Religiosity Scale (CRS)—Original German versions. Dimension Items for both the basic and interreligious versions Basic CRS versions Additional Items for the interreligious versions only Interreligious CRSi versions Intellect 01: Wie oft denken Sie über religiöse Fragen nach? CRS-5 CRS-10 CRS-15 CRSi-7 CRSi-14 CRSi-20 Ideology 02: Wie stark glauben Sie daran, dass Gott oder etwas Göttliches existiert? Public practice 03: Wie häufig nehmen Sie an Gottesdiensten teil? Private practice 04: Wie häufig beten Sie? 04b: Wie häufig meditieren Sie? Experience 05: Wie oft erleben Sie Situationen, in denen Sie das Gefühl haben, dass Gott oder etwas Göttliches in Ihr Leben eingreift? 05b: Wie oft erleben Sie Situationen, in denen Sie das Gefühl haben, mit Allem Eins zu sein? 1 Furthermore, in the two ideological item 02 and 07 the reference to the probability of the existence of God or of an afterlife was replaced by the reference to belief (see [1–3]). This was undertaken in order to ease the understanding of the questions, as research on probabilities has shown that many people do not understand the concept and may not properly deal with probabilities (e.g., [21]). The new versions thus provide more unambiguous references and enhance reliability specifically in respondents with low numeracy. 4.1. Length of the CRS Versions Private practice 09: Wie wichtig ist für Sie das persönliche Gebet? 09b: Wie wic Experience 10: Wie oft erleben Sie Situationen, in denen Sie das Gefühl haben, dass Gott oder etwas Göttliches Ihnen etwas sagen oder zeigen will? 10b: Wie oft Gefühl haben werden? Intellect 11: Wie oft informieren Sie sich durch Radio, Fernsehen, Internet, Zeitschriften oder Bücher über religiöse Fragen? Ideology 12: Wie hoch ist Ihrer Ansicht nach die Wahrscheinlichkeit, dass es eine höhere Macht gibt? Public practice 13: Wie wichtig ist Ihnen die Verbindung zu einer religiösen Gemeinschaft? Private practice 14: Wie oft richten Sie mitten in Ihrem Alltag ein kurzes Gebet an Gott? 14b: Wie oft göttlichen Kra Experience 15: Wie oft erleben Sie Situationen, in denen Sie das Gefühl haben, dass Gott oder etwas Göttliches anwesend ist? he Centrality of Religiosity Scale (CRS)—Original German versions. s versions Basic CRS versions Additional Items for the interreligious versions only Interreligious CRSi versions n nach? CRS-5 CRS-10 CRS-15 CRSi-7 CRSi-14 CRSi-20 oder etwas en teil? 04b: Wie häufig meditieren Sie? en Sie das ches in Ihr 05b: Wie oft erleben Sie Situationen, in denen Sie das Gefühl haben, mit Allem Eins zu sein? afür, mehr über Leben nach dem Auferstehung von Gottesdiensten? Gebet? 09b: Wie wichtig ist für Sie Meditation? en Sie das Gefühl nen etwas sagen 10b: Wie oft erleben Sie Situationen, in denen Sie das Gefühl haben, dass Sie von einer göttlichen Kraft berührt werden? dio, Fernsehen, Internet, gen? hrscheinlichkeit, dass es ng zu einer religiösen tag ein kurzes Gebet an 14b: Wie oft suchen Sie mitten in Ihrem Alltag Kontakt zu einer göttlichen Kraft? n Sie das Gefühl haben, st? 14b: Wie oft suchen Sie mitten in Ihrem Alltag Kontakt zu einer göttlichen Kraft? Religions 2012, 3 719 Religions 2012, 3 4.2. Application to Different Religious Traditions Recently, the scale was modified and extended in order to enhance its scope of applicability [3,4]. The scale was originally developed to measure religiosity in the context of the Abrahmitic tradition with a monotheistic concept of God (Judaism, Christianity, and Islam). In these religions a dialogic pattern of spirituality in which God is perceived as a counterpart of man is dominant. However, in the realm of eastern religious traditions and new western forms of spirituality meditation and mystical experience are of greater relevance. This reflects a more participative pattern of spirituality in which the divine is perceived of as an inherent principle of the self as well as of the universe. Therefore, two types of modifications were undertaken. First, in the basic CRS items in which the concept of ‗God‗ is mentioned it was replaced by the more general expression ‗God or something divine‘.1 This is the case in the items 02, 05, 10, and 15. It is noteworthy that this modification was undertaken for all versions of the CRS, including the basic versions. Second, specifically for the interreligious versions of the CRS additional items reflecting a participative pattern of spirituality were included for the measurement of the dimensions of private practice and experience. These are items 04b and 05b for CRSi-7, items 09b and 10b for CRSi-14, and item 14b for CRSi-20. In the scaling procedure alternatively the basic (e.g., 04) or of the additional item (e.g., 04b) is used, with the item with the higher score entering the total score of the respective CRSi-version. This allows the measurement of the respective dimension (and the centrality value) adaptive to the specific spirituality pattern of the individual respondent. We assume that the respective dimension is expressed according to the higher value of the respective two items. The CRSi-7 reached a high internal consistence of 0.84 (Cronbach‘s Alpha) in the Religion Monitor [4]. Religions 2012, 3 Religions 2012, 3 experience. Here, many Muslim respondents may perceive the idea of a direct contact with God as a violation of the Islamic concept of the absolute sovereignty of God. To avoid possible irritations we recommend a more reserved description of divine actions in items 05 and 10: Instead of ―intervenes in your life‖ (item 05) we recommend ―allows for an intervention in your life‖, and instead of ―wants to communicate or to reveal something to you‖ (item 10) we recommend ―lets something be communicated or revealed to you‖. 4.3. Special Items for Different Religious Groups In surveys with Buddhists, Hindus, and Muslims, specific modifications of the CRS were developed. For Buddhists or Hindus—reflecting their openness for polytheistic concepts and practices (at least in the respective religious folk traditions)—the term ‗God or something divine‘ in items 02, 05, 10, and 15 should be extended to ‗God, deities or something divine‘. This enhances the scope of personal religious constructs that can be measured in the ideological and experiential dimensions. For Muslims, two specific modifications are recommended: First, in Islam there is a differentiation between obligatory prayer (Salat) and private prayer (Du‘a). To ensure the comparability of the frequency and importance of the private prayer dimension with the respective measurements for other religions, in surveys investigating Muslims the value for (Du‘a) should be considered in the calculation of the centrality score. However, it is important first to ask for the frequency and importance of the obligatory prayer (Salat) in the questionnaire, and only thereafter for the private prayer (Du‘a) in order to make the item unambiguous. The second modification concerns the dimension of religious 720 4.4. Coding of Frequencies of Religious Behaviors The items scores are 1 to 5 for these with 5 answer options, for the items concerning prayer, meditation, and religious services the answers are coded according to Table 3. In the calculation of the CRS score, the item sum score is divided through the number of scored scale items. This allows for a range of the CRS score between 1.0 and 5.0. For the categorization of the groups of the ―highly-religious‖, ―religious‖, and ―non-religious‖ we propose the following thresholds: 1.0 to 2.0: not-religious, 2.1 to 3.9: religious, 4.0 to 5.0: highly-religious. For an extensive theoretical and empirical discussion of the categorization see [1], pp. 257–264; [2], pp. 93–99; [3], pp. 220–227; [4]. Alternatively, the thresholds can be understood in an intuitive manner derived from the wording of the five levels of the frequency and intensity response scales. These are related to the presence of religious constructs in the personal religious construct system (see Table 4). Table 3. Recoding of objective frequencies of the items concerning prayer, meditation, and religious services into five score levels. Objective frequencies of prayer (personal and obligatory) and meditation Recoding into five levels Objective frequencies of participation in religious services Recoding into five levels A) Several times a day 5 A) More than once a week 5 B) Once a day B) Once a week C) More than once a week 4 C) One or three times a month 4 D) Once a week 3 D) A few times a year 3 E) One or three times a month E) Less often 2 F) A few times a year 2 F) Never 1 G) Less often H) Never 1 Table 4. Hermeneutics of the wording a five level answer scale. Score Wording Hermeneutics (presence of personal constructs in personality) Frequency Importance Categories of a five-level answer-scale 5 very often very much so Clear presence 4 often quite a bit 3 occasionally moderately Transition area: background presence 2 rarely not very much No or only marginal presence 1 never not at all Table 3. Recoding of objective frequencies of the items concerning prayer, meditation, and religious services into five score levels. 5. Norm Values and Reliabilities in the Religion Monitor Table 5 shows representative norm values for the CRSi-7 in 21 countries. For Germany four columns are displayed, for the general survey in Germany in total with subsamples for the western and the eastern part, and for a separate sample of the Muslim population in Germany. All norm values are derived from the international Religion Monitor which was conducted in the year 2007 [6]. The internal consistency of the CRS-5 in the total sample of the Religion Monitor is 0.85, that of the CRS-10 is 0.93, and that of the CRSi-7 is 0.84 (Cronbach‘s Alphas). Religions 2012, 3 The first two response levels indicate that religious constructs are barely present in an individual. As a result, religious constructs should not become psychologically relevant. For individuals who on average answer within this range thus it is plausible that they can be assigned to the category of the ―non-religious‖. In contrast, the wording of the response categories four ("often" / ―quite a bit‖) and five ("very often‖ / ―very much so") express that religious constructs are clearly present in a personal religious construct system. Consequently, we should assume that they are highly relevant psychologically for that individual's religious experience and behavior and that they exert an influence on non-religious constructs, allowing a categorization of the individual as ―highly religious‖. The intermediate response category, however, represents the transitional range between absence and clear presence of religious constructs. Responses at this level indicate that religious constructs are present in an individual's life horizon but also that they are not activated very frequent and intense. We thus suggest categorizing these individuals as ‗religious‘. 4.4. Coding of Frequencies of Religious Behaviors Objective frequencies of prayer (personal and obligatory) and meditation Recoding into five levels Objective frequencies of participation in religious services Recoding into five levels A) Several times a day 5 A) More than once a week 5 B) Once a day B) Once a week C) More than once a week 4 C) One or three times a month 4 D) Once a week 3 D) A few times a year 3 E) One or three times a month E) Less often 2 F) A few times a year 2 F) Never 1 G) Less often H) Never 1 Table 3. Recoding of objective frequencies of the items concerning prayer, meditatio religious services into five score levels. Table 4. Hermeneutics of the wording a five level answer scale. 721 6. Conclusions The Centrality of Religiosity Scale (CRS) is a measure of the centrality, importance or salience of religious meanings in personality. It has been applied yet in different versions in a multitude of studies in sociology of religion, psychology of religion and religious studies in various countries. It measures the general intensities of the five theoretical defined core dimensions of religiosity, public practice, private practice, religious experience, ideology, and the intellectual dimension. They can together be considered as representative for the total of religious live. The paper presents the theoretical basis and rationale of its construction. Different revised versions of the CRS are presented varying in extension. Furthermore, we describe modifications that were developed for studies with Buddhists, Hindus and Muslims in order to provide a comprehensive basis for the application of the CRS also in interreligious studies. 722 Religions 2012, 3 Table 5. Norm values of CRSi-7 (21 countries). Country DE AT CH IT FR ES GB PL RU IL TR MA NG ID IN TH KR AU US GT BR Total West East Musl. 6. Conclusions N 959 759 200 1945 978 965 959 965 971 885 924 909 885 885 966 993 963 1048 990 971 979 977 965 985 CRS-score Percentile rank “not-religious“ 1.0 3 1 10 3 1 12 2 8 6 2 7 5 1.2 7 4 19 7 3 1 17 3 12 12 4 14 10 1 1.4 12 6 37 1 11 5 2 26 6 17 1 18 7 20 15 2 1.6 18 9 50 3 15 7 3 32 8 22 2 26 10 1 25 18 3 1.8 21 13 54 5 20 11 5 39 12 29 4 34 14 2 29 23 5 1 2.0 28 19 63 7 25 15 7 43 18 35 5 43 17 1 4 35 28 8 2 “religious" 2.2 35 26 67 10 31 21 10 47 22 41 7 48 22 1 2 7 41 34 11 3 2.4 42 33 76 13 37 28 13 53 27 46 9 55 27 2 1 1 2 11 46 38 13 1 4 2.6 47 39 78 17 43 34 15 58 33 51 12 63 32 4 2 2 4 19 50 44 16 2 5 2.8 53 46 82 22 49 40 19 63 41 56 17 68 37 6 4 3 6 28 53 49 18 2 7 3.0 59 52 84 29 56 47 25 68 46 62 21 73 43 9 6 5 10 40 57 55 21 4 8 3.2 65 60 86 36 61 55 29 73 52 67 27 78 48 13 10 8 16 52 60 59 23 5 11 3.4 69 64 89 44 68 63 36 77 58 71 35 83 53 21 16 1 12 25 65 63 64 27 8 14 3.6 75 71 90 54 73 69 43 82 64 76 43 88 59 31 23 4 20 36 77 66 69 32 12 18 3.8 80 77 92 64 78 75 52 85 71 80 52 91 65 42 32 7 29 48 87 70 73 36 21 26 “highly- religious“ 4.0 85 82 93 71 84 81 63 89 77 83 63 94 71 55 44 12 41 61 93 75 77 42 34 35 4.2 90 88 96 81 88 86 73 93 83 88 73 97 78 67 56 21 57 74 96 81 82 53 48 48 4.4 93 92 97 88 92 90 82 96 87 92 85 98 82 78 69 36 74 83 98 85 87 65 65 61 4.6 97 96 99 93 95 95 92 98 92 95 95 99 89 91 80 55 88 93 99 90 92 75 87 78 4.8 99 98 100 96 98 98 98 99 97 97 99 100 93 95 90 76 96 96 100 94 95 87 95 90 5.0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 M 2.84 3.05 2.08 3.53 2.93 3.15 3.66 2.50 3.20 2.74 3.69 2.45 3.29 3.96 4.13 4.58 4.12 3.88 3.24 2.88 2.97 3.92 4.23 4.18 SD 1.10 0.99 0.98 0.86 1.06 0.97 0.89 1.13 1.04 1.15 0.81 0.96 1.12 0.63 0.61 0.38 0.55 0.62 0.62 1.30 1.19 0.99 0.52 0.70 Legend: DE = Germany (Musl. Religions 2012, 3 Religions 2012, 3 References and Notes 1. Stefan Huber. Zentralität und Inhalt: Ein neues multidimensionales Messmodell der Religiosität. Opladen: Leske and Budrich, 2003. 1. Stefan Huber. Zentralität und Inhalt: Ein neues multidimensionales Messmodell der Religiosität. Opladen: Leske and Budrich, 2003. 2. Stefan Huber. ―Zentralität und Inhalt. Eine Synthese der Messmodelle von Allport und Glock‖. In Religiosität: Messverfahren und Studien zu Gesundheit und Lebensbewältigung. Neue Beiträge zur Religionspsychologie, edited by Christian Zwingmann, and Helfried Moosbrugger. Münster: Waxmann, 2004, 79–105. 2. Stefan Huber. ―Zentralität und Inhalt. Eine Synthese der Messmodelle von Allport und Glock‖. In Religiosität: Messverfahren und Studien zu Gesundheit und Lebensbewältigung. Neue Beiträge zur Religionspsychologie, edited by Christian Zwingmann, and Helfried Moosbrugger. Münster: Waxmann, 2004, 79–105. 3. Stefan Huber. ―Are religious beliefs relevant in daily life?‖ In Religion Inside and Outside Traditional Institutions, edited by Heinz Streib. Lieden: Brill Academic Publishers, 2007, 211–230. 3. Stefan Huber. ―Are religious beliefs relevant in daily life?‖ In Religion Inside and Outside Traditional Institutions, edited by Heinz Streib. Lieden: Brill Academic Publishers, 2007, 211–230. 4. Stefan Huber. ―Kerndimensionen, Zentralität und Inhalt. Ein interdisziplinäres Modell der Religiosität.‖ Journal für Psychologie 16 (2008): Article 05. www.journal-fuer- psychologie.de/jfp-3-2008-05.html. 5. Stefan Huber. ―Religion Monitor 2008: Structuring principles, operational constructs, interpretive strategies.‖ In What the World Believes: Analysis and Commentary on the Religion Monitor 2008, edited by Bertelsmann-Stiftung. Gütersloh: Verlag Bertelsmann-Stiftung, 2009, 17–51. 6. Bertelsmann Stiftung, ed. What the World Believes: Analysis and Commentary on the Religion Monitor 2008. Gütersloh: Verlag Bertelsmann-Stiftung, 2009. 7. Charles Y. Glock. ―On the study of religious commitment.‖ Review of Recent Research Bearing on Religious and Character Formation. Research supplement to the July-August 1962, issue of Religious Education, S-98–S-110. 8. Charles Y. Glock. Religion in Sociological Perspective: Essays in the Empirical Study of Religion. Belmont, CA: Wadsworth Publishing Co., 1973. 9. Richard Stark, and Charles Y. Glock. American Piety: The Nature of Religious Commitment. Los Angeles, CA: Berkeley University Press, 1968. 10. Andrew J. Weigert, and Darwin L. Thomas. ―Religiosity in 5-D: A critical note.‖ Social Forces 48 (1969): 260–263. 11. Joseph E. Faulkner, and Gordon F. DeJong. ―Religiosity in 5-D: an empirical analysis.‖ Social Forces 45 (1966): 246–254. 12. Stefan Huber. Dimensionen der Religiosität. Skalen, Messmodelle und Ergebnisse einer empirisch orientierten Religionspsychologie. Bern: Verlag Hans Huber, 1996. 13. Stefan Huber. Die Semantik des empirischen Systems. Archimedischer Punkt und Achillesverse der sozialwissenschaftlichen Religionsforschung. In Theologie im Gespräch mit empirisch arbeitenden Wissenschaften, edited by Matthias Petzoldt. Leipzig: Evangelische Verlagsanstalt, 2012, 13–34. 6. Conclusions = Muslims in Germany); AT = Austria; CH = Switzerland; IT = Italy; FR = France; ES = Spain; GB = Great Britain; PL = Poland; RU = Russian Federation; IL = Israel; TR = Turkey; MA = Morocco; NG = Nigeria; ID = Indonesia; IN = India; TH = Thailand; KR = South Korea; AU = Australia; US = United States; GT = Guatemala; BR = Brazil. 723 16. George C. Homans. Social Behavior: Its Elementary Forms. New York: Harcourt Brace Jovanovich, 1974. References and Notes 14. Dale W. Wimberley. ―Religion and role-identity: A structural symbolic interactionist conceptualisation of religiosity.‖ The Sociological Quarterly 30 (1969): 125–142. 15. Sheldon Stryker. Symbolic Interactionism: A Social Structural Version. 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https://openalex.org/W2130951036	https://europepmc.org/articles/pmc3944870?pdf=render	English	null	Comparison of Imported<i>Plasmodium ovale curtisi</i>and<i>P. ovale wallikeri</i>Infections among Patients in Spain, 2005–2011	Emerging infectious diseases	2,014	cc-by	6,420	Comparison of Imported Plasmodium ovale curtisi and P. ovale wallikeri Infections among Patients in Spain, 2005–2011 Gerardo Rojo-Marcos, José Miguel Rubio-Muñoz, Germán Ramírez-Olivencia, Silvia García-Bujalance, Rosa Elcuaz-Romano, Marta Díaz-Menéndez, María Calderón, Isabel García-Bermejo, José Manuel Ruiz-Giardín, Francisco Jesús Merino-Fernández, Diego Torrús-Tendero, Alberto Delgado-Iribarren, Mónica Ribell-Bachs, Juan Arévalo-Serrano, and Juan Cuadros-González Sequencing data from Plasmodium ovale genotypes co-circulating in multiple countries support the hypothesis that P. ovale curtisi and P. ovale wallikeri are 2 separate species. We conducted a multicenter, retrospective, com- parative study in Spain of 21 patients who had imported P. ovale curtisi infections and 14 who had imported P. ovale wallikeri infections confirmed by PCR and gene sequenc- ing during June 2005–December 2011. The only significant finding was more severe thrombocytopenia among patients with P. ovale wallikeri infection than among those with P. ovale curtisi infection (p = 0.031). However, we also found nonsignificant trends showing that patients with P. ovale wallikeri infection had shorter time from arrival in Spain to onset of symptoms, lower level of albumin, higher median maximum core temperature, and more markers of hemo- lysis than did those with P. ovale curtisi infection. Larger, prospective studies are needed to confirm these findings. Diagnosis of P. ovale malaria can be difficult because of low parasitemia levels, mixed infections with other Plas- modium species, and false negatives from malaria rapid di- agnostic tests (RDTs) (1). However, recent epidemiologic studies conducted by using PCR techniques have found P. ovale infections in most of sub-Saharan Africa, South- east Asia, and the Indian subcontinent (2–5), including prevalence as high as 15% according to results of cross- sectional studies conducted in rural Nigeria (6) and Papua New Guinea (7). In addition, severe complications such as spleen rupture, severe anemia, or acute respiratory distress syndrome (ADRS) (8) may occur in patients with P. ovale malaria. Thus, the global burden of P. ovale infection might have been underestimated. On the basis of differences in its gene sequences, P. ovale was considered to be dimorphic or to comprise 2 sub- species (2,3,9,10). This difference has hampered molecular diagnosis in some cases because of lack of DNA ampli- fication by PCR with gene-specific primers for the small subunit ribosomal RNA (ssrRNA) (10). These subspecies had been named classic and variant P. ovale, but a compre- hensive study recently described differences between these subspecies in at least 6 genes (4). These findings demon- strate that P. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Microbiological Diagnosis The initial diagnosis of imported Plasmodium spp. in- fection was made by thick and/or thin smears and/or by using the second-generation RDT NOW Malaria Test Kit (Binax Inc., Scarborough, ME, USA) for histidine-rich pro- tein 2 antigen of P. falciparum and aldolase enzyme com- mon to all Plasmodium spp. Blood smears were stained by a standard technique with Giemsa solution for 30 min and were reviewed by an expert microbiologist. Parasite count was measured by determining the proportion of parasitized erythrocytes or the number of trophozoites per microliter. P. ovale Subtype Characterization and Confirmation Partial sequencing of the ssrRNA gene was used to differentiate P. ovale curtisi from P. ovale wallikeri. ssr- RNA amplification was performed by using a nested PCR specific for Plasmodium. The first reaction included UNR (5′-GACGGT ATCTGATCGTCTTC-3′) and PLF (5′- AGTGTGTATCCAATCGAGTTTC-3′) primers, which correspond to the first reaction of the seminested multiplex malaria PCR. The second reaction incorporated the prod- ucts of the first reaction, along with NewPLFsh (5′-CTAT- CAGCTTTTGATGTTAG-3′) and NewRevsh (5′-CCTTA- ACTTTCGTTCTTG-3′) primers. Infection with different malaria species yielded products of 710–740 bp. Growth in international travel and migration has in- creased the incidence of imported malaria in industrialized countries. P. ovale infection may represent up to 8% of im- ported malaria cases, according to some published series of patients primarily from West Africa (16,17), where the proportion of sub-Saharan immigrants is high and PCR has been systematically performed. Yet, it is difficult to gather a substantial number of cases with clinico-epidemiologic cor- relation and molecular data. To identify clinical or analytical differences between P. ovale wallikeri and P. ovale curtisi infections and expand data on these infections, we conducted a multicenter, retrospective, comparative study of imported P. ovale infections diagnosed in Spain during 2005–2011. The PCR mixture in both reactions consisted of 75 mmol/L Tris-HCl (pH 9.0), 2 mmol/L MgCl2, 50 mmol/L KCl, 20 mmol/L (NH4)2SO4, 200 μmol/L dNTP, 0.075 μmol/L of the corresponding PCR primers, 1.25 units Taq DNA polymerase (Biotools B&M Labs, S.A., Madrid, Spain), and the template DNA in a reaction volume of 50 μL. The amount of template was 5 μL of DNA extracted by using a QIAamp DNA Blood Mini Kit (QIAGEN). For the second reaction mixture, 2 μL of the PCR product of the first reaction was used as template. For both reactions, a GeneAmp PCR System 2700 thermal cycler (Applied Bio- systems, Foster City, CA, USA) was used, beginning with 7 min at 94°C, followed by 40 cycles of 20 s at 94°C, 20 s at 62°C, and 30 s at 72°C for the first round; or 35 cycles of 20 s at 94°C, 20 s at 53°C, and 20 s at 72°C for the second round. The final cycle was followed by an extension time of 10 min at 72°C.ii Sample Selection During June 2005–December 2011, blood samples from all patients with positive PCR results for imported infection with P. ovale were sent from public hospitals in Spain to the reference Malaria & Emerging Parasitic Dis- eases Laboratory of the National Centre of Microbiology in Madrid. The samples were shipped to the laboratory 1) to confirm the diagnosis of malaria and the species or 2) to study fever, anemia, or suspected malaria in patients with negative results on thick and/or thin smears and RDTs who were considered at high risk for malaria (i.e., immigrants and travelers to malaria-endemic areas). The amplified products were purified by using Illustra DNA and Gel Band Purification Kit (GE Healthcare, Buck- inghamshire, UK) and sequenced by using the Big Dye Terminator v3.1 Cycle Sequencing Kit and ABI PRISM 3700 DNA Analyzer (Applied Biosystems). All amplified products were sequenced in both directions twice. To con- firm P. ovale subtyping, a nested PCR amplification plus sequencing targeting cytochrome (Cyt) b was performed (3) in 3 samples of each group, by using a unique second amplification (nested) reaction with primers Cyt b 2F and Cyt b 2R. Comparison of Imported Plasmodium ovale curtisi and P. ovale wallikeri Infections among Patients in Spain, 2005–2011 ovale actually consists of 2 subspecies that co- circulate in Africa and Asia and that are unable to recom- bine genetically; the differences seem to be explained by real biological factors, rather than ecologic or geographic factors (11). P. ovale curtisi and P. ovale wallikeri were the names proposed for these species (4). M alaria caused by Plasmodium ovale infection has been considered a low-prevalence disease with lim- ited geographic distribution, benign clinical course, and easy treatment; therefore, little attention has been paid to it. Author affiliations: Príncipe de Asturias University Hospital, Madrid, Spain (G. Rojo-Marcos, J. Arévalo-Serrano, J. Cuadros-González,); Instituto de Salud Carlos III, Madrid (J.M. Rubio-Muñoz); Carlos III Hospital, Madrid (G. Ramírez-Olivencia); La Paz University Hospi- tal, Madrid (S García-Bujalance); Doctor Negrín University Hospital, Las Palmas de Gran Canaria, Spain (R. Elcuaz-Romano); Ramón y Cajal Hospital, Madrid (M. Díaz-Menéndez); Gregorio Marañón University Hospital, Madrid (M. Calderón); Getafe University Hospi- tal, Madrid (I. García-Bermejo); University Hospital of Fuenlabrada, Madrid (J. M. Ruiz-Giardín); Severo Ochoa University Hospital, Madrid (F.J. Merino-Fernández); University General Hospital of Alicante, Alicante, Spain (D. Torrús-Tendero); University Hospital Fundación Alcorcón, Madrid (A. Delgado-Iribarren); and Hospital General de Granollers, Barcelona, Spain (M. Ribell-Bachs). M Scant information is available on differences in clini- cal and analytical features, relapse profile, or accuracy of RDT results between these proposed species. Relatively high parasitemia levels were found in some patients with P. ovale wallikeri infection in Thailand (12), Vietnam (13), and Flores Island (14). A study published from a disease-endem- ic area of Bangladesh reported on the clinical features and degree of parasitemia in 13 patients with P. ovale wallikeri DOI: http://dx.doi.org/10.3201/eid2003.130745 409 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 RESEARCH infection and 10 with P. ovale curtisi infection (5). These infections were diagnosed by PCR; only 4 of the 23 patients were symptomatic. Another recent study compared parasit- emia levels, RDT results, and patient country of origin for 31 patients from Côte d’Ivoire and the Comoros Islands with imported P. ovale wallikeri infection and 59 with P. ovale curtisi infection, but no clinical data were provided (15). Clearly, information on these infections is limited. Hilden, Germany), according to the manufacturer’s proto- col. P. ovale molecular diagnosis was confirmed by using seminested multiplex malaria PCR (18), which enables the discernment of the 4 most prevalent human malaria species by amplified fragments of DNA in 2 sequential PCRs. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Isolation of Parasite DNA and Molecular Diagnosis Confirmation Hospitals that submitted PCR-confirmed and P. ovale subtype–identified samples were invited to collaborate in the study. A database was designed and completed after the retrospective review of medical reports and laboratory DNA isolation from whole blood was performed by using the QIAamp DNA Blood Mini Kit (QIAGEN, 410 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Imported Plasmodium ovale Infections, Spain samples from 28 patients and P. ovale wallikeri in 24 sam- ples from to 22 patients. registries. Patient data collected included sex, age, ethnicity, underlying diseases, time living in non–malaria-endemic countries, dates and purpose of travel, countries visited, ma- laria chemoprophylaxis, date of admission and diagnosis, symptoms and clinical signs, physical examinations, and complications of severe malaria according to World Health Organization criteria (19). The closest possible date of in- fection was defined as the day of departure from a malaria- endemic area. The time between date of arrival in Spain and onset of illness or diagnosis was calculated once asymp- tomatic patients were excluded. Patients were classified as early immigrant if they had stayed in a country without ma- laria for <1 year before diagnosis and as visiting friends and relatives if they had traveled to a malaria-endemic country after 1 year living in a non–malaria-endemic area. Recent Plasmodium infection was defined as probable or definite malaria infection in the 3 months before P. ovale infection was diagnosed. Twelve hospitals agreed to participate in the study and provided complete epidemiologic, microbiological, biochemical, clinical, and therapeutic information for 35 of the 50 patients for which P. ovale genetic characteriza- tion was available. Of these, 21 patients had P. ovale curtisi infection and 14 had P. ovale wallikeri infection. Table 1 shows the demographic and epidemiologic data for these 35 patients. Patient age and sex were virtually the same for patients with P. ovale curtisi and P. ovale wallikeri infec- tions and corresponded mostly to young persons from Af- rica who traveled to visit their friends and relatives for a long period or immigrants who had recently arrived. The lapses between time of arrival in Spain, onset of illness, and diagnosis were longer for patients with P. ovale curtisi infection than for those with P. ovale wallikeri in- fection, but not significantly. Most travelers did not take any malaria prophylaxis or did not adhere to the full regi- men. Isolation of Parasite DNA and Molecular Diagnosis Confirmation All but 2 infections were acquired in West Africa, and both Plasmodium species were found in patients from Ni- geria, Equatorial Guinea, Ghana, and Guinea-Conakry. A high rate of underlying disease was found among patients in both parasite groups; the P. ovale curtisi group included 3 early immigrants who were chronically infected with HIV, hepatitis B virus, and hepatitis C virus and carried filarial and intestinal parasites. Recorded laboratory results included microbiological data; complete blood count with leukocytes, hemoglobin, and platelet levels; levels of creatinine, albumin, trans- aminases, lactate dehydrogenase, and direct bilirubin in plasma; and glucose-6-phosphate dehydrogenase activity in erythrocytes. Abdominal ultrasonography and serologic studies to detect infection with HIV and hepatitis A, B, and C viruses were reviewed. Recorded treatments and compli- ance, clinical and microbiological evolution, and duration of hospital stay for those admitted were included. Microbiological and laboratory data for the patients are shown in Table 2. Statistically significant worse levels of thrombocytopenia were found among patients with P. ovale wallikeri infection compared with those who had P. ovale curtisi infection, but no other significant difference was found. One mixed infection with P. falciparum was found among each patient group. The RDT technique used showed a low sensitivity (<30%) for detecting P. ovale once mixed infections were excluded. Statistical Analysis Differences of proportions were evaluated by χ2 test or Fisher exact test, as appropriate for sample size. Means between groups were calculated by using the Student t-test for independent samples if the normal distribution could be assumed; we used the Levene test for homogeneity of variances. If normality was not valid, we used the Mann- Whitney U test for nonparametric variables. Clinical and therapeutic data for the patients are shown in Table 3. All 3 asymptomatic patients had P. ovale curtisi infection; 1 infection was detected as an incidental finding in a blood smear for sickle cell disease, and the other 2 were found during studies of anemia after negative results were found by thick film examination and RDT results. The remaining 7 patients with negative thick smear and positive PCR results reported at least fever. Symptomatic patients showed no difference in clinical signs and symptoms, but those with P. ovale wallikeri did have a higher mean num- ber of symptoms (3.5 per patient) than did those with P. ovale curtisi (2.7). The number of complications was simi- lar in both groups; 3 cases of severe anemia occurred, 2 of them related to sickle cell disease, and 1 case of ADRS oc- curred in a patient with P. ovale wallikeri infection. To test for normality, we used either the Shapiro- Wilks test for small samples or the Kolmogorov-Smirnov test with the Lilliefors correction for large samples. Val- ues were reported as means and SDs or, for nonparametric distributions, medians and interquartile ranges (IQRs). A 2-sided p value of <0.05 was considered to indicate statisti- cal significance. Statistical analyses were performed by us- ing SPSS version 15 (SPSS Inc., Chicago, IL, USA). Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Results During June 2005–December 2011, a total of 102 samples positive by PCR for P. ovale were analyzed at the reference laboratory; of these, we were able to amplify and genotype 55 samples. Poor quality of long-term stored DNA prevented amplification of the other samples. Genetic analyses of the cytb gene identified P. ovale curtisi in 31 Most patients were admitted to a hospital and received inpatient treatment. Roughly half the patients in each group 411 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 RESEARCH Table 1. Demographic and epidemiologic characteristics of patients with imported Plasmodium ovale curtisi or P. ovale wallikeri infections, Spain, 2005–2011* Characteristic P. ovale curtisi, n = 21 P. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 ns (n = 2), Loa loa (n = 1). (n = 3), hookworms (n = 2), Ascaris lumbricoides (n = 2), Strongyloides stercoralis (n = 1), Entamoeba hystolitica (n = 1). Results ovale wallikeri, n = 14 p value Patient sex 0.332 M 10 (47.6) 9 (64.3) F 11 (52.4) 5 (35.7 ) Patient age, y, median (IQR) 36.50 (23.04–52.66) 38.33 (11.79–45.27) 0.377 Age <15 3 (14.3) 4 (28.6) 0.401 Ethnicity 0.721 Black 15 (71.4) 9 (64.3) White 6 (28.6) 5 (35.7) Type of patient 0.260 Early immigrant 6 (28.6) 4 (28.6) Traveler 14 (66.7) 10 (71.4) Reason for travel Visiting friends and relatives 9 (42.8) 7 (50.0) Tourism 1 (7.1) Work 3 (14.3) 2 (14.3) Cooperation 2 (9.5) Unknown 1 (4.8) Duration of travel, d, median (IQR) 75 (23.25–91.50) 23 (15.00–81.50) 0.279 Country of infection 0.486 Equatorial Guinea 12 (57.1) 7 (50.0) Nigeria 2 (9.5) 3 (21.4) Equatorial Guinea or Cameroon 1 (4.8) 0 Ghana 1 (4.8) 1 (7.1) Ethiopia 1 (4.8) 0 Guinea-Conakry 1 (4.8) 0 Liberia 1 (4.8) 0 Angola 1 (4.8) 0 Guinea-Bissau 1 (4.8) 0 Guinea-Conakry or Senegal 0 1 (7.1) Côte d’Ivoire 0 1 (7.1) Mozambique 0 1 (7.1) Chemoprophylaxis 0.627 No prophylaxis 17 (81.0) 13 (92.9) Mefloquine, incomplete 1 (4.8) 1 (7.1) Mefloquine 1 (4.8) 0 Doxycycline 1 (4.8) 0 Atovaquone/proguanil 1 (4.8) 0 Days from arrival to onset of symptoms, median (IQR) 94.5 (12.5–297.2) 9.5 (2.7–58.2) 0.077 Days from onset of symptoms to diagnosis, median (IQR) 8 (2.7–16.5) 3.5 (2.0–7.7) 0.206 Recent Plasmodium infection 3 (14.3) 3 (21.4) >0.999 Other infections Hepatitis B virus >0.999 Active 1/11 (9.1) 0/10 Cured or vaccinated 6/11 (54.5) 5/10 (50.0) Negative 4/11 (36.4) 5/10 (50.0) Hepatitis C virus 1/7 (14.3) 0/10 0.412 HIV 1/7 (14.3) 0/10 0.412 Filariasis† 3/6 (50.0) 0/4 0.200 Intestinal parasites‡ 3/6 (50.0) 1/4 (25.0) 0.571 Other underlying conditions 9 (42.8) 6 (42.8) >0.999 Diabetes mellitus 2 (9.5) 1 (7.1) Drepanocytosis 2 (9.5) 0 Hypertension 4 (19.0) 2 (14.3) Obesity 1 (4.8) 0 Acute pancreatitis 0 1 (7.1) Policystosis and nephrectomy 0 1 (7.1) Oligoarthritis 0 1 (7.1) Glucose-6-phosphate dehydrogenase deficiency 2/14 (14.3) 0/8 0.515 Pregnancy 1 (4.8) 0 >0.999 Values are no. (%) patients or no. positive/total no. (%) patients unless otherwise indicated. IQR, interquartile range. †Mansonella perstans (n = 2), Loa loa (n = 1). ‡Trichiuris trichiura (n = 3), hookworms (n = 2), Ascaris lumbricoides (n = 2), Strongyloides stercoralis (n = 1), Entamoeba hystolitica (n = 1). ics of patients with imported Plasmodium ovale curtisi or P. ovale wallikeri nd epidemiologic characteristics of patients with imported Plasmodium ovale curtisi or P. Results ovale wallikeri 2011 412 Imported Plasmodium ovale Infections, Spain Table 2. Microbiological characteristics of patients with imported Plasmodium ovale curtisi or P. ovale wallikeri infections, Spain, 2005– 2011* Characteristic P. ovale curtisi, n = 21 P. ovale wallikeri, n = 14 p value Positive thick smear, no. (%) patients 16 (76.2) 10 (71.4) >0.999 Positive by PCR only, no. (%) patients 5 (23.8) 4 (28.6) >0.999 Parasitemia, L 2,800 (773.25–5,484.25) 1,243.50 (337.75–6,200.00) 0.699 Mixed infection, no. (%) patients 1† (4.8) 1† (7.1) >0.999 Rapid diagnostic test result, no. positive/total no. patients (%) Common antigen positive 4/16 (25.0) 4/12 (33.3) 0.691 P. falciparum antigen positive 1/15 (6.7) 2/12 (16.6) 0.569 Leukocyte count,  109 cells/L 7.2 (4.9–8.7) 5.5 (4.2–8.2) 0.309 Hemoglobin, g/dL 11.6 (9.7–13.6) 10.9 (9.6–12.1) 0.364 Platelet count,  109 cells/L 126 (106.0–182.5) 91.5 (54.7–117.7) 0.031 Albumin, g/dL 3.7 (3.3–4.1) 3.4 (2.8–3.7) 0.063 Creatinine, mg/dL 0.88 (0.6–1.1) 0.97 (0.5–1.1) 0.730 Lactate dehydrogenase, IU/L 434.5 (358.7–807.7) 563 (462.5–731.7) 0.200 Aspartate aminotransferase, IU/L 24.5‡ (20.0–40.2) 31 (22–41) 0.624 Alanine aminotransferase, IU/L 25.5‡ (16.0–49.7) 23 (18.5–47.0) 0.785 Total bilirubin level, mg/dL 0.68‡ (0.6–1.2) 0.87 (0.6–1.4) 0.426 Values are median (interquartile range) unless otherwise indicated. Boldface indicates significance. †P. falciparum was second infection for both patients. ‡One patient had active hepatitis B virus infection. among those with P. ovale wallikeri infection, a symptom that was absent among those with P. ovale curtisi infec- tion. However, because most cases were asymptomatic or had mild to moderate clinical features, finding significant differences within this narrow clinical spectrum may be especially difficult and might require a much larger study. received chloroquine alone. Almost all patients showed good tolerance to treatment and favorable clinical evolu- tion. More than a quarter (33.3% of P. ovale curtisi and 28.6% of P. ovale wallikeri) did not receive primaquine for radical cure, 2 because of glucose-6-phosphate dehydroge- nase deficiency. One patient was lost to follow-up and did not receive any treatment or monitoring. Because the time of infection is more likely to be accu- rately known, patients with imported malaria might make a better group for study of the epidemiologic and clinical characteristics of different Plasmodium species than those living in malaria-endemic countries. Moreover, signs or symptoms may be less affected by other tropical infections, including mixed Plasmodium infections, or by patient im- munity; imported malaria occurs among a larger number of nonimmune patients and patients who are visiting friends and relatives. Results Differentiation among primary infection or relapse continues to be practically impossible, but the lon- ger the time of latency, the more probable a relapse. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Discussion 3, March 2014 RESEARCH rapeutic characteristics of patients with imported Plasmodium ovale curtisi or P. ovale wallikeri infections, Table 3. Clinical and therapeutic characteristics of patients with imported Plasmodium ovale curtisi or P. ovale wallikeri infections, Spain, 2005–2011 Characteristic P. ovale curtisi, n = 21 P. ovale wallikeri, n = 14 p value Asymptomatic 3 (14.3) 0 0.259 Fever 18 (85.7) 14 (100.0) 0.259 Tertian fever 1 (4.8) 3 (21.4) 0.279 Maximum temperature, ºC, median (IQR) 38.4 (37.5–40.0) 39.7 (38.9–40.5) 0.088 Chills 3 (14.3) 3 (21.4) 0.664 Sweating 0 1 (7.1) 0.400 Headache 6 (28.6) 4 (28.6) >0.999 Nauseas 0 3 (21.4) 0.056 Vomitus 0 3 (21.4) 0.056 Astenia 2 (9.5) 3 (21.4) 0.369 Epigastralgia 2 (9.5) 0 0.506 Arthralgia 5 (23.8) 3 (21.4) >0.999 Myalgia 6 (28.6) 4 (28.6) >0.999 Diarrhea 1 (4.8) 1 (7.1) >0.999 Chest pain 1 (4.8) 1 (7.1) >0.999 Cough 4 (19.0) 3 (21.4) >0.999 Dyspnea 0 1 (7.1) 0.400 Dizziness 2 (9.5) 0 >0.999 Splenomegaly 5 (23.8) 3 (21.4) >0.999 Complications or severe malaria 2 (9.5) 2 (14.3) >0.999 Hemolytic crisis 1 (4.8) 0 Severe anemia, hemoglobin <7 g/dL 1 (4.8) 1 (7.1) Acute respiratory distress syndrome 0 1 (7.1) Admission to hospital 13 (61.9) 13 (92.9) 0.056 Duration of hospitalization, d, median (IQR) 4 (3.0–7.5) 5 (3.5–7.5) 0.390 Treatment 0.563 Chloroquine 12 (57.1) 7 (50.0) Other treatment 8 (38.1) 7 (50.0) Quinine + doxycycline 3 (14.3) 4 (28.6) Atovaquone/proguanil 3 (14.3) 1 (7.1) Quinine + clindamycin + chloroquine/proguanil 1 (4.8) 0 Quinine + clindamycin + chloroquine 0 1 (7.1) Mefloquine 0 1 (7.1) Atovaquone/proguanil + chloroquine 1 (4.8) 0 No treatment 1 (4.8) 0 Primaquine 14 (66.7) 10 (71.4) >0.999 Compliance 19/21 (90.5) 13/13 (100.0)† 0.513 *Values are no. (%) patients or no. positive/total no. (%) patients unless otherwise indicated. IQR, interquartile range. †One patient was lost to follow-up. related to a greater severity of illness (25). Some studies also suggest an inverse correlation between the level of parasitemia and platelet count (26). Plasmodium species were asymptomatic; in our study, the 2 patients who had mixed infections with P. falciparum had at least fever. The number of P. ovale monoinfections in our study confirmed by PCR is high, unlike the number in malaria-endemic areas, where most infections are mixed (5,7,23) and a high rate of submicroscopic carriage occurs (24). Discussion Our comparative study of the epidemiologic and clini- cal characteristics of patients with P. ovale curtisi and P. ovale wallikeri infection found only 1 statistically signifi- cant result, a higher rate of severe thrombocytopenia among patients with P. ovale wallikeri infection (p = 0.031). Nevertheless, we noted nonsignificant results, including a shorter time from arrival to onset of symptoms in travelers who acquired P. ovale wallikeri infection (p = 0.077). This finding fits with findings from a recently published larger series from the United Kingdom in which a significantly shorter latency was found for P. ovale wallikeri compared with P. ovale curtisi infection (20). We also found a trend toward a shorter stay in Africa and shorter interval be- tween onset of symptoms and diagnosis among patients with P. ovale wallikeri infection, which could reflect easier transmission, shorter latency, or higher relapse rates. This finding might also mean that slightly more severe illness, including higher median fever (39.7°C vs. 38.4°C) and a greater number of symptoms (3.5 vs. 2.7 per symptomatic patient), led more patients with P. ovale wallikeri infection than those with P. ovale curtisi infection to seek medical attention earlier. However, the higher percentage of travel- ers who took at least partial prophylaxis among the P. ovale curtisi group (19.2%) might also explain a longer time of onset. A high frequency of nausea and vomiting was found Although criteria for admission to each hospital in- volved in the study were different, P. ovale wallikeri pa- tients were admitted more frequently. The number of com- plications was similar in both groups; 3 cases of severe anemia were reported, 2 related to sickle cell disease, and 1 case of ADRS occurred in a patient infected with P. ovale wallikeri (8). Sickle cell trait was described as a risk factor for infection with P. ovale in Senegal (21), and a recent series showed 3 of 16 patients with P. ovale infection were homozygous for sickle cell disease (22), a clearly dispro- portionate number. Regarding symptomatic patients, the Bangladesh study (5) showed a trend toward a larger number of asymptom- atic P. ovale curtisi infections (90% vs. 75% for P. ovale wallikeri), which would be consistent with our results. In that study, all 13 mixed infections of P. ovale with other 413 erging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Discussion In part, our finding may result from previous anti- malarial treatment or prophylaxis in a number of patients, which could have minimized the number of Plasmodium parasites in the blood. i We found that indirect parameters of hemolysis, such as hemoglobin, lactate dehydrogenase, and bilirubin levels, were less impaired among patients with P. ovale curtisi in- fection than those with P. ovale wallikeri infection, even including 2 patients with sickle cell disease, who had more severe anemia. Albumin values, diminished by other multi- ple types of infections, also tend to be lower in patients with P. ovale wallikeri infection. For transaminases, if we were to exclude a patient with chronic hepatitis B and hypertrans- aminasemia, we would also find higher values for patients with P. ovale wallikeri infection. These data collectively raise the hypothesis that P. ovale wallikeri is slightly more pathogenic, which warrants further investigation.i Among the laboratory results, the only significant difference was found in platelet count, with more severe thrombocytopenia seen among patients with P. ovale wal- likeri infection than among those with P. ovale curtisi infection. Thrombocytopenia is a common finding in pa- tients with malaria; a previous series found 10 (66.6%) of 15 patients with imported P. ovale infection had platelet counts of <140,000/mL (22). The mechanisms that pro- duce thrombocytopenia in malaria are not known but seem Parasitemia levels were not significantly different be- tween the 2 groups (p = 0.699). In 2 recent studies of P. ovale infection that included determination of parasitemia 414 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Imported Plasmodium ovale Infections, Spain patient selection was not systematically planned but was done on the basis of decisions of physicians from many hospitals who sent samples to the reference laboratory and subsequently agreed to participate in this study. Fourth, the study’s retrospective design led to gaps in the informa- tion collected. Fifth, only strains of P. ovale from Africa were analyzed, and patients were from Africa and Europe; a study of infections and patients from Asia or Oceania might show different results. Last, more diversity in the geographic origin of the strains and a mix of nonimmune and semiimmune patients would lead to more heteroge- neous study groups. levels and genetic analysis (5,15), no differences were de- scribed. Case reports of high parasitemia levels in patients with P. References In industrialized countries, improved clinical and mi- crobiological control after treatment and radical cure with primaquine can be achieved for malaria. P. ovale seems to remain sensitivity to chloroquine and other antimalarial drugs. Patients in our study showed good clinical response that could be followed up without any relapse, including among those who had complications. The treatment dif- ferences reflect the different hospital managing protocols, patient age and pregnancy status, or difficulty in identifying P. ovale initially. 1. Mueller I, Zimmerman PA, Reeder JC. Plasmodium malariae and Plasmodium ovale—the “bashful” malaria parasites. Trends Parasitol. 2007;23:278–83. http://dx.doi.org/10.1016/j.pt.2007.04.009 1. Mueller I, Zimmerman PA, Reeder JC. Plasmodium malariae and Plasmodium ovale—the “bashful” malaria parasites. Trends Parasitol. 2007;23:278–83. http://dx.doi.org/10.1016/j.pt.2007.04.009 2. Tachibana M, Tsuboi T, Kaneko O, Khuntirat B, Torii M. Two types of Plasmodium ovale defined by SSU rRNA have distinct sequences for ookinete surface proteins. Mol Biochem Parasitol. 2002;122:223–6. http://dx.doi.org/10.1016/S0166-6851(02)00101-9 2. Tachibana M, Tsuboi T, Kaneko O, Khuntirat B, Torii M. Two types of Plasmodium ovale defined by SSU rRNA have distinct sequences for ookinete surface proteins. Mol Biochem Parasitol. 2002;122:223–6. http://dx.doi.org/10.1016/S0166-6851(02)00101-9 p g ( ) 3. Win TT, Jalloh A, Tantular IS, Tsuboi T, Ferreira MU, Kimura M, et al. Molecular analysis of Plasmodium ovale variants. Emerg Infect Dis. 2004;10:1235–40. http://dx.doi.org/10.3201/eid1007.030411 3. Win TT, Jalloh A, Tantular IS, Tsuboi T, Ferreira MU, Kimura M, et al. Molecular analysis of Plasmodium ovale variants. Emerg Infect Dis. 2004;10:1235–40. http://dx.doi.org/10.3201/eid1007.030411 4. Sutherland CJ, Tanomsing N, Nolder D, Oguike M, Jennison C, Pukrittayakamee S, et al. Two non-recombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally. J Infect Dis. 2010;201:1544–50. http://dx.doi.org/10.1086/652240 4. Sutherland CJ, Tanomsing N, Nolder D, Oguike M, Jennison C, Pukrittayakamee S, et al. Two non-recombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally. J Infect Dis. 2010;201:1544–50. http://dx.doi.org/10.1086/652240 Our study has limitations. First, the small number of patients may lack sufficient statistical power to show dif- ferences between infections with different Plasmodium species. Second, the low performance of genetic amplifica- tion may have caused some sample selection bias (e.g., a higher number of samples that were stored short term or came from patients with higher parasitemia levels). Third, 5. Fuehrer H-P, Habler VE, Fally MA, Harl J, Starzengruber P, Swo- boda P, et al. Discussion ovale wallikeri infection in Southeast Asia are iso- lated and noncomparative (12–14). Sooner and better access to health care in industrial- ized countries might provide a broader range of diagnostic tools for malaria, including RDT, thick and thin film ex- amination, or PCR. Current techniques of RDT still show a low sensitivity for detecting P. ovale (27,28). This prob- lem is usually explained by the genetic variability of the 2 subspecies and the low levels of parasitemia detected. After discarding mixed infections, in our study, the aldol- ase-based RDT used as a common antigen of Plasmodium obtained 20% sensitivity for detection of P. ovale curtisi and 27.27% for P. ovale wallikeri. Results are poorer than those recently published by Bauffe et al., who found a high- er false-negative rate of infection for P. ovale curtisi than for P. ovale wallikeri (60% vs. 43%, respectively) and no significant differences in parasitemia levels (15). In summary, after comparing epidemiologic, clinical, and analytic data for patients with P. ovale wallikeri and P. ovale curtisi infections, we found significantly more marked thrombocytopenia among patients with P. ovale wallikeri infection, but we found no other significant differ- ences. However, some trends toward slightly greater patho- genicity were observed for P. ovale wallikeri infection. The description of both genotypes occurring in sympatry without hybrid forms in an increasing number of countries supports the idea of 2 well-defined species. Larger prospec- tive studies should be conducted to more fully explore this hypothesis. Although circulation of P. ovale was known in these countries, our study provides PCR confirmation for P. ovale curtisi infections from Guinea-Conakry, Ethiopia, and Li- beria and P. ovale wallikeri infections from Mozambique. Both species have been described as sympatric in time and space in Equatorial Guinea, Republic of Congo, Uganda, Bangladesh, and Angola (5,11,29), maintaining genetic differentiation, which supports the hypothesis of 2 distinct subspecies. Moreover, 2 cases of P. ovale curtisi and P. ovale wallikeri co-infection have been reported (5,29). Acknowledgments We thank Francisco Javier Vilar Izquierdo for his assistance in the translation and critical review of this manuscript. Dr Rojo-Marcos is a consultant in tropical and travel medicine with the Department of Internal Medicine, Príncipe de Asturias Uni- versity Hospital, Alcalá de Henares, Madrid, Spain. His research interests include malaria, leishmaniasis, schistosomiasis, imported tropical diseases, and social and health problems of immigration. We found a high rate of underlying chronic diseases among the patients in this study, especially homozygous sickle cell anemia and diabetes mellitus. Previous stud- ies have shown that carriage of the sickle cell trait confers increased susceptibility to P. ovale infection (20) and that diabetes and HIV infection confer increased susceptibility to P. falciparum infection (30). References Plasmodium ovale in Bangladesh: genetic diversity and the first known evidence of the sympatric distribution of Plas- modium ovale curtisi and Plasmodium ovale wallikeri in southern Asia. Int J Parasitol. 2012;42:693–9. http://dx.doi.org/10.1016/j. ijpara.2012.04.015 415 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 RESEARCH 6. May J, Mockenhaupt FP, Ademowo OG, Falusi AG, Olumese PE, Bienzle U, et al. High rate of mixed and subpatent malarial infec- tions in southwest Nigeria. Am J Trop Med Hyg. 1999;61:339–43. 19. World Health Organization. Severe and complicated malaria. Trans R Soc Trop Med Hyg. 1990;84 Suppl 2:1–65. http://dx.doi. org/10.1016/0035-9203(90)90363-J 7. Mehlotra RK, Lorry K, Kastens W, Miller SM, Alpers MP, Bockarie M, et al. Random distribution of mixed species malaria in- fections in Papua New Guinea. Am J Trop Med Hyg. 2000;62:225–31. 20. Nolder D, Oguike MC, Maxwell-Scott H, Niyazi HA, Smith V, Chiodini PL, et al. An observational study of malaria in British travellers: Plasmodium ovale wallikeri and Plasmodium ovale curtisi differ significantly in the duration of latency. BMJ Open. 2013;3:e002711. http://dx.doi.org/10.1136/bmjopen-2013-002711 8. Rojo-Marcos G, Cuadros-González J, Mesa-Latorre JM, Culebras-López AM, de Pablo-Sánchez R. Acute respiratory distress syndrome in a case of Plasmodium ovale malaria. Am J Trop Med Hyg. 2008;79:391–3. 21. Faye FB, Spiegel A, Tall A, Sokhna C, Fontenille D, Rogier C, et al. Diagnostic criteria and risk factors for Plasmodium ovale malaria. J Infect Dis. 2002;186:690–5. http://dx.doi.org/10.1086/342395 9. Li J, Wirtz RA, McConkey GA, Sattabongkot J, Waters AP, Rogers MJ, et al. Plasmodium: genus-conserved primers for species identification and quantitation. Exp Parasitol. 1995;81: 182–90. http://dx.doi.org/10.1006/expr.1995.1107 22. Rojo-Marcos G, Cuadros-González J, Gete-García L, Gómez- Herruz P, López-Rubio M, Esteban-Gutierrez G. Plasmodium ovale infection: description of 16 cases and a review [in Spanish.]. Enferm Infecc Microbiol Clin. 2011;29:204–8. http://dx.doi.org/10.1016/ j.eimc.2010.09.004 10. Calderaro A, Piccolo G, Perandin F, Gorrini C, Peruzzi S, Zuelli C, et al. Genetic polymorphisms influence Plasmodium ovale PCR detection accuracy. J Clin Microbiol. 2007;45:1624–7. http://dx.doi. org/10.1128/JCM.02316-06 23. Kawamoto F, Liu Q, Ferreira MU, Tantular IS. How prevalent are Plasmodium ovale and P. malariae in East Asia? Parasitol Today. 1999;15:422–6. http://dx.doi.org/10.1016/S0169-4758(99) 01511-2 11. Oguike MC, Betson M, Burke M, Nolder D, Stothard JR, Kleinschmidt I, et al. Plasmodium ovale curtisi and Plasmodium ovale wallikeri circulate simultaneously in African communities. Int J Parasitol. 2011;41:677–83. http://dx.doi.org/10.1016/j.ijpara. 2011.01.004 24. Bruce MC, Macheso A, Kelly-Hope LA, Nkhoma S, McConnachie A, Molyneux ME. References Effect of transmission setting and mixed species infections on clinical measures of malaria in Malawi. PLoS ONE. 2008;3:e2775. http://dx.doi.org/10.1371/journal.pone.0002775 12. Win TT, Lin K, Mizuno S, Zhou M, Liu Q, Ferreira MU, et al. Wide distribution of Plasmodium ovale in Myanmar. Trop Med Int Health. 2002;7:231–9. http://dx.doi.org/10.1046/j.1365-3156.2002.00857.x 25. Lacerda MV, Mourão MP, Coelho HC, Santos JB. Thrombocytopenia in malaria: who cares? Mem Inst Oswaldo Cruz. 2011;106(Suppl 1):52–63. http://dx.doi.org/10.1590/S0074-02762011000900007 13. Kawamoto F, Miyake H, Kaneko O, Kimura M, Nguyen TD, Nguyen TD, et al. Sequence variation in the 18S rRNA gene, a target for PCR-based malaria diagnosis in Plasmodium ovale from southern Vietnam. J Clin Microbiol. 1996;34:2287–9. 26. Saravu K, Docherla M, Vasudev A, Shastry BA. Thrombocytopenia in vivax and falciparum malaria: an observational study of 131 patients in Karnataka, India. Ann Trop Med Parasitol. 2011;105:593– 8. http://dx.doi.org/10.1179/2047773211Y.0000000013 14. Win TT, Tantular IS, Pusarawati S, Kerong H, Lin K, Matsuoka H, et al. Detection of Plasmodium ovale by the ICT Malaria P.f/P.v. immunochromatographic test. Acta Trop. 2001;80:283–4. http://dx.doi.org/10.1016/S0001-706X(01)00155-3 27. Moody A. Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev. 2002;15:66–78. http://dx.doi.org/10.1128/ CMR.15.1.66-78.2002 15. Bauffe F, Desplans J, Fraisier C, Parzy D. Real-time PCR assay for discrimination of Plasmodium ovale curtisi and Plasmodium ovale wallikeri in the Ivory Coast and in the Comoros Islands. Malar J. 2012;11:307. http://dx.doi.org/10.1186/1475-2875-11-307 28. Bigaillon C, Fontan E, Cavallo JD, Hernandez E, Spiegel A. Ineffectiveness of the Binax NOW malaria test for diagnosis of Plasmodium ovale malaria. J Clin Microbiol. 2005;43:1011. http://dx.doi.org/10.1128/JCM.43.2.1011.2005 29. Fançony C, Gamboa D, Sebastião Y, Hallett R, Sutherland C, Sousa-Figueiredo JC, et al. Various pfcrt and pfmdr1 geno- types of Plasmodium falciparum cocirculate with P. malariae, P. ovale spp., and P. vivax in northern Angola. Antimicrob Agents Chemother. 2012;56:5271–7. http://dx.doi.org/10.1128/ AAC.00559-12 16. Rojo-Marcos G, Cuadros-González J, Gete-García L, Prieto-Ríos B, Arcos-Pereda P. Imported malaria in a general hospital in Madrid [in Spanish.]. Enferm Infecc Microbiol Clin. 2007;25:168–71. http://dx.doi.org/10.1157/13099367 17. Calderaro A, Gorrini C, Peruzzi S, Piccolo G, Dettori G, Chezzi C. An 8-year survey on the occurrence of imported malaria in a nonen- demic area by microscopy and molecular assays. Diagn Microbiol Infect Dis. 2008;61:434–9. http://dx.doi.org/10.1016/j.diagmicrobio. 2008.03.016 30. Danquah I, Bedu-Addo G, Mockenhaupt FP. Type 2 diabetes mellitus and increased risk for malaria infection. Emerg Infect Dis. 2010;16:1601–4. http://dx.doi.org/10.3201/eid1610.100399 18. Rubio JM, Post RJ, van Leeuwen WM, Henry MC, Lindergard G, Hommel M. References Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the semin- ested multiplex malaria PCR (SnM-PCR). Trans R Soc Trop Med Hyg. 2002;96 Suppl 1:S199–204. http://dx.doi.org/10.1016/S0035- 9203(02)90077-5 G, Hommel M. Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the semin- ested multiplex malaria PCR (SnM-PCR). Trans R Soc Trop Med Hyg. 2002;96 Suppl 1:S199–204. http://dx.doi.org/10.1016/S0035- 9203(02)90077-5 Address for correspondence: Gerardo Rojo-Marcos, Department of Internal Medicine, Príncipe de Asturias University Hospital, Ctra de Meco s/n, 28805 Alcalá de Henares, Madrid, Spain; email: grojo.hupa@salud.madrid.org Zombies—A Pop Culture Resource for Public Health Awareness Reginald Tucker reads an abridged version of the Emerging Infectious Diseases Another Dimension, Zombies— A Pop Culture Resource for Public Health Awareness. http://www2c.cdc.gov/podcasts/player.asp?f=8628220 Emerging Infectious Diseases Journal Podcasts Address for correspondence: Gerardo Rojo-Marcos, Department of Internal Medicine, Príncipe de Asturias University Hospital, Ctra de Meco s/n, 28805 Alcalá de Henares, Madrid, Spain; email: grojo.hupa@salud.madrid.org Zombies—A Pop Culture Resource for Public Health Awareness Reginald Tucker reads an abridged version of the Emerging Infectious Diseases Another Dimension, Zombies— A Pop Culture Resource for Public Health Awareness. http://www2c.cdc.gov/podcasts/player.asp?f=8628220 Emerging Infectious Diseases Journal Podcasts Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 416 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014
https://openalex.org/W2990908788	https://www.shs-conferences.org/10.1051/shsconf/20197006006/pdf	English	null	The investigation of environmental specifics of an autistic child’s family system	SHS web of conferences	2,019	cc-by	3,161	The investigation of environmental specifics of an autistic child’s family system Tatyana Ozerova 1, Elena Vorobyova2 Tatyana Ozerova 1, Elena Vorobyova2 1 Southern Federal University, Academy of psychology and pedagogy, 344006 Rostov-on- Don, Russia y , y 1 Southern Federal University, Academy of psychology and pedagogy, 344006 Rostov-on- Don, Russia 1 Southern Federal University, Academy of psychology and pedagogy, 344006 Rosto Don, Russia 2 Don state technical university, Department of Clinical Psychology and Psychophysiology, 344000 Rostov-on-Don, Russia. Abstract. The paper is devoted to the actual nowadays problem of environmental specifics of an autistic child’s family system. The relevance of the chosen theme is grounded in the paper. Particular attention is paid to the analysis of of related to the problem fundamental theoretical concepts of Russian and translated authors. The paper also presents the outcomes of empirical research and displays statistically significant distinction between psychological indicators of the environment of autistic children’s families and those of families with ordinary children according to such parameters as: roles, rules, control, cooperation, discipline, acceptance/rejection, emotional bond. Considerable distinction between the type of family cohesion and the type of family adaptation of an autistic child’s family system in comparison to the family system of an ordinary child is described. Statistical reliability of correlation between an autistic child’s and an ordinary child’s family system and characteristics of the type of the family (dysfunctional, semifunctional, functional) is analyzed. The paper provides the analysis of distinction between the level of marriage satisfaction in autistic child’s families and in ordinary child’s families. As far back as in the middle of the last century autism was considered to be a rare disorder. Nowadays the number of people suffering from it is increasing. According to the World Health Organization in 2016 every 68th child was born with an autism spectrum disorder and it’s important to note that the number is still growing by 13% a year. According to the World Health Organization experts, autistic disorder is a worldwide phenomenon and this is evident from the fact that people on all continents, in every country, suffer from it regardless of their sex, socio-economic status and race. One of the most pressing concerns https://doi.org/10.1051/shsconf/20197006006 https://doi.org/10.1051/shsconf/20197006006 SHS Web of Conferences 70, 06006 (2019) ICTDPP-2019 * Corresponding author: ozta@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). SHS Web of Conferences 70, 06006 (2019) ICTDPP-2019 https://doi.org/10.1051/shsconf/20197006006 in child psychiatry now is the problem of childhood autism. It can be explained by a number of reasons: in child psychiatry now is the problem of childhood autism. It can be explained by a number of reasons: - first, by certain difficulties in timely diagnosis - first, by certain difficulties in timely diagnosis - second, by the frequency of the development of the disorder - second, by the frequency of the development of the disorder - third, by the fact that the causes of the disorder are not fully identified. - third, by the fact that the causes of the disorder are not fully identified Modern psychiatry nowadays puts forward the idea of an impact of certain factors on forming an autism spectrum disorder. The supporters of the theory of psychogenesis who share the idea that the origin of autism is the impact of adverse psychological factors, have expressed their view ever since the time of Kanner’s discovery of autism. Today the scientific community hasn’t arrived at a common view on the issue, which is another proof of the relevance of the study. Bruno Bettelheim’s monograph “The Empty Fortress: Infantile Autism and the Birth of the Self” has won broad acceptance among specialists and the author is considered to be one of the most faithful adherents of the theory of psychogenic origin of autism [2]. The works of canonical classics of family therapy S.Minuchin, M. Bowen, N. Ackerman, C.Whitaker, I. Boszormenyi-Nagy, the Palo Alto Group and others facilitated the emergence of a new paradigm of symptomatic behavior and under the influence of Bertalanffy’s general systems theory instead of focusing on each individual family member and further summing up the information to get an overall picture, an idea appeared, that understanding the process of functioning of all the family members as a whole provides an opportunity to understand its individual member. The process during which the co- functioning of all family members takes place is called the family system [1]. In the life of an individual person, as well as of the society as a whole, the family plays a large and significant role. * Corresponding author: ozta@mail.ru In the development of each person, the family not only provides personal development, but also plays a leading role in mental disorders and pathogenic situations as it is an important environmental factor for personality development [5]. In the works of Russian and translated family systems psychotherapists, autism is described as a family system symptom along with other symptoms, such as: separation anxiety in a child when entering school, reduced learning capacity at school age, home theft, child’s aggressive behavior, marital conflicts, unmotivated anxiety, depression, sexual dysfunction, anorexia, schizophrenia, bronchial asthma, etc. In the systems approach, the family is treated as a social system that unites a group of people and their relationships, that is, it is a complex of elements and their characteristics, related to each other and having dynamic correlations between them. The family system is prior to its components [3]. The life of a family system is subject to two laws: the law of homeostasis and the law of the development. In case a law is broken, a symptom appears in the system. The carrier of the symptom in the family can be both a child and an adult. A symptom in the family system fulfills certain functions: a) the morphostatic function is the preservation of the family system in its current state, that is, the preservation of family homeostasis, a) the morphostatic function is the preservation of the family system in its current state, that is, the preservation of family homeostasis, b) the morphogenetic function of the symptom is the transformation of the family system (the law of the development). b) the morphogenetic function of the symptom is the transformation of the family system (the law of the development). Russian family psychotherapists describe the parameters and structural elements of the family system: the numerical and personal composition of the family, rules, roles, subsystems, boundaries, myths and legends, interaction stereotypes, stabilizers of the family system, family histories. Breaching one of them can become a reason for appearance of a symptom in one or in several family members [4]. * Corresponding author: ozta@mail.ru On the basis of the conducted examination and summing up the principal points of family systems theories of Russian and translated authors from various approaches and scholarly traditions, we can conclude that dysfunctions of the following family system parameters constitute the specific features of a family system as an environmental factor for 2 SHS Web of Conferences 70, 06006 (2019) ICTDPP-2019 https://doi.org/10.1051/shsconf/20197006006 autistic children: rigid or blurred boundaries of family subsystems; rigid, pathologizing roles; role conflicts, intergenerational coalitions; family's difficulties while passing a life- cycle stage, or difficulty in moving from one stage of the life-cycle to another; having a family extrajection; intergenerational transmission of interaction patterns, repressed and inherited emotions; signs of the "anniversary syndrome"; family myths; family legends; dysfunctional stabilizers; lack of clear rules and regulations. But also dysfunctional: emotional bonds, peculiarities of children upbringing, parent- child relationships, marital relations, family adaptation, low level of satisfaction with family life. In view of the above, in today's society, the study of the characteristics of the family system of autistic children is of particular importance and relevance, and that is the focus of our study, in which 60 families got involved, 30 of which are families with autistic children living in the city of Azov, Rostov region and visiting the Center for psychological, pedagogical, medical and social assistance "Countenance”. As well as 30 families with healthy children living in the city of Novocherkassk, Rostov Region, visiting the Development and Leisure Center. p As a hypothesis, we assumed the following: • in families with autistic children such environmental features as a high level of parental control and a reduced level of emotional acceptance and cooperation may be representative characteristics • in families with autistic children such environmental features as a high level of parental control and a reduced level of emotional acceptance and cooperation may be representative characteristics • in families with autistic children, a dysfunctional type of family can take place, which is characterized by incoherence of family members and chaotic nature of adaptation. • in families with autistic children, a dysfunctional type of family can take place, which is characterized by incoherence of family members and chaotic nature of adaptation. • the level of satisfaction with marriage in families with autistic children may differ from the level of satisfaction with marriage in families with an ordinary child. * Corresponding author: ozta@mail.ru • the level of satisfaction with marriage in families with autistic children may differ from the level of satisfaction with marriage in families with an ordinary child. In the present study the following psychodiagnostic techniques were used: “FACES-3 Family Adaptation and Cohesion Scale”; marriage satisfaction test by V.V. Stolin, T.L. Romanova, G.P. Butenko; parental attitude questionnaire by A.Y. Vargi, V.V. Stolin. Mathematical data processing included the use of the following methods: compilation of contingency tables; nonparametric criteria (Mann-Whitney U-test and Kruskal-Wallace criterion); multivariate analysis of variance (DA). For data processing, traditional software programs for statistical data analysis were used: "STATISTICA 10 RU" and "IBM SPSS Statistics 21". The study revealed the following data: The study revealed the following data: The environmental characteristics of an autistic children's family system have statistically significant differences (p <0.05) from the environmental characteristics of families with an ordinary child in such parameters as: roles, rules, control, cooperation, discipline, acceptance / rejection, emotional bond. The data obtained are presented in the comparative diagram in Figure 1 Fig. 1. Statistically significant differences in the environmental characteristics of the family system. Comparative diagram, in points Fig. 1. Statistically significant differences in the environmental characteristics of the family system. Comparative diagram, in points 3 3 https://doi.org/10.1051/shsconf/20197006006 SHS Web of Conferences 70, 06006 (2019) ICTDPP-2019 SHS Web of Conferences 70, 06006 (2019) ICTDPP-2019 SHS Web of Conferences 70, 06006 (2019) ICTDPP-2019 Statistically significant differences became apparent through the use of the “Parental Relationship Questionnaire” technique (A.Y. Varga, V.V. Stolin). As seen from the comparative diagram, the statistical significance of the differences was verified by the non- parametric U Mann-Whitney test. As illustrated in the comparative diagram, in the control group there are considerably more families with a high level of “Acceptance / Rejection” and “Cooperation”. The experimental group is characterized by an increased level of control and a decreased level of acceptance and cooperation. It is important to note that with increased control, an adult behaves too authoritatively towards the child, imposing strict disciplinary restrictions, demanding unconditional obedience, almost entirely imposing his will on the child. The low level of acceptance and cooperation is a sign that an adult takes little interest in what the child is interested in, does not encourage the child's independence and initiative, does not assess the ability of the child. p y Using “The Family Adaptation and Cohesion Questionnaire (FACES-3)” (D.K. Olson, J. Portner, I. Lavi) technique, we studied the levels of cohesion and adaptation of the family system, which revealed the presence of statistically significant differences in the distribution in the control and experimental groups. In the autistic children's family system, a disengaged type of family cohesion statistically (p <0.05) prevails, thus distinguishing this system from an ordinary child's family system, in which the connected type of family system predomonates. The results are shown by the diagram in Figure2 Fig. 2. Distribution according to the level of the family system cohesion, in percentage correlation. Fig. 2. Distribution according to the level of the family system cohesion, in percentage correlation. We found that in an autistic child's family system, about half of the families have a chaotic type of adaptation, while approximately the same number of families with an ordinary child have a structured type of adaptation. We can observe this tendency of distribution of the level of adaptation in groups in the diagram in Figure 3. Fig. 3. Distribution of adaptation of the family system according to the questionnaire by D.Kh. Olson, J. Portner, I. Lavi, in percentage correlation Fig. 3. Distribution of adaptation of the family system according to the questionnaire by D.Kh. Olson, J. Portner, I. Lavi, in percentage correlation 4 4 https://doi.org/10.1051/shsconf/20197006006 SHS Web of Conferences 70, 06006 (2019) ICTDPP-2019 SHS Web of Conferences 70, 06006 (2019) ICTDPP-2019 The analysis of the distribution of the level of satisfaction with marriage in families of different types showed that the largest percentage of “bad marriages” is in families of a functional type (62.5%); the largest percentage of “transitory marriages” is represented in 5 5 https://doi.org/10.1051/shsconf/20197006006 SHS Web of Conferences 70, 06006 (2019) ICTDPP-2019 semi-functional families (64.3%); the highest percentage (55%) of “good marriages” was found in dysfunctional families; the perception of one’s own marriage as “absolutely successful” is more often expressed in semi-functional type families (44.4%). The study also revealed the following tendencies: g 1) in families of the dysfunctional type, the level of the control factor is statistically higher in the families in which parents are satisfied with the family system; 2) in semi-functional type of families, the level of the control factor is statistically higher in the families in which parents are not satisfied with family adaptation; 3) in functional type of families (and they are in the minority), the level of the control factor is the highest. 4) in the experimental group in the families of semi-functional type, along with dissatisfaction with family adaptation, we can observe an almost complete absence of symbiosis. 5) in the experimental group in the families of dysfunctional and semi-functional types, along with dissatisfaction with family adaptation, a decrease in the level of emotional acceptance of the child by the parents can be observed. Thus, we can state that all the hypotheses put forward have found their confirmation. The reliability of the results obtained is confirmed through the use of diagnostic methods and techniques that are adequate to the purpose and objectives of the study, the correct use of methods of mathematical statistics. The practical significance of this work consists in the following: - The outcome of the study can be used for teaching disciplines revealing the nature of childhood autism. - The results obtained can be used in practical, counseling, therapeutic work of psychologists. - The results of the study can be used in the work of social workers. - The results of the study can serve as the basis for the development of a progra therapeutic and preventive work with families with children having an autistic disorde - The results of the study can serve as the basis for the development of a program for therapeutic and preventive work with families with children having an autistic disorder. SHS Web of Conferences 70, 06006 (2019) ICTDPP-2019 The disengaged type of family system cohesion is characterized by alienated relations, members of such a family are often emotionally distant, have little attachment to each other. The chaotic type of adaptation of the family system has unstable or limited leadership. Decisions made in the family are often ill-conceived, roles are unclear and often shift from one family member to another. A large number of changes leads to unpredictability of what is happening in the system. Such a family system is not resilient to stress factors. A disengaged and chaotic family system is referred to a dysfunctional type of family - it is a family in which performing functions is impaired, as a result of which the goals of the family as a whole and family members in particular are not achieved in the marital, parental, material, household and other spheres of life, which interferes with the personal growth and blocks the possibility of self-actualization of family members. It was also revealed that in the autistic children's family system, a dysfunctional type of a family statistically predominates, while semi-functional and functional types of families predominate in the family system with an ordinary child. For clarity, the diagram is shown in Figure 4. Fig. 4. Proportioning of family types by groups, in percentage correlation. Fig. 4. Proportioning of family types by groups, in percentage correlation. In the autistic children's family system, the number of families satisfied with the family system is almost equal to the number of families dissatisfied with the family system. At the same time, in a family system with an ordinary child, the number of people satisfied with the family system is significantly higher than the number of families dissatisfied with the family system (Fig. 5). Fig. 5. Distribution of the level of satisfaction with the family system by groups, in percentage correlation. Fig. 5. Distribution of the level of satisfaction with the family system by groups, in percentage correlation. SHS Web of Conferences 70, 06006 (2019) ICTDPP-2019 Thus, we can conclude that the environmental specifics of the family system can have an impact on a child with autism. In this regard, it can be assumed that family therapy (working with a family symptom) could become one of the possible forms of therapy, as well as of the prevention of autistic disorder. As a direction of further research, a deeper analysis of the family system is planned to be performed, with a possibly increased number of participants of the examination and the development of a program for therapeutic and preventive work with autistic children's families. References 1. M.A. Bebchuk, E. Zhuykova Help to the family: psychology of decisions and changes (Independent firm “Class”, Moscow, 2015) (Independent firm “Class”, Moscow, 2015) 2. B. Bettelheim, The empty fortress. Childhood Autism and Birth (Academic Project, Moscow, 2013) 3. A.Y. Varga, T.S. Drabkina, Systems family therapy. Short lecture course (Speech, St. Petersburg, 2001) 4. A.V. Chernikov, Systems family therapy: An integrative diagnostic model (Independent firm “Class”, Moscow, 2001) 5. E. Eidemiller, V. Justickis, Psychology and psychotherapy of the family. (Peter, St. Petersburg, 2009) 2. B. Bettelheim, The empty fortress. Childhood Autism and Birth (Academic Project, Moscow, 2013) Moscow, 2013) 3. A.Y. Varga, T.S. Drabkina, Systems family therapy. Short lecture course (Speech, St. Petersburg, 2001) 4. A.V. Chernikov, Systems family therapy: An integrative diagnostic model (Independent 3. A.Y. Varga, T.S. Drabkina, Systems family therapy. Short lecture course (Speech, St. Petersburg, 2001) 4. A.V. Chernikov, Systems family therapy: An integrative diagnostic model (Independent firm “Class”, Moscow, 2001) 5. E. Eidemiller, V. Justickis, Psychology and psychotherapy of the family. (Peter, St. 4. A.V. Chernikov, Systems family therapy: An integrative diagnostic model (Independ firm “Class”, Moscow, 2001) 5. E. Eidemiller, V. Justickis, Psychology and psychotherapy of the family. (Peter, St. Petersburg, 2009) 6
https://openalex.org/W4288246270	https://zenodo.org/records/4285714/files/GSCBPS-2019-0144.pdf	English	null	Practical identification key of Digitaria ciliaris (Retz.) Koeler and Digitaria horizontalis Willd. (Poaceae): two harmful weeds of crops in Senegal	Zenodo (CERN European Organization for Nuclear Research)	2,019	cc-by	2,268	1. Introduction Digitaria ciliaris (Retz.) Koeler and D. horizontalis Willd. are considered as highly aggressive and reported as a problem in 60 countries around the world, infesting more than 30 crops economically important [1]. In Senegal, they are found in all phytogeographic zones [2] and remain among the most important weeds in agriculture. Indeed, D. ciliaris is one of the most common weeds and the most harmful for many crops such as groundnuts, millet [3] and corn [4]. However, its advantage is that it is usually more nutritious than most other warm-season grasses [5]. As for D. horizontalis, it poses a major challenge in favorable environments where it is able to grow vigorously and multiply rapidly [6] despite its strategic role as a livestock feed at the beginning of the rainy season at the time where the dry season straw stock is exhausted [7]. However, on farms, the precise identification of D. horizontalis is not easy because it is most often confused with D. ciliaris. The two species, so similar vegetatively and reproductively, have often been a source of confusion among taxonomists and agronomists. At the local level, any determination key has been developed. [8]’s work has been of great help in providing identification keys for Digitaria species. However, a single character, insufficient, is often used for the discrimination of the two species. The discrimination of these species, potential sources of genes used for the improvement of fonio (Digitaria exilis Stapf), the cultivated species, deserves to be made because of their different relationships between Digitaria exilis. This present work aims to define the morphological differences between D. ciliaris and D. horizontalis and to provide a practical key facilitating their identification. (RESEARCH ARTICLE) ublication history: Received on 30 July 2019; revised on 09 August 2019; accepted on 20 August 20 Article DOI: https://doi.org/10.30574/gscbps.2019.8.2.0144 Abstract Digitaria ciliaris and D. horizontalis are among the most common and harmful weed species of crops in Senegal. Their identification by taxonomists and agronomists is not easy because of their close similarity. The objective of this study is to define their morphological differences and to provide a practical identification key. The results of the study show that Digitaria ciliaris and D. horizontalis are differentiated from three characters in the vegetative and reproductive systems. An illustrated table of these discriminating traits, serving as a key of practical determination, is proposed. Keywords: Discriminant traits; Key; Digitaria ciliaris; Digitaria horizontalis; Weeds  Corresponding author E-mail address: Copyright © 2019 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons GSC Biological and Pharmaceutical Sciences, 2019, 08(02), 101–105 GSC Biological and Pharmaceutical Sciences, 2019, 08(02), 101–105 GSC Biological and Pharmaceutical Sciences, 2019, 08(02), 101–105 Copyright © 2019 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0  Corresponding author E-mail address: Copyright © 2019 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0 Copyright © 2019 Author(s) retain the copyright of this article. This article is published unde 2. Material and methods The plant material was provided from the Herbaria DAKAR and IFAN and from personal collections. A detailed description of the mature plant was made and completed if necessary with some manuals [8, 9]. A total of 41 morphological characters (qualitative and quantitative) have been described and recorded in Table 1. These characters are derived from culm, leaf (sheath, ligule and limb), racemes, spikelets, glumes, flowers and fruit. All the Copyright © 2019 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0 Ngom et al. / GSC Biological and Pharmaceutical Sciences 2019, 08(02), 101–105 characters of each species described have been compared in order to obtain the most discriminating characters differentiating at best the two species studied. p p g ifferentiating at best the two species studied. able 1 Morphological traits studied for the distinction of D. ciliaris and D. horizontalis N° Components Traits D. ciliaris D. 3.1. The discriminating morphological characters Of the 41 traits studied, thirthy one (31) are common to both species. The remaining ten characters, which are discriminant, are related to the ligule, racemes, spikelet, lower glume, upper glume, upper lemma and fruit. The most diagnostic of these are length and pubescence of the ligule, racemes disposition, shape of the lower glume and the relative length of the upper glume. In species of the genus Digitaria, the use of characters from the vegetative system for their distinction is less frequent and uncertain because they are often quite variable within the same species creating transition forms that make identification difficult. However, this study reveals important vegetative characters for the recognition of the two studied species. Indeed, the ligule is most developed in D. ciliaris (0.5-3 mm) than D. horizontalis (1-1.5 mm). The spikelet characters are still the most used for distinguishing species and are of great taxonomic importance, making it much easier to identify species of the genus. In this study, the shape of the lower glume and the relative length of the upper glume are the most discriminating characters for distinguishing D. ciliaris from D. horizontalis. In fact, D. ciliaris has a well-developed lower glume ovate and an upper glume equal or longer than half the length of the spikelet. Contrariwise, in D. horizontalis, the lower glume is either absent or reduced to scales, ring or membranous; the upper glume not exceeding half the length of the spikelet. As for the upper glume, the great variability of most of its characters makes them highly discriminating. These results are in correlation with those of [10] who states that D. horizontalis is distinguished from D. ciliaris by the upper glume (1/3 to ½ as long as the spikelet). The upper glume of D. ciliaris is ½-¾ as long as the spikelet. The arrangement of the racemes on the main axis is also a character of important value to distinguish these two species [11, 12, 13]. D. ciliaris is characterized by its racemes often digitated whereas in D. horizontalis they are often digitated or subdigitated and arranged along a common main axis. 2. Material and methods horizontalis 1 Culm Habit annual annual 2 Height 25 cm ˃ x < 200 cm 25 cm ˃ x < 200 cm 3 Nodes pubescence glabrous glabrous 4 internodes pubescence glabrous glabrous 5 Sheath Pubescence glabrous or pubescent glabrous or pubescent 6 Ligule Apex truncate truncate 7 Length 0.5-3 mm 1-1.5 mm 8 Pubescence glabrous, presence of long periligular hairs glabrous, presence of long periligular hairs 9 Limb Form linear to lanceolate linear to lanceolate 10 Apex attenuated in a peak attenuated in a peak 11 Base rounded rounded 12 Pubescence glabrous or pubescent glabrous or pubescent 13 Margin roughness scabrous scabrous 14 Margin pubescence glabrous glabrous 15 Racemes Disposition often digitate often subdigitate along a central axis 16 Rhachis triquetrous, not winged triquetrous, not winged 17 Spikelet Disposition by 2 by 2 18 Form elliptic to lanceolate lanceolate 19 Pubescence pubescent pubescent 20 Type of hairs apressed apressed 21 Pedicels roughness scabrous scabrous 22 Pedicels pubescence glabrous glabrous 23 Lower glume Form present and ovate absent or reduced to scales, ring or membranous 24 Apex acute truncate 25 Upper glume Form lanceolate to linear Lanceolate to linear 26 Apex acute acute 27 Relative length ½ to ¾ of the spikelet length ≤ ½ of the spikelet length 28 Number of veins 3-veined 3-veined 29 Pubescence pubescent pubescent 30 Lower lemme Form elliptic elliptic 31 Relative length equal to the spikelet length equal to the spikelet length 32 Apex acute acute 33 Number of veins 7- veined 7- veined 34 Pubescence pubescent pubescent 35 Upper lemma Form lanceolate to oblong lanceolate 36 Apex acute acute 37 Relative length equal to the spikelet length equal to the spikelet length 38 Fruit Form oblong oblong 39 Apex acute acuminate or acute 40 Length ˃ 2 mm 1 mm to 2 mm 41 Color brown beige Table 1 Morphological traits studied for the distinction of D. ciliaris and D. horizontalis 102 Ngom et al. / GSC Biological and Pharmaceutical Sciences 2019, 08(02), 101–105 4. Conclusion In this study, 10 discriminant characters was identified among which three (racemes disposition, lower glume form and relative length of the upper glume) can be used for the distinction of D. ciliaris and D. horizontalis. However, it would be more prudent to use simultaneously these three characters. Compliance with ethical standards Acknowledgments We are thankful to the Curators of the Herbaria DAKAR and IFAN for providing herbarium materials. Disclosure of conflict of interest Disclosure of conflict of interest The authors hereby declare that there is no conflict of interest. Ngom et al. / GSC Biological and Pharmaceutical Sciences 2019, 08(02), 101–105 Ngom et al. / GSC Biological and Pharmaceutical Sciences 2019, 08(02), 101–105 Figure 1 Determinative characters distinguishing D. ciliaris from D. horizontalis. Sources: A from https://www.eeob.iastate.edu; C and E from [16]; D and F from [8]. Figure 1 Determinative characters distinguishing D. ciliaris from D. horizontalis. Sources: A from https://www.eeob.iastate.edu; C and E from [16]; D and F from [8]. Figure 1 Determinative characters distinguishing D. ciliaris from D. horizontalis. Sources: A from https://www.eeob.iastate.edu; C and E from [16]; D and F from [8]. [1] Souza RC, Dias AC, Figueiredo MRA, Obara FEB and Christoffoleti PJ. (2012). Growth of the crabgrass species Digitaria ciliaris and Digitaria nuda. Planta Daninha, 30 (2), 317-325. 3.2. The proposed determination key Three determinative characters were retained for the construction of the key (Figure 1). In the proposed key, the first criterion chosen is the relative length of the upper glume. Although the spikelet appears at a late stage and its components delicately appreciable, this key is more reliable. Indeed, according to [14], it is considered as a reliable character by most researchers in the genus but very precise measurements are necessary. By the way, they recommended using relative lengths such as from the upper glume in key construction. As for their study, the present results show that it gave the best indication of the phenetic relationship between D. ciliaris and D. horizontalis. Another character, no less important, to take into account in the distinction of the two species is the presence or not of the lower glume which is absent or reduced to scales, ring or membranous in D. horizontalis while rather developed and ovate in D. ciliaris. Thereby, some authors such as [12] and [15] considered this character of some value to distinguish species while [13] indicated a separation of closely related species on the basis of the length of the lower glume. 103 Ngom A, Mbaye MS, Gueye M, Camara AA and Noba K. (2019). Practical identification key of Digitaria ciliaris (Retz.) Koeler and Digitaria horizontalis Willd. (Poaceae): two harmful weeds of crops in Senegal. GSC Biological and Pharmaceutical Sciences, 8(2), 101-105. Ngom et al. / GSC Biological and Pharmaceutical Sciences 2019, 08(02), 101–105 [2] Ngom A, Mbaye MS, Barnaud A, Gueye MC, Camara AA, Gueye M, Diop BM and Noba K. (2019). Ecological distribution, diversity and use of the genus Digitaria Haller (Poaceae) in Senegal. International Journal of Biodiversity and Conservation, 11(1), 8-17. [3] Noba K. (2002). Weed flora in the southern Groundnut Basin (Senegal): structure, dynamics and impact on the production of millet and groundnuts. State Doctorate Thesis, Cheikh Anta Diop University of Dakar, Senegal, 64- 102. [4] Bassene C, Mbaye MS, Kane A, Diangar S and Noba K. (2012). Weed flora of maize (Zea mays L.) in the southern groundnut basin (Senegal): structure and harmfulness of species. Journal of Applied Biosciences, 59, 4307- 4320. [5] Dalrymple RL, Mitchell R, Flatt B, Dobbs W, Ingram S and Coleman SW. (1999). Crabgrass for Forage: management from the 1990s. The Noble Foundation, Ardmore, OK. [6] Johnson DE and Kent RJ. (2002). The impact of cropping on weed species composition in rice after fallow across a hydrological gradient in West Africa. Weed Research 42, 89-99. [7] Sanou KF, Ouedraogo S, Nacro S, Ouedraogo M and Kaboré-Zoungrana C. (2016). Sustainability of supply and nutritional value of marketed fodder in urban areas of Bobo-Dioulasso, Burkina Faso. Cahiers Agricultures, 25, 15002. [8] Poilecot P. (1999). Poaceae of Niger. Boissiera, 56, 448-469. [8] Poilecot P. (1999). Poaceae of Niger. Boissiera, 56, 448-469. Poilecot P. (1999). Poaceae of Niger. Boissiera, 56, 448-469. [9] Berhaut J. (1967). Flora of Senegal, Second edition. Clairafrique, Dakar, Senegal. [10] Clayton WD. (1974). Notes on the Genus Digitaria Haller Studies in the Gramineae: XXXVI. Kew Bulletin, 29 (3), 517-525. (1950). Monograph of the genus Digitaria.: Universitaire Pers Leiden, Leiden. [11] Henrard JT. (1950). Monograph of the genus Digitaria.: Universitaire Pers Leiden, Leiden. [12] Veldkamp JF. (1973). A revision of Digitaria in Malesia. Blumea, 21, 1-80. [13] Webster RD. (1983). A revision of the genus Digitaria Haller (Paniceae: Poaceae) in Australia. Brunonia, 6, 131- 216. [14] Kok PDF, Robbertse PJ and van Wyk AE. (1988). Systematic study of Digitaria section Digitaria (Poaceae) in southern Africa. South African Journal of Botany, 55(2), 141-153. [15] Bor NL. (1955). The genus Digitaria Heist in India and Burma. Webbia, 2, 301-367. [16] Verloove F. (2008). Studies within the genus Digitaria Haller (Poaceae, Panicoideae) in southwestern Europe. Candollea, 63, 227-233. References [1] Souza RC, Dias AC, Figueiredo MRA, Obara FEB and Christoffoleti PJ. (2012). Growth of the crabgrass species Digitaria ciliaris and Digitaria nuda. Planta Daninha, 30 (2), 317-325. 104 Ngom et al. / GSC Biological and Pharmaceutical Sciences 2019, 08(02), 101–105 How to cite this article How to cite this article 105
https://openalex.org/W1608292591	https://cdr.lib.unc.edu/downloads/cz30q296g	English	null	Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT	Journal of biological chemistry/The Journal of biological chemistry	1,991	cc-by	8,095	Escherichia coli DNA Helicase I Catalyzes a Site- and Strand-specific Nicking Reaction at the F Plasmid oriT * (Received for publication, March 27,1991) (Received for publication, March 27,1991) Steven W. MatsonSgIl, and Brad S . Morton$ From the $Department of Biology and §Curriculum in Genetics, University of North Carolina, Chapel Hill, North Carolina 27599 of regions of duplex DNA in excess of 100 kb’ has been observed (9) suggesting that the enzyme is capable of unwind- ing long regions of duplex DNA in a processive reaction. A site- and strand-specific nick, introduced in the F plasmid origin of transfer, initiates conjugal DNA transfer during bacterial conjugation. Recently, mo- lecular genetic studies have suggested that DNA heli- case I, which is known to be encoded on the F plasmid, may be involved in this nicking reaction (Traxler, B. A., and Minkley, E. G., Jr. (1988) J. Mol. Biol. 204, 206-209). We have demonstrated this site- and strand- specific nicking event using purified helicase I in an in vitro reaction. The nicking reaction requires a super- helical DNA substrate containing the F plasmid origin of transfer, Mg2+ and helicase I. The reaction is protein concentration-dependent but, under the conditions used, only SO-70% of the input DNA substrate is con- verted to the nicked species. Genetic data (Everett, R., and Willetts, N. (1980) J. Mol. Biol. 136, 129-150) have also suggested the involvement of a second F- encoded protein, the Tray protein, in the oriT nicking reaction. Unexpectedly, the in vitro nicking reaction does not require the product of the F plasmid tray gene. The implications of this result are discussed. g g g p p The tral gene encoding helicase I lies near the distal end of the 33-kb F plasmid tray2 operon; its complete DNA se- quence and precise map position are now known (10, ll). Genetic studies indicate that the tral gene product is required for both physical transfer of the F plasmid from the donor to the recipient cell during bacterial conjugation and for donor conjugal DNA synthesis (12, for reviews see Refs. 13-15). Since the discovery that helicase I is encoded on the F plasmid (l), it has been assumed that helicase I utilizes its DNA unwinding activity to play a role in conjugal DNA transfer. The processivity and the polarity of the unwinding reaction make helicase I an attractive candidate for the unwinding of the F plasmid to generate single-stranded DNA (ssDNA) for transfer to a recipient bacterium. Escherichia coli DNA Helicase I Catalyzes a Site- and Strand-specific Nicking Reaction at the F Plasmid oriT * It has been suggested that the unwinding reaction catalyzed by helicase I may act as the motive force that drives strand transfer (13-15). ( ) The initial DNA processing event in conjugal DNA transfer is the site- and strand-specific nicking of the F plasmid at the origin of transfer (oriT). This is followed by the unwinding of the F plasmid, presumably by helicase I, with the 5‘ end of the nicked strand entering the recipient bacterium first (re- viewed in Refs. 13-15). The site- and strand-specific nick is believed to be introduced in oriT by the action of a two subunit endonuclease composed of the products of the tray and traZ genes (16). The tray gene has been cloned, its protein product overproduced and purified, and it has been shown to bind specifically to a region on oriT that lies 60 bp upstream of the nick site (17).* Tray protein alone does not catalyze a nicking reaction within the oriT region (17).* The traZ gene was never precisely mapped, due to a lack of point mutations, although it was localized at the distal end of the F plasmid tra operon (16,18). Minicell experiments suggest the existence of a coding region, distal to tral, responsible for a protein product called 2b with a molecular mass of 78-79 kDa (18). This was thought to be the traZ gene (16,18). Recent mapping and sequencing of the distal end of the tra operon indicates that there is insufficient room to encode a 79-kDa protein between the end of the tral gene and the IS3 sequence which defines the end of the traYZ operon (11). The 79-kDa 2b protein observed in minicells appears, in fact, to be a restart product of the tr$ gene (11). Additional studies have sug- gested that the nicking function originally assigned to traZ may be dependent on tral sequences. Traxler and Minkley (19) have shown that insertions into and deletions of the tral sequence abolish nicking in an in vivo nicking assay, suggest- ing that helicase I may be involved in the nicking reaction. In g p The phosphodiester bond interrupted by helicase I has been shown to correspond exactly to the site nicked in vivo suggesting that helicase I is the site- and strand- specific nicking enzyme that initiates conjugal DNA transfer. * This investigation was supported by American Cancer Society Grant MV-435 (to S. W. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. n Recipient of an American Cancer Society Faculty Research Award. THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology. Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology. Inc. Vol. 266, No. 24, h u e of August 25. pp. 16232-16237.1991 Printed in U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochem The abbreviations used are: kb, kilobase pair(s); ssDNA, single- stranded DNA; oriT, origin of transfer; SDS, sodium dodecyl sulfate; bp, base pair(s); nt, nucleotide(s). E. E. Lahue and S. W. Matson, unpublished observations. This is an Open Access article under the CC BY license. p, p ( ); , ( ) E. E. Lahue and S. W. Matson, unpublished observations. Escherichia coli DNA Helicase I Catalyzes a Site- and Strand-specific Nicking Reaction at the F Plasmid oriT * Thus, helicase 1 is a bifunctional protein which catalyzes site- and strand-strand specific nick- ing of the F plasmid in addition to the previously char- acterized duplex DNA unwinding (helicase) reaction. Escherichia coli DNA helicase I is the product of the F plasmid tral gene (1) and is required for conjugal DNA transfer from a donor to a recipient bacterium (2). This enzyme was the first E. coli DNA helicase to be identified and characterized on the basis of its ability to separate the two strands of duplex DNA in an energy-requiring reaction (3-5). The enzyme, which has been extensively characterized both as a DNA-dependent ATPase (3, 6) and as a helicase (4-8) catalyzes the processive unwinding of duplex DNA migrating in the 5’ to 3’ direction with respect to the strand of DNA on which it is bound, This reaction requires the concomitant hydrolysis of a NTP; there is no apparent preference as to which NTP (dNTP) is utilized in this reaction (6). Unwinding 16232 16232 This is an Open Access article under the CC BY license. Helicase I Nicking at F oriT 16233 16233 was subsequently soaked in 1.5 M Tris-HCI (pH 7.4)/3.0 M NaCl for 30 min, and the DNA was transferred to nylon membranes. The probe was a 540-bp DNA fragment containing the oriT region isolated from pBSoriT and radioactively labeled using a random oligonucleo- tide labeling kit (U. S. Biochemicals). was subsequently soaked in 1.5 M Tris-HCI (pH 7.4)/3.0 M NaCl for 30 min, and the DNA was transferred to nylon membranes. The probe was a 540-bp DNA fragment containing the oriT region isolated from pBSoriT and radioactively labeled using a random oligonucleo- tide labeling kit (U. S. Biochemicals). this view, helicase I would have a dual function in conjugation; site- and strand-specific nicking at oriT, presumably in con- junction with Tray protein and unwinding of the F plasmid. Although the helicase activity of TraI protein has been clearly demonstrated, a biochemical demonstration of an oriTnickin activity has never been accomplished. g ( ) Other Methods-Protein concentrations were determined using the Bio-Rad protein assay; bovine serum albumin was the standard. DNA concentrations were determined by directly reading the absorbance at 260 nm and are expressed in nucleotide equivalents. DNA sequenc- ing was by the dideoxynucleoside 5'-triphosphate chain termination method (23) using Sequenase (U. S. Biochemicals) as described by the supplier. Materials Enzymes-DNA helicase I was purified as previously described (6) from an overproducing strain of E. coli containing pMP8 (19) which was kindly provided by Dr. T. Lohman (Washington University, St. Louis, MO). The purified enzyme (M, = 180,000) was greater than 95% homogeneous as determined on polyacrylamide gels run in the presence of sodium dodecyl sulfate (SDS). Restriction endonucleases were purchased from New England Biolabs, U. S. Biochemicals, or Life Technologies, Inc. and used according to the suppliers' instruc- tions. Proteinase K was from Boehringer Mannheim and eukaryotic topoisomerase I was from Life Technologies, Inc. p yp p Incubation of purified helicase I with pBSoriT resulted in the formation of a DNA species that migrated with nicked circular DNA on an agarose gel (Fig. 1, lanes 1-3). Analysis of the product of this reaction on agarose gels run in the presence or absence of ethidium bromide indicated that this was a nicked molecule and not covalently closed relaxed circular DNA (data not shown). It should be noted that the formation of this nicked DNA species does not require the addition of Tray protein. p g DNAs and Nucleotides-The Bluescript vector (Stratagene) (pBS) was grown in E. coli HBlOl and purified by alkaline/SDS lysis (20) followed by banding in CsCl/ethidium bromide gradients. The plas- mid pBSoriT is a derivative of pBS containing the F plasmid oriT region. It was constructed by cloning the 529-bp BstYIISalI fragment from pED806 (21) into pBS cleaved with BamHI and SalI. The construction was confirmed by DNA sequencing. This 529-bp DNA fragment contains the F oriT region from the BglII site at position 1 on the recent map of the tra operon to the SalI site at position 529 (22). To determine whether the reaction was specific for plasmid DNA containing the F oriT region, helicase I was incubated with an identical plasmid (pBS) lacking the F oriT region (Fig. 1, lanes 7-9). In this case no nicked DNA was formed suggesting that the nick introduced into pBSoriT by helicase I was located within the oriT region. In addition, incubations of helicase I with pBSoriT were carried out in the absence of MgC12 (data not shown) and in the presence of EDTA (Fig. 1, lanes 4-6). Under these conditions there was no nicking of the plasmid DNA indicating that the reaction required the presence of M P . Materials ( ) The oligonucleotides used as primers in DNA sequencing reactions were synthesized on an Applied Biosystems oligonucleotide synthe- sizer and have the following sequence: primer 1, 5'dAATACGA- CTCACTATAGB'; primer 2,5'dACCACCCCTACAAAACGG3'. Nu- cleoside 5"triphosphates were purchased from P-L Biochemicals, Inc., and concentrations were determined by directly reading absorb- ance at the appropriate wavelength. [a-:'?P]dCTP and [a-"SjdATP were from Amersham. The experimental protocol utilized in the experiments de- RESULTS er Helicase I Nicks a Plasmid Containing oriT-Recently, in- sertion and deletion mutagenesis studies have suggested that helicase I might be one component of the enzyme responsible for site- and strand-specific nicking at F oriT (19). In an effort to directly demonstrate a site- and strand-specific nick- ing activity associated with this enzyme we tested purified helicase I, in the presence and absence of purified Tray protein, in a nicking assay using a plasmid which contained a cloned copy of F oriT (pBSoriT). Both enzymes were purified to greater than 95% homogeneity from F- overproducing strains of E. coli and should, therefore, be free of any other F-encoded polypeptide. Escherichia coli DNA Helicase I Catalyzes a Site- and Strand-specific Nicking Reaction at the F Plasmid oriT * In this report we demonstrate the oriT site- and strand- specific nicking reaction catalyzed by purified helicase I. The nicking reaction occurs within the oriT region on the F plasmid at the site shown to be nicked in vivo. The reaction requires a supercoiled DNA substrate and MgC12 but does not require the addition of ATP. Interestingly, the in vitro reac- tion does not require the product of the tray gene. Based on these results we suggest that helicase I is, in fact, the site- and strand-specific endonuclease that initiates strand trans during bacterial conjugation. Methods Nicking Assay-Reaction mixtures (16 pl) contained 50 mM Tris- HCI (pH 7.5), 1 mM MgCI,, 50 mM NaC1,l mM dithiothreitol, 30 pg/ ml bovine serum albumin, 13-16% glycerol, 330 ng of plasmid DNA (Form I) and helicase I (TraI protein) as indicated. Incubation was at 37 "C for 30 min (unless otherwise indicated) followed by the addition of 4 pl containing 50 mM EDTA, 0.5% SDS, 500 pg/ml proteinase K. Incubation was continued for 30 min at 37 "C. The entire reaction mixture was loaded onto 1.0% agarose gels containing 0.5 pg/ml ethidium bromide. Electrophoresis was at 2 V/cm at 4 "C for 12-16 h. The running buffer was 89 mM Tris, 89 mM borate, 2 mM EDTA (TBE). To determine whether this step was necessary to generate nicked DNA, reactions were stopped using EDTA, EDTA and SDS, or EDTA, SDS, and proteinase K (data not shown). The results of these experi- ments suggested that proteinase K and SDS were not required for the conversion of supercoiled DNA to nicked DNA. How- ever, in the absence of protein denaturants a large fraction of the nicked DNA migrated as a smear, more slowly than nicked molecules, suggesting the presence of helicase I bound to the nicked DNA. The addition of SDS alone reduced the intensity of the nicked DNA smear but did not eliminate the complex. Based on these results we are not able to conclusively deter- mine whether a covalent DNA-protein complex exists. The ability of helicase I to nick a covalently closed relaxed circular plasmid was tested using pBSoriT DNA that had been relaxed with the eukaryotic topoisomerase I (Fig. 3). As expected, helicase I converted supercoiled pBSoriT to a nicked species in the presence of MgC12 (Fig. 3, lanes 1-4). The enzyme failed to nick pBSoriT DNA that had been previously relaxed using calf thymus topoisomerase I (Fig. 3, lanes 5-8). The relaxed molecule migrates slightly faster than supercoiled plasmid on this agarose gel run in the presence of ethidium bromide due to intercalation of the dye. We were unable to detect any conversion of the relaxed plasmid to a nicked form suggesting that the nicking reaction catalyzed by helicase I required a superhelical DNA substrate. When supercoiled and relaxed plasmids were mixed and incubated with helicase I there was conversion of the supercoiled substrate to nicked DNA (Fig. 3, lane 9), although with somewhat lower efficiency than in the absence of the relaxed plasmid. Apparently the presence of a relaxed plasmid containing the oriT region does not markedly inhibit the interaction between helicase I and its supercoiled substrate. Moreover, preparation of the relaxed plasmid has not introduced any inhibitors of the nicking reaction into the reaction mixture. p p The nicking reaction catalyzed by helicase I was shown to be protein concentration-dependent (Fig. 2). At a ratio of DNA molecu1es:helicase I monomers of 1:l approximately 10% of the plasmid DNA was nicked by the enzyme in a 30- min incubation (Fig. 2, lane 2). At the highest concentration of helicase I tested over 50% of the DNA substrate was specifically nicked by the enzyme. Increasing the length of the incubation did not dramatically increase the fraction of substrate converted to nicked molecules (data not shown). Moreover, when residual supercoiled DNA was isolated sub- sequent to the nicking reaction it could be converted to a nicked DNA species by helicase I in a second incubation (data not shown). This rules out the possibility of an inhibitory structure present in some fraction of the substrate DNA. Thus, at a 32-fold molar excess of helicase I monomers over the DNA substrate the reaction failed to go to completion. At present we do not have an explanation for this observation. It may be due to a low affinity of helicase I for its binding site on pBSoriT. We are not able to demonstrate a stable protein DNA interaction using linear DNA fragments containing oriT and helicase I in a gel-retardation assay (17, data not shown). However, this latter result may be due to the use of linear DNA molecules instead of a supercoiled substrate (see below). Alternatively, the nicked DNA species may compete with the supercoiled substrate for binding of helicase I. These two possibilities are not mutually exclusive and both may contrib- ute to prevent quantitative conversion of the supercoiled substrate to a nicked DNA molecule. Location of the Nick Site within the F oriT Region-The experiment presented in Fig. 1 strongly suggests that the nick introduced into pBSoriT DNA by helicase I is located within the F oriT region. To further define the site of the nick, and to demonstrate the existence of a single, unique nick, the experiment diagrammed in Fig. 4A was performed. pBSoriT DNA that had been nicked by helicase I was isolated on an agarose gel, cut with a restriction endonuclease to produce a nicked, linear molecule, and the individual DNA strands resolved on an alkaline agarose gel. If the nick introduced by helicase I is both site- and strand-specific this strategy is expected to resolve three bands of ssDNA; a linear full length strand and two shorter molecules resulting from the nicked strand. The expected products after restriction with XmnI are DNA fragments of about 3490 nucleotides (nt), 2360 and 1130 nt in length if the nick site in oriT is at the position found in vivo (22). As can be seen in the alkaline agarose gel stained with ethidium bromide (Fig. -Linear pBSoriT -Linear DES ( ) Alkaline Agarose Gel Electrophoresis-pBSoriT DNA nicked by helicase I in a standard reaction mixture (increased to 80 pl) was isolated on 0.8% agarose gels run in Tris/acetate/EDTA buffer and purified free of agarose using Geneclean (Bio-101, Inc.). Nicked DNA was incubated with the indicated restriction enzyme for 30-60 min at 37 "C, phenol/CHCl:,-extracted, precipitated with ethanol, and resuspended in 25 pl of 100 mM NaOH, 1 mM EDTA, 15% glycerol, 0.5% loading dyes. Alkaline agarose gels (1.0%) were cast in 50 mM NaCl, 1 mM EDTA, 100 mM NaOH as described (20); the gel running buffer was 100 mM NaOH, 1 mM EDTA. Electrophoresis was at 2 V/ cm for 14-18 h at 4 "C. Gels were neutralized in 0.5 M Tris-HCI (pH 8) followed by soaking in TBE. DNA was visualized by staining with ethidium bromide (1 pg/ml) in TBE. For Southern blotting the gel FIG. 1. Helicase I nicks F oriT-contai, t.,, I';', t i ' '5. Nicking reactions were as described under "Exper,.:,. ' ( . , u r d ' using either 330 ng of supercoiled pBSoriT DNP ' 221 ng of supercoiled pBS DNA (lanes 7-9). In lunr. ' ' ! I mM) was substituted for 1 mM MgCI, in the reactiot . . c .. l , . f Olphoresis was on an 0.8% agarose gel run in the abrc .I a i i I . & 8 1 bromide. The DNA was subsequently visualized by .:t .'. ' --.: 0.5 pg/ml ethidium bromide. Lanes 1,4, and 7,O ng of h 5 ' .: .. . , m v s 2,5, and 8, 100 ng of helicase I; lunes 3, 6, and 9, 400 I I ~ o I . . i ,ase I; lane 10, 330 ng of pBSoriT DNA + 321 ng of pBS LNs ai to completion with XbuI to yield linear DNA molecules. The posiions of supercoiled pBSoriT (sc) and nicked pBSoriT (nc) have been indicated. Helicase I Nicking at F oriT 16234 scribed above included an incubation in the presence of SDS and proteinase K after incubation with helicase I. This step was incorporated to trap nicked complexes generated by hel- icase I that might be analogous to the relaxosomes observed at the plasmid RP4 oriT (24, 25). 1 2 3 4 5 6 7 6 9 1 0 1 2 3 4 5 6 7 6 9 1 0 nc -L sc + “I C C C C 1 2 3 4 5 6 7 0 FIG. 2. Protein concentration dependence of the nicking reaction. Nicking reactions were as described under “Experimental Procedures” using 330 ng of pBSoriT DNA. Electrophoresis was on a 1.0% agarose gel run in the presence of 0.5 pg/ml ethidium bromide. Lanes 1-7 contained 0,25,50,100,200,400, and 800 ng of helicase I, respectively. Lane 8 contains marker pBSoriT DNA converted to linear and nicked species using HincII in the presence of ethidium bromide (33). The positions of nicked circular DNA (nc), linear DNA ( l ) , and supercoiled DNA (sc) have been indicated. 4B, lane 4), the products observed correspond to those expected suggesting that the 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 FIG. 3. Helicase I does not nick relaxed covalently closed circular DNA. Nicking reaction mixtures were as described under “Experimental Procedures” using either 260 ng of supercoiled pBSoriT DNA (lanes 1-4) or 250 ng of relaxed covalently closed pBSoriT DNA (lanes 5-8). Electrophoresis was on a 1.0% agarose gel run in the presence of 0.5 pg/ml ethidium bromide. Lanes 1 and 5, 0 ng of helicase I; lanes 2 and 6,400 ng of helicase I; lanes 3 and 7,800 ng of helicase I; lanes 4 and 8, 1.6 pg of helicase I; lane 9, 260 ng of supercoiled pBSoriT DNA + 250 ng of relaxed covalently closed pBSoriT DNA and 800 ng of helicase I; lane 10, pBSoriT DNA cut with XbaI in a partial reaction to provide markers. The positions of nicked circular pBSoriT (nc), supercoiled pBSoriT (sc), linear pBSoriT ( l ) , and relaxed covalently closed circular pBSoriT (ccc) have been indicated. FIG. 2. Protein concentration dependence of the nicking reaction. Nicking reactions were as described under “Experimental Procedures” using 330 ng of pBSoriT DNA. Electrophoresis was on a 1.0% agarose gel run in the presence of 0.5 pg/ml ethidium bromide. Lanes 1-7 contained 0,25,50,100,200,400, and 800 ng of helicase I, respectively. Lane 8 contains marker pBSoriT DNA converted to linear and nicked species using HincII in the presence of ethidium bromide (33). The positions of nicked circular DNA (nc), linear DNA ( l ) , and supercoiled DNA (sc) have been indicated. FIG. 2. Protein concentration dependence of the nicking reaction. Nicking reactions were as described under “Experimental Procedures” using 330 ng of pBSoriT DNA. Electrophoresis was on a 1.0% agarose gel run in the presence of 0.5 pg/ml ethidium bromide. Lanes 1-7 contained 0,25,50,100,200,400, and 800 ng of helicase I, respectively. Lane 8 contains marker pBSoriT DNA converted to linear and nicked species using HincII in the presence of ethidium bromide (33). The positions of nicked circular DNA (nc), linear DNA ( l ) , and supercoiled DNA (sc) have been indicated. Helicase I Nicking at F oriT 16235 Helicase I Ni 1 xmnr B kb - 2.95 - 2.51 - 1.92- 1.0 - 0.45 - 1 2 3 4 C kb - * 2.95- 2.51 - 1.92 - 4 1.0 - 1 1 2 3 FIG. 4. Alkaline agarose gel analysis of nicked pBSoriT. 1 2 3 4 5 6 7 0 This is due to the fact that the template strand is interrupted at this site forcing the DNA polymerase to cease synthesis. A strong stop was seen in all four lanes (Fig. 5, lanes 13-16) when primer 1 was utilized in reactions contain- ing nicked pBSoriT DNA as the template. The site of the strand interruption was on the same strand and in precisely the same position as the nick site located by a similar method on plasmids isolated from the cell (22). A control experiment was done using primer 2 to direct synthesis on the opposite strand through the region expected to contain the nick site. In this case there was no strong stop (Fig. 5, lanes 5-8) indicating no interruption of the DNA strand. We conclude that purified helicase I makes a site- and strand-specific nick in the F oriT region under our in vitro conditions, and in the absence of any additional proteins, at the same site nicked in vivo. nick. This is due to the fact that the template strand is interrupted at this site forcing the DNA polymerase to cease synthesis. A strong stop was seen in all four lanes (Fig. 5, lanes 13-16) when primer 1 was utilized in reactions contain- ing nicked pBSoriT DNA as the template. The site of the strand interruption was on the same strand and in precisely the same position as the nick site located by a similar method on plasmids isolated from the cell (22). A control experiment was done using primer 2 to direct synthesis on the opposite strand through the region expected to contain the nick site. In this case there was no strong stop (Fig. 5, lanes 5-8) indicating no interruption of the DNA strand. We conclude that purified helicase I makes a site- and strand-specific nick in the F oriT region under our in vitro conditions, and in the absence of any additional proteins, at the same site nicked in vivo. 1 2 3 4 5 6 7 0 When a different restriction endonuclease was used to produce the nicked, linear DNA the sizes of the two DNA fragments resulting from the nicked strand were altered as expected (Fig. 4C, lane 3). In this case, SulI was used to produce the nicked, linear DNA molecule, and, due to the small size of one of the predicted DNA strands, the DNA was transferred to a nylon membrane and probed with oriT DNA sequences to locate the three DNA fragments. The expected products after restriction with Sal1 are DNA fragments of 3490, 3100, and 390 nt in length. The autoradiograph shown in Fig. 4C (lane 3 ) demonstrates three DNA fragments of the expected sizes. As a control, nicked DNA cut with XmnI (Fig. 4c, lane 2) was included in this experiment and, as expected, DNA fragments of 3490,2360, and 1130 nt in length were observed. Together these data suggest that the nick introduced into pBSoriT is unique and at, or very near, the site that is nicked in vivo to initiate DNA strand transfer during bacterial con- jugation. position of the nick introduced in vitro is at or near the position of the nick site identified in vivo. These data also indicate the existence of a single, unique nick site. When a different restriction endonuclease was used to produce the nicked, linear DNA the sizes of the two DNA fragments resulting from the nicked strand were altered as expected (Fig. 4C, lane 3). In this case, SulI was used to produce the nicked, linear DNA molecule, and, due to the small size of one of the predicted DNA strands, the DNA was transferred to a nylon membrane and probed with oriT DNA sequences to locate the three DNA fragments. The expected products after restriction with Sal1 are DNA fragments of 3490, 3100, and 390 nt in length. The autoradiograph shown in Fig. 4C (lane 3 ) demonstrates three DNA fragments of the expected sizes. As a control, nicked DNA cut with XmnI (Fig. 4c, lane 2) was included in this experiment and, as expected, DNA fragments of 3490,2360, and 1130 nt in length were observed. Together these data suggest that the nick introduced into pBSoriT is unique and at, or very near, the site that is nicked in vivo to initiate DNA strand transfer during bacterial con- jugation. nick. 1 2 3 4 5 6 7 0 Helicase I Nicking at F oriT 1 xmnr B kb - 2.95 - 2.51 - 1.92- 1.0 - 0.45 - 1 2 3 4 C kb - * 2.95- 2.51 - 1.92 - 4 1.0 - 1 1 2 3 FIG. 4. Alkaline agarose gel analysis of nicked pBSoriT. A, strategy for determining the site of the nick using alkaline agarose gels. See text for details. B, alkaline agarose gel stained with ethidium bromide. The markers were pBS DNA digested to completion with XbaI ( l a n e 1 ) and pBS DNA digested to completion with PuuI (lane 2) or PuuII (lane 3). Lane 4, approximately 1 pg of nicked pBSoriT DNA cut with XmnI. C, an autoradiograph of an alkaline gel trans- ferred to a nylon membrane and probed with a 540-bp oriT DNA fragment is presented. Lane I, nicked pBSoriT DNA; lune 2, nicked pBSoriT DNA cut with XmnI; lane 3, nicked pBSoriT DNA cut with SalI. The markers (not shown) were the same as in B. 1 2 3 4 A nick G A T C G A T C " ". - 5 6 7 8 9101112 1 FIG. 5. The site nicked by helicase I in that observed in vivo. Nicked pBSoriT described under "Experimental Procedures." A relative positions and polarities of the primer site and strand specificity of the nick introdu autoradiograph of DNA sequencing reactions of pBSoriT DNA (lunes 1-4, 9-12) or nicked 5-8,13-16) and either primer 2 (lanes 1-8) or The strong stop in lanes 13-16 indicates th sequence of this region has been indicated on 1 2 3 4 A nick G A T C G A T C " ". - 5 6 7 8 9101112 G A T C 13 14 15 16 1 xmnr C kb - 2.95- 2.51 - 1.92 - 1.0 - 1 1 2 3 B kb - 2.95 - 2.51 - 1.92- 1.0 - 0.45 - 1 2 3 4 * 4 C B 1 2 3 13 14 15 16 9101112 1 2 3 4 1 2 3 4 5 6 7 8 FIG. 4. Alkaline agarose gel analysis of nicked pBSoriT. A, strategy for determining the site of the nick using alkaline agarose gels. See text for details. B, alkaline agarose gel stained with ethidium bromide. 1 2 3 4 5 6 7 0 A, strategy for determining the site of the nick using alkaline agarose gels. See text for details. B, alkaline agarose gel stained with ethidium bromide. The markers were pBS DNA digested to completion with XbaI ( l a n e 1 ) and pBS DNA digested to completion with PuuI (lane 2) or PuuII (lane 3). Lane 4, approximately 1 pg of nicked pBSoriT DNA cut with XmnI. C, an autoradiograph of an alkaline gel trans- ferred to a nylon membrane and probed with a 540-bp oriT DNA fragment is presented. Lane I, nicked pBSoriT DNA; lune 2, nicked pBSoriT DNA cut with XmnI; lane 3, nicked pBSoriT DNA cut with SalI. The markers (not shown) were the same as in B. Helicase I Nicking at F oriT 16235 1 xmnr B kb - 2.95 - 2.51 - 1.92- 1.0 - 0.45 - 1 2 3 4 C kb - * 2.95- 2.51 - 1.92 - 4 1.0 - 1 1 2 3 FIG. 4. Alkaline agarose gel analysis of nicked pBSoriT. A, strategy for determining the site of the nick using alkaline agarose 1 2 3 4 A nick G A T C G A T C " ". - 5 6 7 8 9101112 G A T C 13 14 15 16 FIG 5 The sitenicked by helicase I in vitrois the same as king at F oriT 16235 1 2 3 4 A nick G A T C G A T C " ". - 5 6 7 8 9101112 G A T C 13 14 15 16 FIG. 5. The site nicked by helicase I in vitro is the same as that observed in vivo. Nicked pBSoriT DNA was isolated as described under "Experimental Procedures." A, diagram showing the relative positions and polarities of the primers used to determine the site and strand specificity of the nick introduced by helicase I. B, an autoradiograph of DNA sequencing reactions performed using 1-2 pg of pBSoriT DNA (lunes 1-4, 9-12) or nicked pBSoriT DNA (lanes 5-8,13-16) and either primer 2 (lanes 1-8) or primer 1 (lanes 9-16). The strong stop in lanes 13-16 indicates the site of the nick; the sequence of this region has been indicated on the right. The solid bar between lanes 8 and 9 denotes the region on the opposite strand complementary to the site nicked by helicase I. 1 2 3 4 5 6 7 0 The markers were pBS DNA digested to completion with XbaI ( l a n e 1 ) and pBS DNA digested to completion with PuuI (lane 2) or PuuII (lane 3). Lane 4, approximately 1 pg of nicked pBSoriT DNA cut with XmnI. C, an autoradiograph of an alkaline gel trans- ferred to a nylon membrane and probed with a 540-bp oriT DNA fragment is presented. Lane I, nicked pBSoriT DNA; lune 2, nicked pBSoriT DNA cut with XmnI; lane 3, nicked pBSoriT DNA cut with SalI. The markers (not shown) were the same as in B. FIG. 5. The site nicked by helicase I in vitro is the same as that observed in vivo. Nicked pBSoriT DNA was isolated as described under "Experimental Procedures." A, diagram showing the relative positions and polarities of the primers used to determine the site and strand specificity of the nick introduced by helicase I. B, an autoradiograph of DNA sequencing reactions performed using 1-2 pg of pBSoriT DNA (lunes 1-4, 9-12) or nicked pBSoriT DNA (lanes 5-8,13-16) and either primer 2 (lanes 1-8) or primer 1 (lanes 9-16). The strong stop in lanes 13-16 indicates the site of the nick; the sequence of this region has been indicated on the right. The solid bar between lanes 8 and 9 denotes the region on the opposite strand complementary to the site nicked by helicase I. FIG. 5. The site nicked by helicase I in vitro is the same as that observed in vivo. Nicked pBSoriT DNA was isolated as described under "Experimental Procedures." A, diagram showing the relative positions and polarities of the primers used to determine the site and strand specificity of the nick introduced by helicase I. B, an autoradiograph of DNA sequencing reactions performed using 1-2 pg of pBSoriT DNA (lunes 1-4, 9-12) or nicked pBSoriT DNA (lanes 5-8,13-16) and either primer 2 (lanes 1-8) or primer 1 (lanes 9-16). The strong stop in lanes 13-16 indicates the site of the nick; the sequence of this region has been indicated on the right. The solid bar between lanes 8 and 9 denotes the region on the opposite strand complementary to the site nicked by helicase I. position of the nick introduced in vitro is at or near the position of the nick site identified in vivo. These data also indicate the existence of a single, unique nick site. DISCUSSION The formation of these nicked molecules was dependent on specific tra gene products. The t r a y mutants utilized in these studies were all deletion mutants which contained deletions of more than just the tray gene. In fact, no point mutants in tray have ever been isolated. In these studies deletion of traJ also abolished the nicking reaction. Since the TraJ protein is thought to be a positive regulator of the tray2 operon, these data were interpreted as an effect on transcription of the tray2 operon resulting in little or no production of Tray protein. Perhaps the tray deletion mutants used in this study have a polar effect on the transcription of t r d . Although this seems unlikely in view of the discovery of a promoter associated with the tral gene (30), it remains a formal possibility. Nevertheless, the in vitro nicking of F oriT does not require Tray protein. Additional roles for Tray protein in the metabolism of F DNA during bacterial conjugation can be envisaged and are likely. How- ever, at this time the role of Tray protein remains unclear. g y y The helicase I nicking reaction has been shown to be protein concentration dependent, but the reaction fails to go to com- pletion under the conditions we have used. At a 32-fold molar excess of helicase I monomers over DNA molecules 50-70% of the plasmid DNA was nicked by helicase I (Fig. 2). Longer incubations or the inclusion of Tray protein in the reaction mixture did not significantly alter this result: It is possible that helicase I is active as a multimer and that a fraction of the purified enzyme is not active. Together these two factors could account for the incomplete reaction. Helicase I has been reported to be active as an aggregate of monomers in duplex DNA unwinding reactions (4). To determine whether this is true of the nicking activity will require further investigation. Alternatively, helicase I may have a low affinity for its binding site on oriT or it may have a high affinity for the nicked DNA species. Nicked DNA-helicase I complexes have been observed as smears migrating more slowly than nicked DNA on agarose gels when SDS/proteinase K treatment is omitted (data not shown). DISCUSSION Interestingly, no Tra- mutants in oriT were isolated which directly involve the nucleotides on either side of the phospho- diester bond cleaved by helicase I. This may be due to the particular methods used to generate or isolate oriT mutants. There have been no efforts as yet to saturate this region in oriT with mutations, and it is likely that additional mutants will be found which affect either binding of helicase I, nicking by helicase I, or both. y g y ( ) The F oriT nicking reaction catalyzed by helicase I requires a superhelical DNA substrate and M F . Presumably the M F interacts with the DNA substrate to make the relevant phos- phodiester bond in the DNA backbone more susceptible to scission by helicase I. The requirement for superhelical DNA is consistent with our previous observation that helicase I fails to interact with linear DNA fragments as determined using gel-retardation assays (17).3 Thus when the supercoiled substrate is replaced with covalently closed relaxed DNA there is no conversion to a nicked DNA species. In view of the fact that helicase I fails to retard DNA fragments con- taining oriT in a gel-shift assay, we suggest that helicase I does not recognize its binding site in oriT except when present in a supercoiled conformation. This result may suggest a reason for the apparent requirement for E. coli gyrase in bacterial conjugation (26, 27). Additional studies will be re- quired to fully appreciate the role of supercoiled DNA in the nicking reaction catalyzed by helicase I. y Perhaps the most surprising result is the fact that this reaction does not depend on Tray protein. It has long been assumed that Tray protein, in conjunction with a second protein, would be responsible for site- and strand-specific nicking at oriT. Indeed, purified Tray protein has been shown to bind specifically to oriT (17) at a site just upstream of, but not including, the nick site? The notion of a TraYZ(1) endo- nuclease is based largely on early work suggesting that the product of the t r a y gene was required for oriT nicking (16). These experiments utilized a X phage containing oriT and Flac plasmids which provided the Tra functions in trans. A fraction of the packaged X phage contained nicked molecules which correspond to a nick in oriT. B. S. Morton and S. W. Matson, unpublished observations. ' Tsai, M.-M., Fu, Y.-H. F., and Deonier, R. C. (1990) J. Bucteriol. 172,4603-4609. E. E. Lahue, B. S. Morton, and S. W. Matson, unpublished observations. DISCUSSION Purified helicase I catalyzes the site- and strand-specific nicking of the F plasmid at oriT, a reaction required prior to unwinding, to initiate DNA strand transfer during bacterial conjugation. Thus this enzyme is likely to have two roles, as previously suggested (19), in conjugal DNA transfer. First, the enzyme nicks the F plasmid (or the chromosome in Hfr strains) in a site- and strand-specific manner at F oriT. To define the site nicked by helicase I at single nucleotide resolution, nicked DNA was isolated and subjected to DNA sequencing reactions using the dideoxynucleotide chain ter- mination method (Fig. 5). When a primer that hybridizes to the strand that has been nicked is utilized in these reactions a strong stop is expected in all four lanes at the site of the Helicase I Nicking at F oriT 16236 Participation of helicase I in this reaction has been inferred (19) but not previously demonstrated biochemically. Second, helicase I is thought to unwind the F plasmid from the nick site to generate the ssDNA transferred into the recipient bacterium. Helicase I has been characterized as a processive helicase I (4-6) that is likely to be capable of catalyzing this unwinding reaction. However, it has been suggested that other helicases found in E. coli may also be capable of catalyzing this unwinding reaction in tral mutants that lack helicase activity but retain nicking activity (19). Participation of helicase I in this reaction has been inferred (19) but not previously demonstrated biochemically. Second, helicase I is thought to unwind the F plasmid from the nick site to generate the ssDNA transferred into the recipient bacterium. Helicase I has been characterized as a processive helicase I (4-6) that is likely to be capable of catalyzing this unwinding reaction. However, it has been suggested that other helicases found in E. coli may also be capable of catalyzing this unwinding reaction in tral mutants that lack helicase activity but retain nicking activity (19). at precisely the same site shown to be nicked in vivo. Cis- dominant Tra- mutations in oriT have been isolated (28, 29) and involve nucleotide changes at positions 4 bases and 9 bases upstream of the nick site we have determined. These may be part of a recognition site for helicase I binding to oriT. E. E. Lahue, B. S. Morton, and S. W. Matson, manuscript in preparation. Acknowledgments-We would like to thank Dr. Dan Bean for critical reading of this manuscript and Susan Whitfield for prepara- tion of the figures. In addition, we would like to thank Dr. Erich REFERENCES 18. Manning, P. A., Kusecek, B., Morelli, G., Fisseau, C., and Acht- man, M. (1982) J. Bacteriol. 150, 76-88 1. Abdel-Monem, M., Taucher-Scholz, G., and Klinkert, M. Q. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,4659-4663 19. Traxler, B. A., and Minkley, E. G., Jr. (1988) J. Mol. Biol. 204, , ( ) , 205-209 2. ( ) Willetts, N. S., and Maule, J. (1979) Mol. Gen. Genet. 169, 325- 336 20. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular C1oning:A Laboratory Manual, Cold Spring Harbor Laboratory, pp. 91-91, Cold Spring Harbor, NY 3. Abdel-Monem, M., and Hoffmann-Berling, H. (1976) Eur. J. Biochem. 65,431-440 4. Abdel-Monem, M., Durwald, H., and Hoffmann-Berling, H. (1976) Eur. J. Bwchem. 65, 441-449 pp , p g , 21. Johnson, D. A., Everett, R., and Willetts, N. S. (1981) J. Mol. h l d i ( ) BWl. 153, 187-202 5. ( ) Abdel-Monem, M., Lauppe, H.-F., Kartenbeck, J., Durwald, H., and Hoffmann-Berling, H. (1977) J. Mol. Biol. 110,667-685 22. Thompson, T. L., Centola, M. B., and Deonier, R. C. (1989) J. Mol. BWl. 207,505-512 23. Tabor, S., and Richardson, C. C. (1987) Proc. Natl. Acad. Sci. U. , S. A. 84,4767-4771 6. Lahue, E. E., and Matson, S. W. (1988) J. Biol. Chem. 263, 3208-3215 24. Pansegrau, W., Ziegelin, G., and Lanka, E. (1990) J. Bwl. Chem. 265,10637-10644 7. Benz, I., and Muller, H. (1990) Eur. J. Biochem. 189, 267-276 8. Wessel, R., Muller, H., and Hoffmann-Berling, H. (1990) Eur. J. Biochem. 189, 277-285 25. Pansegrau, W., Balzer, D., Kruft, V., Lurz, R., and Lanka, E. , 25. Pansegrau, W., Balzer, D., Kruft, V., Lurz, R., and La (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6555-6559 9. Kuhn, B., Abdel-Monem, M., Krell, H., and Hoffmann-Berling, H. (1979) J. Biol. Chem. 254,11343-11350 26. Barbour, S. D. (1967) J. Mol. Biol. 28,373-376 ( ) , , ( ) , 27. Bouck, N., and Adelberg, E. A. (1970) J. Bacteriol. 102,688-701 ( ) 27. Bouck, N., and Adelberg, E. A. (1970) J. Bacteriol. 10. ( ) Bradshaw, H. D., Jr., Traxler, B. A., Minkley, E. G., Jr., Nester, E. W., and Gordon, M. P. (1990) J. Bacteriol. 172, 4127-4131 28. Everett, R., and Willetts, N. (1982) EMBO J. 1, 747-753 29. Thompson, R., Taylor, L., Kelly, K., Everett, R., and Willetts, N. (1984) EMBO J. 3, 1175-1180 11. Traxler, B. Helicase I Nicking at F oriT 16237 Lanka, Dr. Richard Deonier, and Jim George for stimulating discus- sions. Lanka, Dr. Richard Deonier, and Jim George for stimulating discus- sions. 15. Willetts, N. S., and Wilkins, B. (1984) Microbiol. Reu. 48, 21-41 , , , ( ) , 16. Everett, R., and Willetts, N. (1980) J. Mol. Biol. 136, 129-150 17. Lahue, E. E., and Matson, S. W. (1990) J. Bacterwl. 172, 1385- 1391 DISCUSSION This suggests that the enzyme has some affinity for the nicked DNA species and this molecule may compete with the supercoiled substrate for initial binding of excess protein. A critical component may also still be missing in the in vitro reaction. Binding sites for integration host factor have been defined within the oriT region on F.5 Perhaps binding of integration host factor to oriT is required for quantitative conversion to the nicked species by helicase 1. At present we are unable to distinguish among these possibilities. It should be noted that reconstitution of the relaxosome and subsequent nicking at oriT in the plasmid RP4 system results in approx- imately 30% of the substrate converted to a nicked species (25). Thus a low nicking efficiency may be an inherent prop- erty of the in vitro reconstitution of these reactions. , y p The F oriT nicking reaction seems remarkably simple com- pared to the in vitro nicking reaction demonstrated using the RP4 plasmid (24, 25). Plasmid RP4 requires the products of two genes in order to form a relaxosome capable of introducing a specific nick in the RP4 oriT (25). In addition, it has been shown that one of these proteins, the product of the RP4 tra1 gene, remains covalently bound to the 5' end of the nicked DNA strand (24, 25). At this point the nature of the nick introduced by helicase I in F oriT is not clear. The data presented here do not allow us to conclude whether or not helicase I remains covalently bound to either the 3' end or the 5' end of the nicked DNA strand. Previous data (31) suggest the formation of a relaxosome, similar to that seen in the RP4 system, on the F plasmid. This suggests the possibil- ity of a protein-DNA complex at the nick site in F oriT. Additional work will be required to determine whether or not such a complex exists, and if so, where helicase I is bound. y We have demonstrated that purified helicase I nicks oriT Acknowledgments-We would like to thank Dr. Dan Bean for critical reading of this manuscript and Susan Whitfield for prepara- tion of the figures. In addition, we would like to thank Dr. Erich Helicase I Nicking at F oriT REFERENCES A., and Minkley, E. G., Jr (1987) J. Bacteriol. 169, 3251-3259 ( ) 30. Mullineaux, P., and Willetts, N. (1985) in Plasmids in Bacteria (Helinski, D. R., Cohen, S. N., Clewell, D. B., Jackson, D. A., and Hollaender, A., eds) pp. 605-614, Plenum Publishing C o p , New York 12. Kingsman, A., and Willetts, N. (1978) J. Mol. Biol. 122, 287- 300 13. Willetts, N., and Skurray, R. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp. 1110-1133, American Society for Microbiology, Wash. D. C. 31. Kline, B. C., and Helinski, D. R. (1971) Biochemistry 10,4975- 4980 14. , y gy, Willetts, N., and Skurray, R. (1980) Annu. Rev. Genet. 14, 41- 76 14. , y gy, Willetts, N., and Skurray, R. (1980) Annu. Rev. Genet. 14, 41- 76 32. Shortle, D., and Botstein, D. (1983) Methods Enzymol. 100,457- 468 76
https://openalex.org/W3005697287	https://ejournal.upi.edu/index.php/IJAL/article/download/23211/11654	English	null	Investigating metacognitive knowledge in reading comprehension: The case of Indonesian undergraduate students	Indonesian Journal of Applied Linguistics	2,020	cc-by-sa	6,315	* Corresponding Author Email: hamiddin@unisma.ac.id Hamiddin1 and Ali Saukah2 g p , y f g , g, y Malang, East Java 65145, Indonesia 1,2Department of English, Faculty of Letters, Universitas Negeri Malang, Jl. Semarang No. 5 Malang, East Java 65145, Indonesia Malang, East Java 65145, Indonesia 1,2Department of English, Faculty of Letters, Universitas Negeri Malang, Jl. Semarang No. 5 Malang, East Java 65145, Indonesia ABSTRACT Myriad studies have conveyed that metacognition is a key to successful reading comprehension in language classes. However, these studies focus on metacognitive strategies, metacognitive awareness, and metacognitive skills in ESL reading contexts. Anchored by the lack of empirical works on the EFL reading situation, this study investigated the metacognitive knowledge of Indonesian undergraduate students in their reading classes. Data were obtained through in-depth interviews with four successful and four less successful EFL students. The results of this study portrayed that successful readers encompass more metacognitive knowledge, awareness, motivation, and behavior if compared to the less successful readers. It is also evidence that students with good cognition, habit, and attitude in reading activities are more successful in their EFL reading, and their cognition, habit, and attitude serve as essential elements in constructing their metacognitive knowledge. Suggestions are also discussed at the end of this paper. Keywords: English as a foreign language student; metacognitive knowledge; reading comprehension comprehension First Received: 24 October 2019 Revised: 20 November 2019 Accepted: 7 December 2019 Final Proof Received: 21 January 2020 Published: 31 January 2020 How to cite (in APA style): Hamiddin, H. & Saukah, A. (2020). Investigating metacognitive knowledge in reading comprehension: The case of Indonesian undergraduate students. Indonesian Journal of Applied Linguistics, 9, 608-615. doi: 10.17509/ijal.v9i3.23211 How to cite (in APA style): Hamiddin, H. & Saukah, A. (2020). Investigating metacognitive knowledge in reading comprehension: The case of Indonesian undergraduate students. Indonesian Journal of Applied Linguistics, 9, 608-615. doi: 10.17509/ijal.v9i3.23211 Heriyawati, Saukah, & Widiati, 2018). These studies uncovered that reading is a central skill in promoting students’ literacy, competence, and academic achievement. It is also an interactive and cognitive process because readers are active (Akkakoson, 2012) in constructing meaning during their reading activities. To construct the meaning, readers bring different kinds of knowledge to interpret and evaluate the meaning of texts. Understanding the meaning of the text will occur if the readers decode the texts based on available cognitive resources (Furnes & Norman, 2015). Indonesian Journal of Applied Linguistics, 9(3), January 2020 Indonesian Journal of Applied Linguistics, 9(3), January 2020 fewer cognitive resources will lead the readers to use other knowledge and strategies such as skimming, skipping, and scanning in comprehending text. Besides, readers are the potential to encounter decoding and cognitive barriers if they do not fully understand texts. This problem can then be solved through sufficient metacognitive knowledge. It is a key factor for successful monitoring and control of reading (Kolić- Vehovec, Zubkovic, & Pahljina-Reinic, 2014). Planning how to approach the text, monitoring comprehension, and evaluating the progress of completing a task are examples of the metacognitive action of the readers (Livingston, 2003). Metacognition helps readers control their cognitive process in acquiring information and the meaning of a text. associated with metacognitive beliefs, metacognitive awareness, metacognitive experiences, metacognitive knowledge, feeling of knowing, the judgment of learning, the theory of mind, metamemory, metacognitive skills, high-order skills, comprehension monitoring, learning strategies, and self-regulation (Wang, 2019). g Given its decent contribution in reading skills enactment, studies on metacognition partly focus on metacognitive awareness of reading strategies (Yüksel & Yüksel, 2012), metacognitive strategies and critical thinking (Altıok, Başer, & Yükseltürk, 2019), metacognitive awareness and the teaching of reading (Zhussupova & Kazbekova, 2016), metacognitive process and intelligence (Taub et al., 2019), and metacognitive awareness of doctoral social science student performance (Yang & Bai, 2019). This evidences that scholars have not considered another essential domain in metacognition, one of which is metacognitive knowledge. g Meanwhile, Oz (2005) considers metacognition as a complex process of knowledge about the cognition domain and its regulation, which consists of mental activities related to thinking, knowing, and remembering. It can be stated that in the EFL reading context, metacognition is a complex process in controlling and regulating cognitive experiences and awareness of the readers and how to activate cognitive knowledge, how to relate the prior knowledge with current texts, and how to solve the problem during reading activities. Metacognitive as knowledge is classified into three main categories: the learner, the learning task, and the process of learning (Wenden, 1998). g g In the context of English as a foreign language teaching in Indonesia, for instance, research on metacognitive knowledge of undergraduate students concerning their reading comprehension seems sparse from the empirical investigation. In fact, different cognitive enterprises (Aryadoust, 2019) and different language settings, as well as culture (Peets, Yim, & Bialystok, 2019), may influence students’ metacognitive knowledge in practice. Indonesian Journal of Applied Linguistics, 9(3), January 2020 To fill this void, this study, therefore, investigated the metacognitive knowledge of undergraduate students of an English department in an Indonesian private university. The purposes were twofold: 1) exploring the metacognitive knowledge of English students in EFL reading and 2) documenting how they demonstrate knowledge, learning tasks, processes, and the use of reading strategies in EFL reading. Metacognitive involves three categories, such as person, task, and strategy (Flavell, 1979). Person category means one’s belief about the intraindividual category, inter-individual category, and universal category. In the Task category, learners’ awareness of characteristics of the specific task is constructed, and how to manage and understand it (Oz, 2005). This category is categorized on task purpose, task type, and task demand (Flavell, 1979). The strategy category, additionally, is awareness of learners in applying metacognitive strategies during attending to a task. The most appropriate strategies for learners will promote successful completion of a task (Oz, 2005), and it includes learners’ knowledge about cognitive and metacognitive strategies, when and where it is appropriate to use such strategies (Livingston, 2003). In this part, Wenden (1998, in Hauck, 2005) adds that knowledge about strategy or strategic knowledge depends on whether the focus is on the learner, the learning task, or the process of learning. INTRODUCTION Heriyawati, Saukah, & Widiati, 2018). These studies uncovered that reading is a central skill in promoting students’ literacy, competence, and academic achievement. It is also an interactive and cognitive process because readers are active (Akkakoson, 2012) in constructing meaning during their reading activities. To construct the meaning, readers bring different kinds of knowledge to interpret and evaluate the meaning of texts. Understanding the meaning of the text will occur if the readers decode the texts based on available cognitive resources (Furnes & Norman, 2015). Scholars have reached a consensus that cognition is a thinking process, and metacognitive knowledge, specifically, refers to how a person realizes this thinking process. In this study, for the sake of a more contextual focus, we use both cognition and cognitive knowledge interchangeably since these two terms encompass shared orientation with different theoretical meanings (Marulis, Baker, & Whitebread, 2020; Moritz & Lysaker, 2018). In recent years, there has been an increasing interest in EFL reading research such as in China (Ke & Chan, 2017; Yu & Reichle, 2017; Zhao et al., 2019), Korea (Kim, Liu, & Cao, 2017; Pae, Kim, Mano, & Wang, 2019), Japan (Takeuchi et al., 2018), Iran (Sadeghi, Khezrlou, & Modirkhameneh, 2017), and, assuredly, Indonesia (Aditomo & Hasugian, 2018; Two aspects of reading, such as decoding and cognitive resources, lead to proficient reading competence and understanding the meaning of the texts. If the decoding process does not work in reading a text, the readers load more cognitive resources to read the words correctly (Furnes & Norman, 2015). However, 608 Indonesian Journal of Applied Linguistics, 9(3), January 2020 Indonesian Journal of Applied Linguistics, 9(3), January 2020 METHOD G d d Geared under an exploratory research paradigm, this study recruited four successful and four less successful EFL students enrolling in an English department of a private university in Malang, East Java, Indonesia. As one of the authors of this study is the reading lecturer, the successful and less successful criteria were mainly based on the students’ reading scores; A for successful and C for less successful students. As can be seen in Table 1, the successful students are coded as A, B, C, and D, while the less successful students are coded as E, F, G, and H (see Table 2). Several studies have suggested that students’ metacognitive knowledge contributes to their successful language learning (Chevalier, Parrila, Ritchie, & Deacon, 2017; Zhang, 2018). Furthermore, Zhang (2018) argued that students’ metacognition or metacognitive knowledge serves as an essential pedagogical inquiry in ESL/EFL and applied linguistics contexts. Metacognitive knowledge also enhances students’ competence and learning autonomy since they can monitor and evaluate their own learning goals (Schiff, Ben-Shushan, & Ben-Atzi, 2017). It is also They were invited for an hour in-depth interview using semi-structured questions on metacognitive knowledge (person, task, and strategy). Prior to doing it, we sent a consent form to the participants. They all agreed to participate in this study. In the interview process, they were inquired about their perceptions of reading activity, such as motivation to read, self- efficacy, emotion, and attitude. Copyright © 2020, authors, e-ISSN: 2502-6747, p-ISSN: 2301-9468 609 Indonesian Journal of Applied Linguistics, 9(3), January 2020 Table 1. Successful students’ demographic data No Name (pseudonym) Gender Semester English learning experiences Reading Score 1 A Male 3 8 years A 2 B Female 3 8 years A 3 C Female 3 8 years A 4 D Female 3 8 years A Table 2. Less Successful Students’ Demographic Data No Name (pseudonym) Gender Semester English learning experiences Reading Score 1 E Male 3 8 years C 2 F Female 3 8 years C 3 G Female 3 8 years C 4 H Female 3 8 years C Table 2. Less Successful Students’ Demographic Data The interview was conducted using participants’ second language, immediately after the participants completed reading English and Indonesian texts. Each participant was asked to read the texts prior to the interview. The process of the interview, which lasted in an hour, was phone-recorded and transcribed in the verbatim model. Person category In the person category aspect, successful readers have sufficient metacognitive knowledge as EFL readers. They are confident and frequently evaluate their reading ability. It is depicted from the following excerpts: Excerpt 7: ”I read my roommate’s English newspaper and books when I did not understand the content, and I discussed it with my roommate to get my understanding.” (Student C). Excerpt 1: ”I really understand the text in which the text related to what I have read.” (Student A) Excerpt 8: ”I read online texts such as sports news and English comic using Apps on my mobile phone.”(Student D). Furthermore, intraindividual, and interest are expressed differently by both successful and less successful readers. The successful readers consistently improve their reading ability by reading online English texts, books, novels, magazines, and comics. It is observed from the excerpts of Student A, B, C, and D. They shared that: Afterward, the data were analyzed based on Miles and Huberman’s (1994) data analysis, namely, data reduction, data display, and conclusion. Eventually, the interview transcripts were classified into person, task, and strategy domains. To achieve ease in the analysis process, the three areas were coded into P (person), T (task), and S (strategy). Excerpt 5: “I read English novels and always read English comics, and it is very helpful for me to improve my English.” (Student A). p “I read English novels and always read English comics, and it is very helpful for me to improve my English.” (Student A). Excerpt 6: ”I like to read English textbooks such as psychology and science, and I also help my roommate to translate their English tasks.” (Student B). METHOD G d d The data obtained from the interview were then translated into English, and to assure its reliability and validity, we confirm the translation results to a professional translator, who is also a teaching staff in our department. In the case of missing information during an interview session or having technical problems with the recorder, we re-interviewed the participants to ensure internal data consistency. means that they recall their prior knowledge (cognitive and metacognitive strategies) when reading new texts. Unlike the successful readers, less successful readers contended that they do not have self-confidence and good ability in reading. They also did not recall their prior knowledge in reading new texts. For instance, one of the less successful students contended that: the less successful students contended that: Excerpt 4: ”I do not have good reading ability. I am aware that my reading ability is low, so I need to read more.”(Student E). Excerpt 4: ”I do not have good reading ability. I am aware that my reading ability is low, so I need to read more.”(Student E). self-efficacy, Strategy category Successful readers demonstrate varied strategies in understanding texts. They are aware that reading strategy helps them complete the reading tasks, for example: texts. Student B, for instance, commented that: Excerpt 11: “I read the Indonesian texts because Indonesian text is easier than English in terms of vocabulary.” texts. Student B, for instance, commented that: Excerpt 11: “I read the Indonesian texts because Indonesian text is easier than English in terms of vocabulary.” Excerpt 18: “I commonly read the English text from the last paragraph to the middle and first paragraph because the last paragraph of the English text is the conclusion, and it describes the content.” (Student F). The Indonesian texts are very familiar to them as well as close to their daily spoken and written communication. The differences between the two texts, according to them, are on the vocabulary. It is noted that vocabulary is essential for successful EFL reading. Knowing more English vocabulary will help them understand the text. In reading the texts, Student B also underlines the essential words, phrases, or sentences. She confirms that the strategies help her understand the English texts easily. Students C, interestingly, has a different way of understanding the texts. She understands the texts by finding out the essential and informational words related to the whole texts. Besides, she also reads the introduction of the texts repeatedly until she obtains the important point from the text. In her opinion, the introduction section gives a brief description of the content of the texts. It is depicted in the interview process: The different task knowledge, both successful and less successful, is on task purpose and task demand. Both successful and less successful readers express a different opinion about the purpose of the reading. In addition to enriching vocabulary, successful readers consider that the purpose of reading is to gain information, understand texts, and find out the main ideas. These facts were depicted from the following excerpts: Excerpt 12: Excerpt 12: Excerpt 19: “I usually read from the first paragraph, like the introduction section. I read it many times. This part commonly gives clues on the main idea or content of the whole text.” (Student C). Excerpt 19: Excerpt 19: Excerpt 19: “I usually read from the first paragraph, like the introduction section. I read it many times. This part commonly gives clues on the main idea or content of the whole text.” (Student C). Excerpt 2: ”I understand the content of the text if the topic is related to my daily lives, such as love, psychology, science, and education.” (Student B). Meanwhile, less successful readers are less interested in improving their English reading ability. Some of them even never read English texts. For instance, Student E shared that: Excerpt 9: “I never read other English texts because the vocabularies are difficult. I just read the Indonesian novel.” Meanwhile, less successful readers are less interested in improving their English reading ability. Some of them even never read English texts. For instance, Student E shared that: Excerpt 9: “I never read other English texts because the vocabularies are difficult. I just read the Indonesian novel.” Excerpt 3: “It is easy for me to know the content from the reading passage if the topics presented related to my daily activities.”(Student C). Excerpt 3: “It is easy for me to know the content from the reading passage if the topics presented related to my daily activities.”(Student C). The three successful readers declared that they have self-confidence in reading any texts, especially texts related to their previous reading activities. It Copyright © 2020, authors, e-ISSN: 2502-6747, p-ISSN: 2301-9468 610 Indonesian Journal of Applied Linguistics, 9(3), January 2020 Furthermore, both successful and less successful readers are different in task demand and how to understand the English texts. They shared that: In other cases, one of them preferred to read online English texts such as quotes and short stories, for example Student G contended that: example, Student G contended that: Excerpt 10: “I like reading quotes on Instagram and English short stories on a website.” example, Student G contended that: Excerpt 10: “I like reading quotes on Instagram and English short stories on a website.” g y Excerpt 15: “When I read English texts, I read the conclusion of texts.”(Student B). Excerpt 16: “I read the text from the last paragraph, continue to the body of a paragraph.” (Student C). The successful readers tend to have self-efficacy, self-motivation, inter-individual characteristics, and interest in improving their English ability, especially reading. Mostly, less successful readers tend to be passive students, less motivated, fewer interests, and less inter-individual characteristics in improving their reading ability, only one of them who has self- motivation, self-efficacy, and interest in enhancing her reading ability. In addition, successful readers have strong knowledge about themselves in terms of self- efficacy, motivation, intra-individual, and interest, and less successful readers do not have. Interestingly, the less successful readers focus on vocabulary and translating the unknown words. It is evident from this excerpt: Excerpt 17: ”If I find the meaning of unfamiliar words in the texts, sometimes I write the words and find the definition in the dictionary using my mobile phone.” (Student F). In summary, the task category of both successful and less successful readers is different. The successful readers have sufficient task understanding rather than the less successful readers. However, successful readers sometimes focus on vocabulary and grammar. On the contrary, the less successful readers frequently list new words to help their understanding of the texts, and it indicates that they have insufficient task understanding. Task category g y In this section, both successful and less successful students perceive that Indonesian and English texts are different in terms of vocabulary. They did not mention the different structures, particularly among the two languages. When we disseminate the reading texts (Indonesian and English versions), both successful and less successful students favored reading the Indonesian texts. Student B, for instance, commented that: Excerpt 11: “I read the Indonesian texts because Indonesian text is easier than English in terms of vocabulary.” In this section, both successful and less successful students perceive that Indonesian and English texts are different in terms of vocabulary. They did not mention the different structures, particularly among the two languages. When we disseminate the reading texts (Indonesian and English versions), both successful and less successful students favored reading the Indonesian Excerpt 14: “I read to get information from the text.”(Students G) Strategy category ”The purpose of reading is to find the topic rather than knowing the meaning of every word. If I do not know some difficult words, I can guess it from the contexts.”(Student A) Excerpt 23: Excerpt 23: “I read all paragraphs in the text, and then I look for the meaning of the text. I don’t have specific strategies in reading, Sir.” (Student G). Excerpt 23: “I read all paragraphs in the text, and then I look for the meaning of the text. I don’t have specific strategies in reading, Sir.” (Student G). Excerpt 23: “I read all paragraphs in the text, and then I look for the meaning of the text. I don’t have specific strategies in reading, Sir.” (Student G). Other important aspects of being successful readers, such as self-efficacy, motivation, intra- individual characteristics, and interest of the students, also affect their reading comprehension. Brown (2007) argued that internal and external motivation encourages the students to succeed in a task. Furthermore, Bruning, Schraw, Norby, and Ronning (2004) adds that self- efficacy will help students perform well in any academic setting. Our findings correspond to what Brown (2007) and Bruning et al. (2004) have documented previously, in which successful readers are more active, highly motivated, have good inter- individual characters, and interest in acknowledging themselves to be successful learners. Thus, person category of successful and less successful readers differ in term of motivation, self-efficacy, self-confidence, inter-individual characteristic, and interest. Both Student G and Student H did reading activities repeatedly to store vocabulary as many as possible, which can later stimulate their cognitive aspects. Reading strategies are not the only way to help the less successful readers in understanding the texts. As revealed by Student G, selecting the time for reading also influenced her understanding. She conveyed that: g y Excerpt 23: “I have a special time for my reading; I could understand the texts when I read it after midnight prayer, before Morning Prayer.” This response shows that less successful readers also employ reading strategies, although these strategies do not directly lead them to understand the texts. Thus, it is categorized as self-efficacy in learning. Empirically, the participants of this study view no differences between the Indonesian and English texts. Thus, the present study shows that both successful and less successful students encountered no variety of tasks. Besides, the participants have different knowledge of task purpose and task understanding. The successful readers use their declarative knowledge and procedural knowledge in understanding the tasks intensively, as depicted in Excerpt 12. Excerpt 23: In this case, Zhang & Ardasheva (2019) believed that readers who have metacognitive awareness interpret a reading task based on context, and they select reading strategies in relation to reading Excerpt 21: The self-confidence of students is a part of self- efficacy, and it affects students’metacognition. The students’ metacognition may depend on their cognitive processes and efforts. It is evident that successful comprehension in EFL reading does not occur automatically (Ferede & Nchindila, 2017), and it rather depends on the directed cognitive effort of the students, referred to as their metacognitive processes, which consists of knowledge about and regulation of cognitive processing (Helmstaedter, Durch, Hoppe, & Witt, 2019). Therefore, the metacognitive knowledge of students will appear if they have good processes and efforts on their cognition. As a result, the input of cognition knowledge influences students’ metacognition, both knowledge and strategies. Less successful readers, however, do not employ specific strategies in understanding the texts. When asked to read the English texts, they performed it without any significant efforts to understand the content. Although they have no specific reading strategies, they are aware that their English vocabularies are insufficient. It can be observed from the excerpts of Student G and Student H. They shared that: Student G and Student H. They shared that: Stude t G a d Stude t . ey s a ed t at: Excerpt 22: “I always try to understand the text from the main idea, Sir……But sometimes, I don’t understand vocabulary. It is hard for me if I don’t understand the words meaning in the text.”(Student H). y Excerpt 22: “I always try to understand the text from the main idea, Sir……But sometimes, I don’t understand vocabulary. It is hard for me if I don’t understand the words meaning in the text.”(Student H). I always try to understand the text from the main idea, Sir……But sometimes, I don’t understand vocabulary. It is hard for me if I don’t understand the words meaning in the text.”(Student H). Excerpt 13: Similarly, Student A also uses a particular strategy in his reading activities. In understanding a text, he uses skimming and scanning strategy, and he reads the sentences containing difficult words and tries to find out the topic. Other successful readers, Student D, reports that retelling is suitable for her. She argued that retelling Excerpt 13: “Besides getting the main idea, I also focused on grammar because knowing the grammar will help me to get the main point of the text.”(Student C) Copyright © 2020, authors, e-ISSN: 2502-6747, p-ISSN: 2301-9468 611 Indonesian Journal of Applied Linguistics, 9(3), January 2020 Strasser, 2019), and intraindividual (Lou & Noels, 2019) are much attributed to reading activity. Successful and less successful readers in this study are basically aware of themselves and how they should enact to be effective readers. strategy is useful in understanding the texts. Similarly, both Student A and Student D respond that their comprehension is getting increased by at least 65% after doing the strategies in 15 minutes. In sum, successful readers use specific reading strategies to understand texts. Their prior knowledge about reading strategy has been activated in reading new texts. The findings of this study also show that from the statements of three successful readers (see Excerpts 12, 13, and 15), they have more self-confidence in reading any texts. They could estimate their understanding of the texts. Zhang (2018) reveals that successful readers believed that self-confidence facilitates learning because higher proficiency levels and self-rapport of the students are reliable indicators of readers’ confidence. Students’ estimation of their comprehension shows that they have a belief in their ability to accomplish a task, and it is considered as self-efficacy (Zhang & Ardasheva, 2019). been activated in reading new texts. Excerpt 20: Excerpt 20: “I like using skimming and scanning techniques. Sometimes, I try to understand the words that seem difficult to understand.” (Student A). Excerpt 20: “I like using skimming and scanning techniques. Sometimes, I try to understand the words that seem difficult to understand.” (Student A). Excerpt 21: “In semester two, I did a lot of retelling activities. I retell the text that I have read to my friends. It makes me understand more.” (Student D). ACKNOWLEDGMENTS Our sincere thanks are addressed to M. Faruq Ubaidillah, Tim Percepatan Publikasi (Center for Scientific Publication), Universitas Negeri Malang, Indonesia, and the anonymous reviewers of Indonesian Journal of Applied Linguistics for their insightful feedback on the earlier version of this paper. The strategies implemented by successful readers are to understand sentences during their reading activities. Razı and Çubukçu (2014) contended that readers’ metacognition will plan the reading task, monitor whether a coherent representation of the text is being maintained, and adopt different processing strategies related to the goals and outcomes of the ongoing reading. However, successful readers in our study show insufficient reading strategies when understanding different tasks. Therefore, the strategy that they used refers to reading strategy awareness rather than strategy category while they are reading a text. REFERENCES Aditomo, A., & Hasugian, E. J. (2018). 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Previous studies have also uncovered that self-efficacy (Aro et al., 2018), motivation (Hwang, 2019), interest (Pezoa, Mendive, & Copyright © 2020, authors, e-ISSN: 2502-6747, p-ISSN: 2301-9468 612 Indonesian Journal of Applied Linguistics, 9(3), January 2020 Indonesian Journal of Applied Linguistics, 9(3), January 2020 Indonesian Journal of Applied Linguistics, 9(3), January 2020 unspecified strategies in reading. As a result, they encounter many hindrances in understanding the reading materials. purposes, task demand, and their cognitive styles. Meanwhile, in completing a task, learners involve declarative knowledge (learners know what factor influence the performance) and procedural knowledge (learners use many reading comprehension strategies such as taking notes, slowing down for relevant information, summarizing the main idea) (Stephanou, & Mpiontini, 2017). This study may be open to some limitations. First, controlling and classifying successful and less successful readers based on the academic reading score may lead to unreliable students’ reading competence. Second, the process of selecting participants remain simple. Therefore, similar studies employing more participants using random sampling techniques are encouraged. Besides, investigating the relationship among components of metacognitive knowledge and strategies in EFL reading across culture, participant, age, and gender is also promising. p ) In terms of strategy category, our study unveils that successful readers use reading strategies in comprehending specific texts such as skimming, scanning, underlying, paying attention to relevant information, and retelling, as conveyed by Student A in the Excerpt 20. It is thus in line with research documenting that successful learners use different strategies from unsuccessful learners (Zhang, Thomas, & Qin, 2019), and ineffective learners are inactive learners (Van Laer & Elen, 2019). In Indonesian academic contexts, most successful learners intensively use strategies in their language learning, especially metacognitive strategies (Cai, King, Law, & McInerney, 2019). CONCLUSION Thi d d This study documented Indonesian undergraduate students’ metacognitive knowledge of reading through three categories (person, task, and strategy) and how they enact the metacognitive knowledge in the reading process. It is observed that successful readers encompass more metacognitive knowledge if compared to the less successful readers. In terms of Person Category, successful readers construed themselves as competent readers and gradually assessed their reading activities. 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https://openalex.org/W3212657768	https://zenodo.org/record/5783951/files/The_Elephant_ProblemDetermining_Bulk_Thermal_Diffusivity.pdf	English	null	The Elephant Problem—Determining Bulk Thermal Diffusivity	Energies	2,021	cc-by	8,041	Robert Beaufait , Sebastian Ammann and Ludger Fischer Competence Center Thermal Energy Storage (CCTES), Lucerne University of Applied Sciences and Arts, Technikumstrasse 21, 6048 Horw, Switzerland; sebastian.ammann@hslu.ch (S.A.); ludger.ﬁscher@hslu.ch (L.F.) Correspondence: robertpeter.beaufait@hslu.ch Abstract: This study investigates a measurement method of thermal diffusivity for samples with arbitrary geometries and unknown material properties. The aim is to curve ﬁt the thermal diffusivity with the use of a numerical simulation and transient temperature measurement inside the object of interest. This approach is designed to assess bulk material properties of an object that has a composite material structure such as underground soil. The method creates the boundary conditions necessary to apply analytical theory found in the literature. It was found that measurements best correlated with theory and simulation at positions between the center and surface of an object. Keywords: thermal diffusivity; measurement method; energy storage 1. Introduction Underground soil for thermal energy storage is becoming more common as it is abundant and a viable storage media. The earth consists of at least two phases, consisting of materials that have very different thermal properties (e.g., humid earth, gravel, and air). The properties of the underground need to be determined to properly design seasonal underground thermal storages. Bore probe samples of underground are composed of inhomogeneous materials and mean soil property values are needed for the analysis and design of the storage performance. Citation: Beaufait, R.; Ammann, S.; Fischer, L. The Elephant Problem—Determining Bulk Thermal Diffusivity. Energies 2021, 14, 7444. https://doi.org/10.3390/en14217444 Academic Editor: Victor Terekhov Received: 6 September 2021 Accepted: 3 November 2021 Published: 8 November 2021 Citation: Beaufait, R.; Ammann, S.; Fischer, L. The Elephant Problem—Determining Bulk Thermal Diffusivity. Energies 2021, 14, 7444. https://doi.org/10.3390/en14217444 Academic Editor: Victor Terekhov Received: 6 September 2021 Accepted: 3 November 2021 Published: 8 November 2021 energies energies 2.1. Materials the simplified geome The material chosen for physical test measurements was polylactic acid (PLA). It was chosen for its low thermal diffusivity, which allowed for a compact test stand and ﬂexibility as a 3D-printable material, i.e., a sphere and an elephant. Materials often used in thermal storages exhibit low thermal diffusivities, making PLA a suitable choice. Tap water was used as the heat transfer ﬂuid in the tank. Table 1 lists the material properties of the materials used. The tank was insulated using Swisspor AG (Boswil, Switzerland) PIR insulation panels. Figure 1 below shows the basic layout of the test stand. 2. Materials, Theory, and Methods 2.1. Materials The material chosen for physical test measurements was polylactic acid (PLA). It was chosen for its low thermal diffusivity, which allowed for a compact test stand and flexi- bility as a 3D-printable material, i.e., a sphere and an elephant. Materials often used in thermal storages exhibit low thermal diffusivities, making PLA a suitable choice. Tap wa- ter was used as the heat transfer fluid in the tank. Table 1 lists the material properties of the materials used. The tank was insulated using Swisspor AG (Boswil, Switzerland) PIR insulation panels. Figure 1 below shows the basic layout of the test stand. Table 1. Thermal properties of materials used in the experimental set-up. Material Thermal Conductivity Speciﬁc Heat Capacity Density Water 0.629 [W/(m·K)] 1 4178.8 [J/(kg·K)] 1 992.1 [kg/m3] 1 Air 0.0267 [W/(m·K)] 1 1006.7 [J/(kg·K)] 1 1.149 [kg/m3] 1 PLA 0.129 [W/(m·K)] 2 1301.5 [J/(kg·K)] 2 1120.7 [kg/m3] 2 PIR 0.02 [W/(m·K)] 3 n/a n/a 1 Referenced from the VDI Heat Atlas @ 40 ◦C for water, 30 ◦C for air. 2 Determined using in-house measurements. 3 Referenced from Swisspor product label. Thermal diffusivity of PLA 8.8442 × 10−8 (m2/s) determined from 2. Table 1. Thermal properties of materials used in the experimental set-up. Material Thermal Conductiv- ity Specific Heat Capacity Density Water 0.629 [W/(m·K)] 1 4178.8 [J/(kg·K)] 1 992.1 [kg/m3] 1 Air 0.0267 [W/(m·K)] 1 1006.7 [J/(kg·K)] 1 1.149 [kg/m3] 1 PLA 0.129 [W/(m·K)] 2 1301.5 [J/(kg·K)] 2 1120.7 [kg/m3] 2 PIR 0.02 [W/(m·K)] 3 n/a n/a 1 Referenced from the VDI Heat Atlas @ 40 °C for water, 30 °C for air. 2 Determined using in-house measurements. 3 Referenced from Swisspor product label. Thermal diffusivity of PLA 8.8442 × 10−8 (m2/s) determined from 2. 2.1. Materials the simplified geome Theory An analytical solution for the transient temperature evolution inside an object exists for a sphere, cylinder, and flat plate [10,11]. Figure 2 below depicts the qualitative temper- ature change for a cool sphere immersed into a warm fluid A tank made of 2 mm carbon nickel steel plate was fabricated to contain a ﬂuid bath in which the objects were to be placed for measurement. 2.1. Materials the simplified geome A tank made of 2 mm carbon nickel steel plate was fabricated to contain a fluid bath in which the objects were to be placed for measurement. Figure 1. Sketch of test stand. 2.2. Theory An analytical solution for the transient temperature evolution inside an object exists Figure 1. Sketch of test stand. A tank made of 2 mm carbon nickel steel plate was fabricated to contain a ﬂuid bath Table 1. Thermal properties of materials used in the experimental set-up. Material Thermal Conductivity Speciﬁc Heat Capacity Density Water 0.629 [W/(m·K)] 1 4178.8 [J/(kg·K)] 1 992.1 [kg/m3] 1 Air 0.0267 [W/(m·K)] 1 1006.7 [J/(kg·K)] 1 1.149 [kg/m3] 1 PLA 0.129 [W/(m·K)] 2 1301.5 [J/(kg·K)] 2 1120.7 [kg/m3] 2 PIR 0.02 [W/(m·K)] 3 n/a n/a 1 Referenced from the VDI Heat Atlas @ 40 ◦C for water, 30 ◦C for air. 2 Determined using in-house measurements. 3 Referenced from Swisspor product label. Thermal diffusivity of PLA 8.8442 × 10−8 (m2/s) determined from 2. Table 1. Thermal properties of materials used in the experimental set-up. Material Thermal Conductiv- ity Specific Heat Capacity Density Water 0.629 [W/(m·K)] 1 4178.8 [J/(kg·K)] 1 992.1 [kg/m3] 1 Air 0.0267 [W/(m·K)] 1 1006.7 [J/(kg·K)] 1 1.149 [kg/m3] 1 PLA 0.129 [W/(m·K)] 2 1301.5 [J/(kg·K)] 2 1120.7 [kg/m3] 2 PIR 0.02 [W/(m·K)] 3 n/a n/a 1 Referenced from the VDI Heat Atlas @ 40 °C for water, 30 °C for air. 2 Determined using in-house measurements. 3 Referenced from Swisspor product label. Thermal diffusivity of PLA 8.8442 × 10−8 (m2/s) determined from 2. A tank made of 2 mm carbon nickel steel plate was fabricated to contain a fluid bath in which the objects were to be placed for measurement. Table 1. Thermal properties of materials used in the experimental set-up. Table 1. Thermal properties of materials used in the experimental set-up. M t i l Thermal Conductiv- Specific Heat D it Figure 1. Sketch of test stand. Figure 1. Sketch of test stand. 2.2. Theory An analytical solution for the transient temperature evolution inside an object exists for a sphere, cylinder, and flat plate [10,11]. Figure 2 below depicts the qualitative temper- ature change for a cool sphere immersed into a warm fluid A tank made of 2 mm carbon nickel steel plate was fabricated to contain a ﬂuid bath in which the objects were to be placed for measurement. 2.2. Citation: Beaufait, R.; Ammann, S.; Fischer, L. The Elephant Problem—Determining Bulk Thermal Diffusivity. Energies 2021, 14, 7444. https://doi.org/10.3390/en14217444 The use of analytical instruments to measure mean property values (e.g., thermal conductivity) of such geological probes is quite complex owing to the geometry and nature of the samples. Geological probes are extracted with rotary drilling equipment, rendering cylindrical samples. Owing to the ﬁnite length of drill rods (<3 m), only sections of earth can be obtained, and they are often fractured or broken during the process of extraction [1]. Extending the theory of heat conduction in solids to measure thermal conductivity requires subjective assumptions treating samples as homogenous materials with uniform water distribution, perfect contact with the measurement device, and no migration of moisture during measurement [2]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. There are two categories of state-of-the art measurement methods (steady-state and transient) for thermal conductivity, which is needed to calculate thermal diffusivity. Each method has its own set of advantages and disadvantages in determining thermal conductivity [2–9]. A new method is developed in this work for the determination of thermal diffusivity for a solid sample of arbitrary shape using a combination of a simple experimental temper- ature measurement in combination with a numerical simulation of a complex geometry and ﬁt of the thermal properties. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). p p To develop the method, ﬁrst, an analytical and numerical solution are evaluated for a sphere as a well-known geometry using a material with known properties. The deﬁnition of a maximum acceptable deviation for thermal diffusivity of 10% is established to constrain the analysis. Second, a simple experiment to employ the necessary boundary conditions to validate the test method and numerical model is designed and developed. The procedure assumes a sufﬁciently high heat transfer rate of approximately 1000 W/(m·K) https://www.mdpi.com/journal/energies Energies 2021, 14, 7444. https://doi.org/10.3390/en14217444 Energies 2021, 14, 7444 2 of 10 over the entire object surface. Finally, tests using an irregular shape (an elephant as a metaphor for sympathy and size of the problem) are analyzed for comparison with the simpliﬁed geometry. 2 of 10 The procedure assumes a sufficiently high heat transfer rate of approximately 1000 2.2. Theory An analytical solution for the transient temperature evolution inside an object exists for a sphere, cylinder, and ﬂat plate [10,11]. Figure 2 below depicts the qualitative temperature change for a cool sphere immersed into a warm ﬂuid. 3 of 10 Figure 2. A cool sphere warming in a warm environment adapted from [11]. Figure 2. A cool sphere warming in a warm environment adapted from [11]. Figure 2. A cool sphere warming in a warm environment adapted from [11]. Figure 2. A cool sphere warming in a warm environment adapted from [11]. Figure 2. A cool sphere warming in a warm environment adapted from [11]. Figure 2. A cool sphere warming in a warm environment adapted from [11]. Energies 2021, 14, 7444 3 of 10 3 of 10 The x-axis indicates the radial position, and the y-axis indicates temperature. The initial temperature of the sphere before entering the ﬂuid is denoted as T0 and the ﬁnal tempera- ture as T∞. Proﬁles t1 to t5 display how the temperature changes as a function of time and radial position within a spherical object. The x-axis indicates the radial position, and the y-axis indicates temperature. The in- itial temperature of the sphere before entering the fluid is denoted as T଴ and the final temperature as Tஶ. Profiles tଵ to tହ display how the temperature changes as a function of time and radial position within a spherical object. T t fil f ti f l i t t b d diti Fi t Temperature proﬁles are a function of several important boundary conditions. First, a symmetric temperature proﬁle assumes a constant value of high heat transfer over the entire surface of the object. Second, the thermal properties of the object must be considered as well as the characteristics of the ﬂuid ﬂow. Third, geometry has a great inﬂuence on the temperature evolution within the object. Figure 3 below illustrates the solution for the transient temperature ﬁeld as an error function approximated with a series of transcendental functions. The plot below is for the center of a sphere of arbitrary material and heat transfer conditions. Temperature profiles are a function of several important boundary conditions. First, a symmetric temperature profile assumes a constant value of high heat transfer over the entire surface of the object. Second, the thermal properties of the object must be considered as well as the characteristics of the fluid flow. 2.2. Theory Third, geometry has a great influence on the temperature evolution within the object. Figure 3 below illustrates the solution for the transient temperature field as an error function approximated with a series of transcen- dental functions. The plot below is for the center of a sphere of arbitrary material and heat transfer conditions. Figure 3. The analytical transient temperature change at the center of a sphere adapted from [11]. Figure 3. The analytical transient temperature change at the center of a sphere adapted from [11]. Figure 3. The analytical transient temperature change at the center of a sphere adapted from [11]. Figure 3. The analytical transient temperature change at the center of a sphere adapted from [11]. The x-axis represents non-dimensional time and is scaled using the thermal diffusiv- ity κ (m2/s) of the chosen material and scaled by the characteristic length, r (m), i.e., the Fourier number: The x-axis represents non-dimensional time and is scaled using the thermal diffusivity κ (m2/s) of the chosen material and scaled by the characteristic length, r (m), i.e., the Fourier number: t Fo = κ ⋅t (1) Fo = κ · t r2 (1) (1) (1) Fo = rଶ (1) The y-axis is defined by a non-dimensional temperature representing the fractional temperature change relative to the range defined by the initial temperature of the sphere, The y-axis is deﬁned by a non-dimensional temperature representing the fractional temperature change relative to the range deﬁned by the initial temperature of the sphere, T0, and the temperature of the water in the tank, T∞, water in the tank, Tஶ, Θ = θ θ = Tሺr, tሻ െ Tஶ T T (2) Θ = θ θ0 = T(r, t) −T∞ T0 −T∞ (2) (2) (2) θ଴ T଴ Tஶ The curves in Figure 3 above represent the evolution of temperature at the center of a sphere, Tሺr, tሻ, where r = 0. They are a family of curves representing the ratio of con- vective heat transfer at the surface, h (W/(m2·K)), to the conductive heat transfer, λ The curves in Figure 3 above represent the evolution of temperature at the center of a sphere, T(r, t), where r = 0. 2.2. Theory They are a family of curves representing the ratio of convective heat transfer at the surface, h (W/(m2·K)), to the conductive heat transfer, λ (W/(m·K)), inside the object scaled by the characteristic length, e.g., the Biot number for a sphere: Bi = h · r λ (3) (3) The Biot number determines the transient temperature proﬁle as an object is heated up or cooled down in an immersed ﬂuid. Generally, a Bi ≪1 indicates relatively poor convective heat transfer and near uniform temperature within the object (a), whereas a Bi ≫1 indicates relatively poor conductive heat transfer and a large temperature gradient within the object. Figure 4 below compares both (a) and (b) scenarios at two different times. Energies 2021, 14, 7444 4 of 10 re grad o diffe (a) (b) Figure 4. Simulated temperature evolution at three positions for (a) t = 30 s and (b) t = 60 s [12]. Figure 4. Simulated temperature evolution at three positions for (a) t = 30 s and (b) t = 60 s [12]. (a) (b) (b) (a) ure 4. Simulated temperature evolution at three positions for (a) t = 30 s and (b) t = 60 s [1 Figure 4. Simulated temperature evolution at three positions for (a) t = 30 s and (b) t = 60 s [12]. The sphere on the right in both panels represents a metal that experiences a sh e in temperature in a very short period. In addition, Figure 3 reveals an exponen proach to the theoretical boundary condition Bi = ∞ with increasing Biot numb he roots of the transcendental equation for Bi = 120 are approximately 0.83% diffe om the boundary condition Bi = ∞. A Bi ≥120 was chosen sufficient to assume undary condition for comparison with experimental measurements. Thus, the met this work relies on high Biot numbers with relative high heat transfer coefficients e selection of a test material with low conductivity was made to allow for more m The sphere on the right in both panels represents a metal that experiences a sharp rise in temperature in a very short period. In addition, Figure 3 reveals an exponential approach to the theoretical boundary condition Bi = ∞with increasing Biot numbers. The roots of the transcendental equation for Bi = 120 are approximately 0.83% different from the boundary condition Bi = ∞. 2.3.1. Procedure . Methods 3.1. Procedure The measurement concept was developed with an experiment to create the bound nditions necessary for comparison of the measurements with the related theory for here and with a numerical model for the elephant. The numerical model was valida r a sphere also using the analytical theory. All measurements were compared using The measurement concept was developed with an experiment to create the boundary conditions necessary for comparison of the measurements with the related theory for the sphere and with a numerical model for the elephant. The numerical model was validated for a sphere also using the analytical theory. All measurements were compared using the ideal boundary condition Bi = ∞. The goal was to measure the elephant and record a position speciﬁc temperature over time to determine the thermal diffusivity by comparison with a numerical model. g ometries. 2.3. Methods g ometries. 2.3. Methods 2.2. Theory A Bi ≥120 was chosen sufﬁcient to assume this boundary condition for comparison with experimental measurements. Thus, the method in this work relies on high Biot numbers with relative high heat transfer coefﬁcients and the selection of a test material with low conductivity was made to allow for more measurements during the change in temperature. y ements during the change in temperature. Geometries of an arbitrary shape take on similarly shaped curves as those foun gure 3. Analytical functions for the transient temperature evolution for arbitrary ge ries do not exist in the literature. A complex surface exhibits a unique set of Biot cur at do not correlate with those of a sphere. Therefore, a numerical simulation is necess t th f i ith th d lt f bit Geometries of an arbitrary shape take on similarly shaped curves as those found in Figure 3. Analytical functions for the transient temperature evolution for arbitrary geometries do not exist in the literature. A complex surface exhibits a unique set of Biot curves that do not correlate with those of a sphere. Therefore, a numerical simulation is necessary to generate the curves necessary for comparison with the measured results for arbitrary geometries. eal boundary condition B osition specific temperatur 2.3.2. Physical Experiment sition specific temperature over time to determine the thermal diffusivity by comp n with a numerical model. Using the stated materials and related theory, an experiment was developed to de- termine the average thermal diffusivity of an arbitrary object. The proposed method prescribes the rapid immersion of a test object into a turbulent isothermal ﬂuid. The test stand shown in Figure 5 consists of an insulated steel tank for the purpose of circulating water across the test object via a pump. A quantity of water weighing 60 kg was used to transfer heat to the cooler object without a signiﬁcant drop or gain in ﬂuid temperature during the test. 5 of 10 p p ng 60 kg i fl id Energies 2021, 14, 7444 (a) (a) (a) (b) Figure 5. Test stand insulation and tank (a) with lid and pump (b). Figure 5. Test stand insulation and tank (a) with lid and pump (b). (a) (b) Figure 5. Test stand insulation and tank (a) with lid and pump (b). (b) (b) (b) (a) Figure 5. Test stand insulation and tank (a) with lid and pump (b). Figure 5. Test stand insulation and tank (a) with lid and pump (b). Test stand insulation and tank (a) with lid and pump (b). A Keysight (Otelfingen, Switzerland) DAQ970A data logger recorded temperature data from calibrated type K, 0.13 mm wire thickness, (∅ = 0.6 mm), PFA-insulated ther- mocouples (Omega (Norwalk, CT, USA): 5SC-TT-KI-36-2M) for the water temperature and temperature inside the object. A measurement sweep was recorded every ~0.2–0.3 s for approximately 1 h. The velocity of the water was regulated (~0.1 m/s) to create a suffi- ciently high and constant heat transfer coefficient at the surface of the objects consistent with a Bi > 120 with the given geometry and thermal diffusivity of the test material. A Keysight (Otelﬁngen, Switzerland) DAQ970A data logger recorded temperature data from calibrated type K, 0.13 mm wire thickness, (∅= 0.6 mm), PFA-insulated thermo- couples (Omega (Norwalk, CT, USA): 5SC-TT-KI-36-2M) for the water temperature and temperature inside the object. A measurement sweep was recorded every ~0.2–0.3 s for ap- proximately 1 h. The velocity of the water was regulated (~0.1 m/s) to create a sufﬁciently high and constant heat transfer coefﬁcient at the surface of the objects consistent with a Bi > 120 with the given geometry and thermal diffusivity of the test material. eal boundary condition B osition specific temperatur 2.3.2. Physical Experiment g g y y Spheres (∅ = 60 mm) and an elephant (l, w, h = 63 mm × 43 mm × 61 mm) of PLA measuring were 3D-printed using an Ultimaker 3 (Utrecht, The Netherlands). A hole o Spheres (∅= 60 mm) and an elephant (l, w, h = 63 mm × 43 mm × 61 mm) of PLA measuring were 3D-printed using an Ultimaker 3 (Utrecht, The Netherlands). A hole of heres (∅ = 60 mm) and an elephant (l, w, h = 63 mm × 43 mm × 61 mm) of PLA ing were 3D-printed using an Ultimaker 3 (Utrecht, The Netherlands). A hole of Spheres (∅ = 60 mm) and an elephant (l, w, h = 63 mm × 43 mm × 61 mm) of PLA measuring were 3D-printed using an Ultimaker 3 (Utrecht, The Netherlands). A hole of ~1 mm was drilled to a prescribed internal point to place a thermocouple and measure the internal temperature of the object over time. The calibrated thermocouples were placed in contact with the objects at the end of the drilled hole. Figure 6 shows an example of hole drilled for measurement. Spheres (∅= 60 mm) and an elephant (l, w, h = 63 mm × 43 mm × 61 mm) of PLA measuring were 3D-printed using an Ultimaker 3 (Utrecht, The Netherlands). A hole of ~1 mm was drilled to a prescribed internal point to place a thermocouple and measure the internal temperature of the object over time. The calibrated thermocouples were placed in contact with the objects at the end of the drilled hole. Figure 6 shows an example of hole drilled for measurement. heres (∅ = 60 mm) and an elephant (l, w, h = 63 mm × 43 mm × 61 mm) of PLA ing were 3D-printed using an Ultimaker 3 (Utrecht, The Netherlands). A hole of was drilled to a prescribed internal point to place a thermocouple and measure the temperature of the object over time. The calibrated thermocouples were placed in with the objects at the end of the drilled hole. Figure 6 shows an example of hole for measurement. (a) (b) Figure 6. Computed tomographic scan of drilled holes inside the sphere (a) and elephant (b). 63 mm 60 mm (a) (b) . Computed tomographic scan of drilled holes inside the sphere (a) and elephant (b). 63 mm 60 mm Figure 6. eal boundary condition B osition specific temperatur 2.3.2. Physical Experiment Computed tomographic scan of drilled holes inside the sphere (a) and elephant (b). (b) 63 mm (b) 63 mm (a) 6 (a) 60 mm (a) (b) Figure 6. Computed tomographic scan of drilled holes inside the sphere (a) and elephant (b) Computed tomographic scan of drilled holes inside the sphere (a) and elephant (b). Figure 6. Computed tomographic scan of drilled holes inside the sphere (a) and elephant (b). eal boundary condition B osition specific temperatur 2.3.2. Physical Experiment Keysight (Otelfingen, Switzerland) DAQ970A data logger recorded temperature m calibrated type K, 0.13 mm wire thickness, (∅ = 0.6 mm), PFA-insulated ther- ples (Omega (Norwalk, CT, USA): 5SC-TT-KI-36-2M) for the water temperature mperature inside the object. A measurement sweep was recorded every ~0.2–0.3 s roximately 1 h. The velocity of the water was regulated (~0.1 m/s) to create a suffi- high and constant heat transfer coefficient at the surface of the objects consistent Bi > 120 with the given geometry and thermal diffusivity of the test material. i a i 0 i e gi e geo e y a e a i u i i y o e e a e ia Spheres (∅ = 60 mm) and an elephant (l, w, h = 63 mm × 43 mm × 61 mm) of PLA measuring were 3D-printed using an Ultimaker 3 (Utrecht, The Netherlands). A hole of ~1 mm was drilled to a prescribed internal point to place a thermocouple and measure the internal temperature of the object over time. The calibrated thermocouples were placed in contact with the objects at the end of the drilled hole. Figure 6 shows an example of hole drilled for measurement. g g y y Spheres (∅= 60 mm) and an elephant (l, w, h = 63 mm × 43 mm × 61 mm) of PLA measuring were 3D-printed using an Ultimaker 3 (Utrecht, The Netherlands). A hole of ~1 mm was drilled to a prescribed internal point to place a thermocouple and measure the internal temperature of the object over time. The calibrated thermocouples were placed in contact with the objects at the end of the drilled hole. Figure 6 shows an example of hole drilled for measurement. heres (∅ = 60 mm) and an elephant (l, w, h = 63 mm × 43 mm × 61 mm) of PLA ing were 3D-printed using an Ultimaker 3 (Utrecht, The Netherlands). A hole of was drilled to a prescribed internal point to place a thermocouple and measure the l temperature of the object over time. The calibrated thermocouples were placed in with the objects at the end of the drilled hole. Figure 6 shows an example of hole for measurement. on of COMSOL Simulation and Theory 3.1. Validation of COMSOL Simulation and Theory 3.1. Validation of COMSOL Simulation and Theory on of COMSOL Simulation and Theory ve use of a COMSOL simulation for arbitrary geometries required a model val- h a geometry compatible with the analytical theory. The comparison was made e at the center (r = 0), r = 14.8 mm, and r =19.3 mm. A sphere of initially 20 °C d to a sudden change of surface temperature of 40 °C. Figure 8 plots the simu- h l i l f i d f 2 h (7200 ) Effective use of a COMSOL simulation for arbitrary geometries required a model validation with a geometry compatible with the analytical theory. The comparison was made for a sphere at the center (r = 0), r = 14.8 mm, and r =19.3 mm. A sphere of initially 20 ◦C was exposed to a sudden change of surface temperature of 40 ◦C. Figure 8 plots the simulated temperatures versus the analytical for a period of 2 h (7200 s). Effective use of a COMSOL simulation for arbitrary geometries required a model val- dation with a geometry compatible with the analytical theory. The comparison was made or a sphere at the center (r = 0), r = 14.8 mm, and r =19.3 mm. A sphere of initially 20 °C was exposed to a sudden change of surface temperature of 40 °C. Figure 8 plots the simu- ated temperatures versus the analytical for a period of 2 h (7200 s). y p ( ) (a) (b) omparison of simulated [12] and theoretical results in (a) non-dimensional form and (b) (a) (b) Figure 8. Comparison of simulated [12] and theoretical results in (a) non-dimensional form and (b) dimensional form. Figure 8. Comparison of simulated [12] and theoretical results in (a) non-dimensional form and (b) dimensional form. ( ) (b) y p (a) (b) (a) (a) (b) mparison of simulated [12] and theoretical results in (a) non-dimensional form and (b) igure 8. Comparison of simulated [12] and theoretical results in (a) non-dimensional form and (b) imensional form. Figure 8. Comparison of simulated [12] and theoretical results in (a) non-dimensional form and (b) dimensional form. p [ ] ( ) form. 3.2. Comparison of Experimental Spheres with COMSOL and Theory p [ ] ( ) form. 3.2. 2.3.3. Numerical Simulation A numerical simulation in COMSOL Multiphysics (Zürich, Switzerland) v5.5 was used to model the temperature evolution of the object placed into the steel tank. The model did not account for the ﬂuid ﬂow. A boundary condition of Bi = ∞was chosen, where the free temperature of the ﬂuid is assumed to be the surface temperature of the object. A sphere was simulated with a 2D axisymmetric transient simulation for comparison with the analytical theory using a maximum element size of 0.6 mm, consisting of 14,176 elements. After validation, a 3D transient model for the elephant served as the point of comparison with measurement data from the elephant test object with a maximum element size of 6.38 mm, consisting of 29,383 elements. Figure 7 below shows both meshes simulated. Energies 2021, 14, 7444of em 6 of 10 point m el- (a) (b) D axisymmetric model of the sphere (a) and 3D model of the elephant (b) [12]. Figure 7. 2D axisymmetric model of the sphere (a) and 3D model of the elephant (b) [12]. simulated. (a) (b) Figure 7. 2D axisymmetric model of the sphere (a) and 3D model of the elephant (b) [12]. (a) simulated. (a) (b) (b) h ( ) d 3D d l f h l h (b) [12] (b) (a) axisymmetric model of the sphere (a) and 3D model of the elephant (b) [12]. Figure 7. 2D axisymmetric model of the sphere (a) and 3D model of the elephant (b) [12]. igure 7. 2D axisymmetric model of the sphere (a) and 3D model of the elephant (b) [12]. 3. Results . Results on of COMSOL Simulation and Theory 3.1. Validation of COMSOL Simulation and Theory 3.1. Validation of COMSOL Simulation and Theory on of COMSOL Simulation and Theory 3.1. Validation of COMSOL Simulation and Theory 3.1. Validation of COMSOL Simulation and Theory Comparison of Experimental Spheres with COMSOL and Theory Consistent with the validation, the same three positions were used for experimen- tal measurements in four spheres of PLA (K2–K5) and their corresponding individual measurements (e.g., K2_#1, K4_#6, and so on). The plot colors reference the positions indicated in Figure 8. Figure 9 below plots an individual measurement at each position and with a list of the remaining measurements below. Thermal diffusivity was determined via curve ﬁtting with the reference point (1 −Θ) = 0.70 as the value to ﬁt the simulated results with no deviation relative to the measured result. The data below the plots represent the measurement deviation of the calculated thermal diffusivity for each test (PLA from Table 1) with theoretical calculations and simulated output. Energies 2021, 14, 7444 7 of 10 eas- e 1) with theoretical calculations and simulated output. r = 0 mm r = 14.8 mm r = 19.3 mm Theory COMSOL Theory COMSOL Theory COMSOL K2_#1 K2_#4 K2_#5 K3_#2 K3_#3 +27.17% +24.51% +24.31% +24.58% +24.23% +30.00% +26.75% +26.85% +26.00% +25.50% K4_#2 K4_#3 K4_#4 K4_#5 K4_#6 +1.52% +1.71% +2.52% +2.62% +2.99% +2.70% +0.00% +3.50% +3.25% +4.25% K5_#2 K5_#3 K5_#4 K5_#5 K5_#6 +0.99% +1.66% +1.28% +2.01% +0.77% +1.35% +1.75% +2.00% +2.65% +2.05% Figure 9. Measurement results of spheres plotted against fitted 2D simulation [12] output for ther- mal diffusivity, and subsequent measurements below for each position. Figure 9. Measurement results of spheres plotted against ﬁtted 2D simulation [12] output for thermal diffusivity, and subsequent measurements below for each position. p r = 0 mm r = 14.8 mm r = 19.3 mm Theory COMSOL Theory COMSOL Theory COMSOL K2_#1 K2_#4 K2_#5 K3_#2 K3_#3 +27.17% +24.51% +24.31% +24.58% +24.23% +30.00% +26.75% +26.85% +26.00% +25.50% K4_#2 K4_#3 K4_#4 K4_#5 K4_#6 +1.52% +1.71% +2.52% +2.62% +2.99% +2.70% +0.00% +3.50% +3.25% +4.25% K5_#2 K5_#3 K5_#4 K5_#5 K5_#6 +0.99% +1.66% +1.28% +2.01% +0.77% +1.35% +1.75% +2.00% +2.65% +2.05% Figure 9. Measurement results of spheres plotted against fitted 2D simulation [12] output for ther- mal diffusivity, and subsequent measurements below for each position. Figure 9. Measurement results of spheres plotted against ﬁtted 2D simulation [12] output for thermal diffusivity, and subsequent measurements below for each position. Figure 9. Measurement results of spheres plotted against fitted 2D simulation [12] output for ther- mal diffusivity, and subsequent measurements below for each position. Figure 9. on of COMSOL Simulation and Theory 3.1. Validation of COMSOL Simulation and Theory 3.1. Validation of COMSOL Simulation and Theory Measurement results of spheres plotted against ﬁtted 2D simulation [12] output for thermal diffusivity, and subsequent measurements below for each position. 3.3. Comparison of Experimental Elephant and COMSOL 3.3. Comparison of Experimental Elephant and COMSOL 4.2. Comparison of Experimental Sphere Measurements, Theory, and COMSOL 4.2. Comparison of Experimental Sphere Measurements, Theory, and COMSOL The accuracy of measurements varied with the position within the sphere. The center (r = 0 mm) measurements proved to be the worst performers. Figure 9 shows relatively large deviations from the theory and model and is an expected result. The center of an object with spherical symmetry is equally affected in space and time by imperfect placing of its tip. The asymmetric nature of the fluid flow, measurement equipment, and the hole drilled into a sphere disrupt the boundary condition of a uniform heat transfer rate at the surface. Thus, the measurements render a poor comparison with model results as the model did not incorporate any asymmetric features. The accuracy of measurements varied with the position within the sphere. The center (r = 0 mm) measurements proved to be the worst performers. Figure 9 shows relatively large deviations from the theory and model and is an expected result. The center of an object with spherical symmetry is equally affected in space and time by imperfect placing of its tip. The asymmetric nature of the ﬂuid ﬂow, measurement equipment, and the hole drilled into a sphere disrupt the boundary condition of a uniform heat transfer rate at the surface. Thus, the measurements render a poor comparison with model results as the model did not incorporate any asymmetric features. model did not incorporate any asymmetric features. For the off-center positions (r = 14.8, 19.3 mm), the results were in good agreement with the model. These two measurement positions were located closer to the direction of p y y For the off-center positions (r = 14.8, 19.3 mm), the results were in good agreement with the model. These two measurement positions were located closer to the direction of ﬂuid ﬂow. The surfaces that inﬂuenced these measurement points exhibit much more consistent surface characteristics and ﬂow regimes, and thus more consistent heat transfer rates. While the results in measurement point r = 19.3 mm were more accurate, it was located only 10.3 mm away from the nearest surface. This excluded a major portion of the material from the measurement. A meaningful bulk material property measurement is representative of the largest possible proportion of the sample tested. Therefore, the best measurement recorded during this study was r = 14.8 mm. 3.3. Comparison of Experimental Elephant and COMSOL 3.3. Comparison of Experimental Elephant and COMSOL A hole was drilled from one side of the elephant to a prescribed internal position as with the spheres (Figure 6b). The thermocouple was place at this position and temperature was recorded over time and compared with the numerical simulation, as symmetry could no longer be applied. Figure 10 below shows two measurements and deviations from 3D simulations with the point of measurement marked with a black dot. A hole was drilled from one side of the elephant to a prescribed internal position as with the spheres (Figure 6b). The thermocouple was place at this position and temperature was recorded over time and compared with the numerical simulation, as symmetry could no longer be applied. Figure 10 below shows two measurements and deviations from 3D simulations with the point of measurement marked with a black dot. 8 (c) (a) (b) Figure 10. Two (E1_#4 (a), E1_#5 (b)) measurement results of elephant plotted against fitted 3D simulation output thermal diffusivity and a lengthwise cross section of E1_#4 (c) [12]. Figure 10. Two (E1_#4 (a), E1_#5 (b)) measurement results of elephant plotted against ﬁtted 3D simulation output for thermal diffusivity and a lengthwise cross section of E1_#4 (c) [12]. (a) (c) (a) (b) (b) Figure 10. Two (E1_#4 (a), E1_#5 (b)) measurement results of elephant plotted against fitted 3D simulation outpu thermal diffusivity and a lengthwise cross section of E1_#4 (c) [12]. Figure 10. Two (E1_#4 (a), E1_#5 (b)) measurement results of elephant plotted against ﬁtted 3D simulation output for thermal diffusivity and a lengthwise cross section of E1_#4 (c) [12]. Energies 2021, 14, 7444 Figure 10. Two thermal diffusiv 8 of 10 or 4.1. Validation of COMSOL Simulation and Theory f y To no surprise, the validation of the COMSO To no surprise, the validation of the COMSOL model using the analytical theory for a sphere proved a success. As shown in Figure 11, the COMSOL model tracked the analytical solution with virtually no deviation at the comparison point of (1 −Θ) = 0.70 or 34 ◦C. p , g y y a sphere proved a success. As shown in Figure 11, the COMSOL model tracked the ana- lytical solution with virtually no deviation at the comparison point of (1 − Θ) = 0.70 or 34 °C. (a) (b) (c) Figure 11. Comparison of numerical results [12] for (a) r = 0 mm; (b) r = 14.8 mm; and (c) r = 19.3 mm with the analytical approximation [10,11]. Figure 11. Comparison of numerical results [12] for (a) r = 0 mm; (b) r = 14.8 mm; and (c) r = 19.3 mm with the analytical approximation [10,11]. (b) (c) (a) (b) (a) (c) Figure 11. Comparison of numerical results [12] for (a) r = 0 mm; (b) r = 14.8 mm; and (c) r = 19.3 mm with the analytical approximation [10,11]. Figure 11. Comparison of numerical results [12] for (a) r = 0 mm; (b) r = 14.8 mm; and (c) r = 19.3 mm with the analytical approximation [10,11]. 4.2. Comparison of Experimental Sphere Measurements, Theory, and COMSOL 4.2. Comparison of Experimental Sphere Measurements, Theory, and COMSOL 4. Discussion 4.1. Validation o 4.1. Validation of COMSOL Simulation and Theory f y To no surprise, the validation of the COMSO 4.1. Validation of COMSOL Simulation and Theory f y To no surprise, the validation of the COMSO It is appa f 5. Conclusions of measurement. This area experiences a very different flow regime from the surfaces tha meet the flow first. In addition, the legs are sinks to heat augmenting the temperatur evolution with surfaces outside the assumption of constant heat transfer. 5. Conclusions The results of this study have found a viable method of determining thermal diffu sivity via a transient temperature measurement inside an object immersed in a movin fluid and comparison with a numerical simulation. The use of 3D-printed material wa The results of this study have found a viable method of determining thermal diffu- sivity via a transient temperature measurement inside an object immersed in a moving ﬂuid and comparison with a numerical simulation. The use of 3D-printed material was particularly challenging as the objects created did not exhibit consistent quality. Nev- ertheless, simulated temperature proﬁles in COMSOL were congruent in shape to the measurements in tests using spheres and the elephant. The next logical step would be to take the related theory and results to support the construction of a test rig capable of measuring a cylindrical geometry. p p particularly challenging as the objects created did not exhibit consistent quality. Never theless, simulated temperature profiles in COMSOL were congruent in shape to the meas urements in tests using spheres and the elephant. The next logical step would be to tak the related theory and results to support the construction of a test rig capable of measurin a cylindrical geometry. The method developed here can now be used as follows with an example of a cylindri- cal bore probe. The bore probe will be packed in foil to prevent drying, and a temperature sensor (or two) will be placed close to the center with known positions. The cylinder shall be stable in temperature (e.g., ambient) and will be placed in a water bath or oven with different and constant temperature. The recorded temperature versus time will be compared to numerical simulations of the same geometry and by ﬁtting the thermal diffu- sivity, matching measurement, and simulation, the effective property is obtained. In case thermal conductivity is required, the speciﬁc heat capacity of the sample and density must be measured with other standard analytic instruments like calorimetry. Establishing the necessary boundary conditions for assumed Bi = ∞is critical. 4.3. Comparison of Experimental Elephant Measurements COMSOL The deviation of measurements from the ideal boundary condition Bi = ∞for the elephant was between the measurements made at the center of spheres (r = 0 mm) and r = 14.8 mm. The deviation from model output indicates effects indicative of an inconsistent boundary condition. The position measured experienced thermal effects from surfaces that did not conform well with the assumption of a constant heat transfer rate at the surface. Figure 12 below shows a widthwise-vertically oriented slice along the axis of the drilled hole. The point of measurement is indicated with a white dot. It is apparent that the region of the lower belly is close enough to inﬂuence the point of measurement. This area experiences a very different ﬂow regime from the surfaces that meet the ﬂow ﬁrst. In addition, the legs are sinks to heat augmenting the temperature evolution with surfaces outside the assumption of constant heat transfer. 9 of 10 t the sur xis of th Energies 2021, 14, 7444 (a) (c) (b) Figure 12. Two (E1_#4 (a), E1_#5 (b)) measurement results of elephant plotted against fitted 3D simulation output for thermal diffusivity and a widthwise cross section of E1_#4 (c) [12]. Figure 12. Two (E1_#4 (a), E1_#5 (b)) measurement results of elephant plotted against ﬁtted 3D simulation output for thermal diffusivity and a widthwise cross section of E1_#4 (c) [12]. (a) (c) (a) (a) (b) (b) Figure 12. Two (E1_#4 (a), E1_#5 (b)) measurement results of elephant plotted against fitted 3D simulation output fo thermal diffusivity and a widthwise cross section of E1_#4 (c) [12]. Figure 12. Two (E1_#4 (a), E1_#5 (b)) measurement results of elephant plotted against ﬁtted 3D simulation output for thermal diffusivity and a widthwise cross section of E1_#4 (c) [12]. It is appa f 5. Conclusions Flow conditions need to be determined with a robust margin to guarantee that Bi ≥120 is achieved and difference between Bi = 120 and Bi = ∞is negligible. Further, a larger object may require either a larger ﬂuid bath or larger ﬂuid stream as the Nusselt number scales with increasing characteristic length. Author Contributions: All authors listed conceived and designed the project and contributed to the design of experiments and in reviewing the paper; R.B. and S.A. designed and built the setup; R.B. performed the experiments; R.B. analyzed the data; R.B. and L.F. wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Energies 2021, 14, 7444 10 of 10 10 of 10 Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. References 1. National Academies of Sciences, Engineering, and Medicine. Manual on Subsurface Investigations; Th Washington, DC, USA, 2019. [CrossRef] 1. National Academies of Sciences, Engineering, and Medicine. Manual on Subsurface Investigations; The National Academies Press: Washington, DC, USA, 2019. [CrossRef] 2 B hi G C P B i P bl i Th l C d ti it M t f S il I t J Th h 2013 34 1962 1974 Washington, DC, USA, 2019. [CrossRef] 2. Bovesecchi, G.; Coppa, P. Basic Problems in Thermal-Conductivity Measurements of Soils. Int. J. Thermophys. 2013, 34, 1962–1974. [CrossRef] Washington, DC, USA, 2019. [CrossRef] 2. Bovesecchi, G.; Coppa, P. Basic Problems in Thermal-Conductivity Measurements of Soils. Int. J. Thermophys. 2013, 34, 1962–1974. [CrossRef] 2. Bovesecchi, G.; Coppa, P. Basic Problems in Thermal-Conductivity Measurements of Soils. Int. J. Thermo [CrossRef] 3. Palacios, A.; Cong, L.; Navarro, M.; Ding, Y.; Barreneche, C. Thermal conductivity measurement techniques for characterizing thermal energy storage materials—A review. Renew. Sustain. Energy Rev. 2019, 108, 32–52. [CrossRef] gy g gy 4. Jannot, Y.; Degiovanni, A. Thermal Properties Measurement of Materials; John Wiley & Sons, Inc.: Hoboken, 4. Jannot, Y.; Degiovanni, A. Thermal Properties Measurement of Materials; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2018. [CrossRef] 4. Jannot, Y.; Degiovanni, A. Thermal Properties Measurement of Materials; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2018. [CrossRef] 5. Maglic, K.D.; Cezairliyan, A.; Peletsky, V.E. Compendium of Thermophysical Property Measurement Methods Vol. 1, Survey of Measurement Techniques; Plenum Press: New York, NY, USA, 1984. [CrossRef] 4. Jannot, Y.; Degiovanni, A. Thermal Properties Measurement of Materials; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2018. [CrossRef] 5. Maglic, K.D.; Cezairliyan, A.; Peletsky, V.E. Compendium of Thermophysical Property Measurement Methods Vol. 1, Survey of 5. Maglic, K.D.; Cezairliyan, A.; Peletsky, V.E. Compendium of Thermophysical Property Measuremen Measurement Techniques; Plenum Press: New York, NY, USA, 1984. [CrossRef] q 6. Maglic, K.D.; Cezairliyan, A.; Peletsky, V.E. Compendium of Thermophysical Property Measurement Methods Vol. 2, Rec Mmended Measurement Techniques and Practices; Springer USA: Boston, MA, USA, 1992. [CrossRef] q ; p g , , , [ ] 7. Zhao, D.; Qian, X.; Gu, X.; Jajja, S.; Yang, R. Measurement Techniques for Thermal Conductivity and Interfacial Thermal Conductance of Bulk and Thin Film Materials. J. Electron. Packag. 2016, 138, 040802. [CrossRef] q p g 7. Zhao, D.; Qian, X.; Gu, X.; Jajja, S.; Yang, R. Measurement Techniques for Thermal Co 7. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: Many thanks go to colleagues who helped make this project possible. First Competency Center Thermal Energy Storage for the opportunity to deepen knowledge empir- ically. Second to the lab facilities whose support for measurements and scans were integral in the process of realizing the test stand and understanding 3D printing as well as the internal structure of the test objects. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Zhao, D.; Qian, X.; Gu, X.; Jajja, S.; Yang, R. Measurement Techniques for Thermal Conductivity and Interfacial Thermal C d f lk d h l l l k [C f] 7. Zhao, D.; Qian, X.; Gu, X.; Jajja, S.; Yang, R. Measurement Techniques for Thermal Co Conductance of Bulk and Thin Film Materials. J. Electron. Packag. 2016, 138, 040802. [CrossR Q J jj g q Conductance of Bulk and Thin Film Materials. J. Electron. Packag. 2016, 138, 040802. [CrossRef] 8. Czichos, H.; Saito, T. Springer Handbook of Materials Measurement Methods; Smith, L., Ed.; Springer: Berlin/Heidelberg, Germany, 2006. [CrossRef] 9. Czichos, H.; Saito, T. Springer Handbook of Metrology and Testing; Smith, L., Ed.; Springer: Berlin/Heidelberg, Germany, 2011. [CrossRef] 10. VDI. VDI Heat Atlas, 2nd ed.; Springer GmbH: Berlin/Heidelberg, Germany, 2010; ISBN 978-3-540-778 p g g y 11. Hetnarski, R.B. Encyclopedia of Thermal Stresses, 2014th ed.; Springer: Berlin/Heidelberg, Germany, 2013; pp. 6186–6198. 12 COMSOL M lti h i ® 5 5 St kh l S d A il bl li l ( d 20 J l 2021) 11. Hetnarski, R.B. Encyclopedia of Thermal Stresses, 2014th ed.; Springer: Berlin/Heidelberg, Germany, 201 .B. Encyclopedia of Thermal Stresses, 2014th ed.; Springer: Berlin/Heidelberg, Germany, 2013; pp. 6186–6198. , y p f , ; p g g, y, ; pp 12. COMSOL Multiphysics®v. 5.5. Stockholm, Sweden. Available online: www.comsol.com (accessed on 20 July 2021). y p f p g g y 12. COMSOL Multiphysics®v. 5.5. Stockholm, Sweden. Available online: www.comsol.com (accessed on ultiphysics®v. 5.5. Stockholm, Sweden. Available online: www.comsol.com (accessed on 20 July 2021).
https://openalex.org/W2070749934	https://bmcinfectdis.biomedcentral.com/track/pdf/10.1186/1471-2334-14-S2-P95	English	null	Prevalence of Human Papilloma Virus in Tunisia	BMC infectious diseases	2,014	cc-by	577	Prevalence of Human Papilloma Virus in Tunisia Ali Mrabet*, MT Khoufi, C Tounsi, R Dhaoui, M Chibani, N Doss From Abstracts from International Symposium HIV and Emerging Infectious Diseases 2014 Marseille, France. 21-23 May 2013 Published: 23 May 2014 Introduction Cervical cancer is still a major health problem in Tunisia and over the world. HPV infection may induce malignant transformation of lesions, especially in case of colonization by oncogenic HPV genotypes. Early detection of viral gen- ome by molecular biology in women with normal cytology in cervical screening test (CST) can be a part of a screen- ing policy to improve cancer prevention. Our aim was to evaluate HPV prevalence and genotype distribution in rou- tine CST. doi:10.1186/1471-2334-14-S2-P95 Cite this article as: Mrabet et al.: Prevalence of Human Papilloma Virus in Tunisia. BMC Infectious Diseases 2014 14(Suppl 2):P95. doi:10.1186/1471-2334-14-S2-P95 Cite this article as: Mrabet et al.: Prevalence of Human Papilloma Virus in Tunisia. BMC Infectious Diseases 2014 14(Suppl 2):P95. Results The mean age was 41.5 ±9.1 years (20-71 y). The mean age of the marriage was 24.8±5 years (16-50 y). The age of the first pregnancy was 24.8±5.7 years (14-48 y). Six percent reported history of genital lesion in their partner. 37% used or are still using Intra Uterine Device and 48% used or are still using contraceptive pills. 58% had never had a routine CST and 27% had one CST. 65% of the women reported a history of vaginal discharge, 25% a genital ulceration and 4.5% condylomas. The cervix examination showed 51% of normal cervix, without any clinical macroscopic anomaly. 41% had a cervix inflam- mation, 18% had leucorrhea, 7% had polypus and 3% had condylomas. HPV prevalence was 3%. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit This is an Open Access article distributed under the terms of the Creative Commons icenses/by/4.0), which permits unrestricted use, distribution, and reproduction in erly cited. The Creative Commons Public Domain Dedication waiver (http:// plies to the data made available in this article, unless otherwise stated. Submit your next manuscript to BioMed Central and take full advantage of: Methods It was a prospective study conducted over a six month period (2012, January to June). 665 women were screened by an examination of the cervix, a cervical screening test and a detection of the HPV DNA by PCR. This detection of the high oncogenic risk HPV was made from the cyto- brush that was used for the spreading of the cervix cells. Mrabet et al. BMC Infectious Diseases 2014, 14(Suppl 2):P95 http://www.biomedcentral.com/1471-2334/14/S2/P95 Mrabet et al. BMC Infectious Diseases 2014, 14(Suppl 2):P95 http://www.biomedcentral.com/1471-2334/14/S2/P95 Open Access Open Access Conclusion Our results showed lower HPV prevalence, probably due to later onset of sexual practice and fewer sexual partners. Tunisian Military Health General Directorate, Tunis, Tunisia Tunisian Military Health General Directorate, Tunis, Tunisia © 2014 Mrabet et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
https://openalex.org/W2157150174	https://europepmc.org/articles/pmc4649453?pdf=render	English	null	Costimulatory T cell engagement via a novel bispecific anti-CD137 /anti-HER2 protein	Journal for immunotherapy of cancer	2,015	cc-by	717	Hinner et al. Journal for ImmunoTherapy of Cancer 2015, 3(Suppl 2):P187 http://www.immunotherapyofcancer.org/content/3/S2/P187 Hinner et al. Journal for ImmunoTherapy of Cancer 2015, 3(Suppl 2):P187 http://www.immunotherapyofcancer.org/content/3/S2/P187 Hinner et al. Journal for ImmunoTherapy of Cancer 2015, 3(Suppl 2):P187 http://www.immunotherapyofcancer.org/content/3/S2/P187 Open Access Open Access Authors’ details 1 1Pieris Pharmaceuticals, Inc., Freising, Germany. 2Stanford University, Stanford, CA, USA. Published: 4 November 2015 Conclusion We report the first bispecific therapeutic protein that targets the potent costimulatory immunoreceptor CD137 in a tumor-target dependent manner, utilizing HER2 as the tumor target. Compared to currently exist- ing CD137-targeting antibodies, this approach has the potential to provide a more localized activation of the immune system with reduced peripheral toxicity. Bispe- cific T cell engagers based on CD137 and HER2 may have utility in HER2-positive cancers where there is a significant unmet medical need. Background CD137 is a potent costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. The receptor, also known as 4-1BB, is mainly expressed on activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells. While multiple lines of evidence show that CD137 is a highly promising therapeutic target, current approaches are not designed to achieve a tumor- target driven activation. Thus, these approaches may dis- play a limited therapeutic window due to peripheral T cell and NK cell activation, leading to unwanted toxicity. To overcome this limitation, we generated a bispecific protein therapeutic binding to CD137 and to a differentially expressed tumor target, HER2. Costimulatory T cell engagement via a novel bispecific anti-CD137 /anti-HER2 protein Marlon J Hinner1*, Rachida-Siham Bel Aiba1, Alexander Wiedenmann1, Corinna Schlosser1, Andrea Allersdorfer1, Gabriele Matschiner1, Christine Rothe1, Ulrich Moebius1, Holbrook E Kohrt2, Shane A Olwill1 From 30th Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2015) National Harbor, MD, USA. 4-8 November 2015 simultaneously bound. Compared to non-engineered tras- tuzumab, binding to human receptors FcgRI and FcgRIII was significantly reduced, while binding to the neonatal Fc receptor (FcRn) was retained. All constructs were found to be fully stable in human and mouse plasma, and in mice displayed pharmacokinetics similar to trastuzumab. HER2- dependent agonistic engagement of CD137 was demon- strated in ex-vivo T cell activation assays utilizing HER2- positive human cell lines. The functional activity of the bispecific constructs was found to be dependent on their geometry. 1Pieris Pharmaceuticals, Inc., Freising, Germany Full list of author information is available at the end of the article Methods Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to CD137 with high affinity and specificity. The CD137-specific Antica- lin protein was genetically fused to a trastuzumab variant at different positions, yielding four different constructs covering a range of distances between the binding sites of the T cell-target and the tumor cell target. To minimize Fcg-receptor interaction of the resulting bispecific and concomitant potential toxicity towards CD137-positive cells, the backbone of trastuzumab was switched from IgG1 to an engineered IgG4 isotype. © 2015 Hinner et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Results All four bispecific constructs bound the targets CD137 and HER2 with a nearly identical affinity compared to the parental building blocks, and both targets could be doi:10.1186/2051-1426-3-S2-P187 Cite this article as: Hinner et al.: Costimulatory T cell engagement via a novel bispecific anti-CD137 /anti-HER2 protein. Journal for ImmunoTherapy of Cancer 2015 3(Suppl 2):P187. doi:10.1186/2051-1426-3-S2-P187 Cite this article as: Hinner et al.: Costimulatory T cell engagement via a novel bispecific anti-CD137 /anti-HER2 protein. Journal for ImmunoTherapy of Cancer 2015 3(Suppl 2):P187. 1Pieris Pharmaceuticals, Inc., Freising, Germany Full list of author information is available at the end of the article © 2015 Hinner et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated.
https://openalex.org/W2745591781	http://www.journalijar.com/uploads/951_IJAR-18606.pdf	English	null	FLOWERING AND FRUITING SEASONAL CHANGES OF SIX ACCESSIONS OF JATROPHA CURCAS L. IN A SEMI-ARID REGION OF SENEGAL.	International journal of advanced research	2,017	cc-by	7,473	Abstract ……………………………………………………………… Jatropha curcas is the priority species for biofuel promotion in Senegal. However the lack of knowledge on floral biology and fruiting of this plant remains a problem to increase its yield and improve farmers’ adoption. The objectives of this study were to assess seasonal changes on flowering and fruiting of six accessions of J curcas in semi-arid area in Senegal. In this study the total number of inflorescences, flowers and fruits were evaluated during six months on four years old plants, installed in the Agricultural Engineering School of Thies. The results showed significant effect of factors (season, accession) and interaction between them on floral and fruiting traits of J. curcas. Due to more favorable climatic conditions, the performance of flowering is better in wet season. During this season, the number of male and female flowers ranged, respectively from 107.47–191.60 and from 2.47–11.87 per inflorescence. Consequently, ripe fruits production was higher in wet season and varied between 66 and 97% of the total production. Two peaks of flowering were observed; the first one occurred in dry season and the second, with the highest number of inflorescences, arrived in wet season. Fruiting patterns had highest concentration between September 15th and October 3rd. For biofuel production in semi-arid areas of Senegal, three accessions were identified due to high seed yields. Copy Right, IJAR, 2017,. All rights reserved. FLOWERING AND FRUITING SEASONAL CHANGES OF SIX ACCESSIONS OF JATROPHA CURCAS L. IN A SEMI-ARID REGION OF SENEGAL. *Ibrahima Diedhiou1, Roger Bayala1, Moustapha Diere Sagna1 and Papa Madiallacke Diedhiou2. 1. Ecole Nationale Supérieure d’Agriculture (ENSA), Université de Thiès (UT), BP A 296, Route de Khombole, Thiès, Sénégal. 2. UFR des Sciences Agronomiques d’Aquaculture et de Technologies Alimentaires (UFR S2ATA)/Université Gaston Berger de Saint-Louis (UGB), Route de Ngallèle BP 234 Saint-Louis, Sénégal. ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 5(7), 2138-2148 ISSN: 2320-5407 ISSN: 2320-5407 ISSN: 2320-5407 Under these conditions improvement of J. curcas yield becomes imperative to enhance profitability of the plantations. To realize this perspective, it is necessary to have reliable data on flowering, fruiting and relationship between them. Indeed, these data are essential to develop and plan strategies selection and breeding (Kaur et al., 2011). In wet regions of Asia such as India, Malaysia and Indonesia, many studies focused on J. curcas phenology characteristics and especially its floral biology. These aspects have so far received little attention in North America, where it is noted exceptionally, the study of Nietsche et al. (2014) carried out in south Florida. It emerged from these investigations that J. curcas is a monoecious and diclinous (with inflorescences of unisexual flowers) species. However, sometimes J. curcas might also produce hermaphrodite flowers (Fresnedo-Ramirez, 2013). Flowering begins in first year if environmental conditions are favorable (Silip et al., 2010). However, flowering is more important in the second and third years after plantation. The flowering cycle of J. curcas can happen the whole year in wet areas (Vidal, 1962). It is noticed that plants from cuttings get flower earlier that those from seeds. The number of flowering peaks depends on moisture and nutrients availability. Flowering characteristics are significantly affected by the season (Nietsche et al., 2014) and accessions origin (Wijaya et al., 2009; Nietsche et al., 2014). The inflorescence of J. curcas is a raceme (Kaur et al., 2011). It carries more male flowers (from 25-215) than female flowers (from 0 - 16.6). (Raju and Ezradanam, 2002; Ghosh and Singh, 2008; Wijaya et al., 2009; Alam et al., 2011; Kaur et al., 2011; Nietzsche et al., 2014 ; Mat et al., 2015). The male to female flower ratio is an important factor because it is significantly correlated to yield (Wijaya et al., 2009). It is between 7.14 and 55.4 (Raju and Ezradanam 2002; Ghosh and Singh, 2008; Rao et al., 2008; Wijaya et al., 2009; Alam et al., 2011; Kaur et al., 2011; Nietzsche et al., 2014). The morphological characteristics of flowers of J. curcas (color, number of petals and sepals, size, number of stamens, features of pistil), have been described by Alam et al. (2011). J. curcas pollination is mainly provided by insects especially bees, butterflies, flies, dragonflies, beetles and ants (Ghosh and Singh, 2008; Kaur et al., 2011). Introduction:- Introduction:- Jatropha curcas L. (J. curcas) is a perennial species belonging to the Euphorbiaceae family and native of Mexico and Central America (Heller, 1996). It was introduced in Africa in the 16th century via the Cape Verde Islands. Nowadays, it is widely cultivated in Asia, Africa and America for its various products and benefits which can support an integrated rural development. The oil extracted from seeds has high-energy value whose properties are similar to diesel oil (Heller, 1996). It is also used for soap or lubricants manufacturing and the seed cakes as manure (Traoré et al., 2015). 2138 (Traoré et al., 2015). However, many studies have reported that the lower yield of this species is one of the major constraints to its integration into agricultural production systems (Wijaya et al., 2009; Diédhiou et al., 2012; Everson et al., 2013). Corresponding Author:- Ibrahima Diedhiou. Address:- Ecole Nationale Supérieure d’Agriculture (ENSA), Université de Thiès (UT), BP A 296, Route de Khombole, Thiès, Sénégal. However, many studies have reported that the lower yield of this species is one of the major constraints to its integration into agricultural production systems (Wijaya et al., 2009; Diédhiou et al., 2012; Everson et al., 2013). 2138 Int. J. Adv. Res. 5(7), 2138-2148 Int. J. Adv. Res. 5(7), 2138-2148 ISSN: 2320-5407 It is usually cross-pollinated types, but the case of pollination between flowers of the same plant happens. Fruiting of J. curcas can starts in the first year. According to Silip et al. (2010), flowering to fruit set occurs within one to eight days. Fruits develop physiologically or reach mature green stage within 21 to 35 days. The species can have until four cyclical fruiting peaks per year (Alam et al., 2011). Fruit to female flower ratio (fruit set) varies from 0.37 to 0.79 (Gosh and Singh, 2008; Kaur et al., 2011; Nietsche et al., 2014). The yield of J. curcas has wide variation (from 32 to 745.72 g/tree) according to origin, age, soil, climate and farming practices (Heller, 1996; Rao et al., 2008; Wijaya et al., 2009; Wani et al., 2012; Diédhiou et al., 2012; Shabanimofrad et al., 2013; Singh et al., 2013). Although J. curcas is considered tolerant to drought, several studies (Kaur et al., 2011; Ma et al., 2016) showed that water has a significant effect on its phenology and particularly fruiting. If J. curcas phenology seems to be well documented in Asia, while in West Africa and especially in the Sudano- Sahelian areas where climate and soil conditions are different, there is little information on this question (Ahoton and Quénum, 2012). In these conditions, the objective of this study was to analyze seasonal changes in flowering and fruiting of six J. curcas accessions in a semi-arid region of Senegal. Experimental Design:- A set of six accessions of J. curcas, where four were from Senegal, one from Tanzania and one from Mozambic was used for the experiment. Accessions codes in ENSA’s collection and the rainfall of their ecological areas are showed in table 1. Seedlings of the six accessions were produced from vigorous seeds in a nursery using polybag containing a mixture of soil and compost in the ratio of 1:1. Four months old seedlings were established in the field during August 2008. Seedlings were irrigated during 90 days after planting and at 15 days intervals. No fertilization and pruning have been done. Weeding was performed as needed, usually in rainy season. The experimental design is a randomized block design (RBD) with six treatments (accessions) and four replicates. The sub-plot was a square of 6 m side and had 16 trees. Planting spacing was 2 m in-row x 2 m between-rows, having 2500 plants per hectare. he study, trees were 42 months old. All accessions had started flowering at the second year after At the time of the study, trees were 42 months old. All accessions had started flowering at the second year after plantation. ions of J. curcas and ecological characteristics of their origin. p Table 1:- Accessions of J. curcas and ecological characteristics of their origin. Table 1:- Accessions of J. curcas and ecological characteristics of their origin. Accession Code Rainfall (mm) Ecological zone Country of collection Plantation 1 CE6 400 Sahelian Senegal Noto 3 CE14 400 Sahelian Senegal Keur Samba Gueye CE50 800 Sudanian Senegal Ngodiba CE95 800 Sudanian Senegal Tanzanie CE97 N/A N/A Tanzania Mozambique CE98 N/A N/A Mozambique (N/A): Not Available Sampling and data Collection:- Sampling and data Collection:- Flowering phenology:- Flowering phenology: The flowering survey has been done during nine months (February- October 2012) on three trees per accession, randomly selected in three blocks of the experimental plot. Quantitative flowering traits were recorded, including the total number of inflorescences per tree, the number of female flowers per inflorescence, the number of male flowers per inflorescence and the male to female flower ratio. The total number of inflorescences per tree was counted every 14 and 7 days respectively in dry season and rainy season. The flowers sex parameters were recorded by simple count method once in May (dry season) and September (wet season). ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 5(7), 2138-2148 Int. J. Adv. Res. 5(7), 2138-2148 Int. J. Adv. Res. 5(7), 2138-2148 The study site lies on a leached and disturbed ferruginous sand soil classified as a ferruginous Oxisol (FAO, 1998). The top horizon (0 - 0.7 m), is sandy with a friable continuous structure and no distinct horizonation with low clay content of about 5%. The organic matter and nitrogen content is about 0.5 and 0.3% respectively and water-retention capacity ranging from 60 to 100 mm (Zanté, 1983). Experimental Design:- In the wet season, this survey was carried out on the six accessions; but in the dry season it was conducted on only four accessions (CE6, CE95, CE6 and CE97) which produced recognizable flowers in this season. Fifteen inflorescences per each of the three selected trees per accession were sampled, so a total of 45 inflorescences were considered per accession. Material and Methods:- Study site:- y The experiment was carried out in research station of National School of Agriculture (ENSA), located at 4 km away from Thiès (14°42’52’’N and 16°28’64’’W), in Senegal. The climate is tropical semi-arid with short rainy season (June to October) and long dry season (November to May). The total annual precipitation is 480 mm (Kizito et al., 2006). During the experiment, the rain was collected with Decagon weather station (Decagon Devices, Inc., Pullman, WA, USA). The rainfall started in the third 10 days of June and become more and more important from July. August and September were wetted months with respectively, 233.6 and 226.8 mm cumulative rainfall. The quantity of precipitation of these months represents 81% of the total annual rainfall (568.2 mm). Overall, there was a good distribution of precipitation during that year. The mean annual minimum ambient temperature is 19°C and a mean annual maximum ambient temperature of 33°C. The minimum and maximum of relative humidity range from 49 to 91% in rainy season and from 27 to 75% in dry season. 2139 ISSN: 2320-5407 Flowering phenology:- g p gy The flowering rhythms of the six J. curcas accessions are summarized in Fig 1. All accessions produced inflorescences continuously throughout the observation period. However the full flowering occurred between April (dry warm season) and July (early in rainy season) for all accessions. Accessions had two peaks of flowering. The first peak occurred in dry season (April, 24th – June 6th) and the second, with the highest number of inflorescences, arrived in wet season (8 - 22 July). Fig 1:- Number of inflorescences per tree according to the time for six J. curcas accessions. 0 50 100 150 200 250 300 350 40024-Feb10-Mar25-Mar9-Apr24-Apr9-May24-May8-Jun23-Jun8-Jul23-Jul7-Aug22-Aug6-Sep21-Sep6-Oct21-Oct Number of inflorescences per tree Dates CE50 CE95 CE6 CE97 CE98 CE14 Fig 1:- Number of inflorescences per tree according to the time for six J. curcas accessions. The effect of the season on number of male flowers per inflorescence, number of female flowers per inflorescence and male to female flower ratio is shown in Table 2. There were significant differences between seasons (p < 0.05) for the number of female flowers per inflorescence but not for the number of male flowers per inflorescence. The number of female flowers per inflorescence was significantly higher in the wet season than in the dry season. Consequently, the male to female flowers ratio was lower in the wet season compared to the dry season. Table 2:- Effect of the season on flowering parameters of J. curcas. Parameters Seasons Statistical parameters Dry season Wet season F P Number of male flowers per inflorescence 143.76a 138.38a 0.58 0.448 Number of female flowers per inflorescence 4.28b 6.84a 13.91 <0.001 Male to female flower ratio 33.59 20.23 - - (-): no analysis of variance; means followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test. Table 2:- Effect of the season on flowering parameters of J. curcas. Parameters Seasons Stat The influence of the accession on number of male flowers per inflorescence, number of female flowers per inflorescence and male to female flower ratio is presented in table 3. There were significant differences between accessions (p < 0.05) for all flowering parameters. Number of male flowers per inflorescence ranged from 127.10 to 149.88 and the number of female flowers per inflorescence ranged between 3.68 and 6.88. Fruiting phenology:- The fruiting survey was performed during 10 months (February- November 2012) on four trees per accession, randomly selected in four blocks of the experimental plot. Observations were made every 14 and seven days respectively in dry season and rainy season. The yield (seed weight per hectare for the density of 2500 plants/ha) and its components (number of fruit per tree, fruit weight per tree, fruit weight and seed weight per tree) were calculated for each season and also for the whole duration of the survey. The number of fruit was recorded by simple count method. The fruit weight and the seed weight were measured by electronic balance in the laboratory. 2140 ISSN: 2320-5407 Int. J. Adv. Res. 5(7), 2138-2148 Data Analysis y The data were submitted to a two ways analysis of variance (ANOVA) using Statistix. Means separation among treatments was performed by Tukey’s test at the 5% probability level based on the F-test of ANOVA. Flowering phenology:- For the number of female flowers per inflorescence, the highest was seen in CE6 (11.58) while the lowest in CE50 (2.18). The accessions CE97 and CE98 had intermediate values for this parameter, 8.31 and 6.98 respectively. Male to female flowers ratio recorded was highest for CE6 (16.23) and lowest for CE50 (47.60). Table 5:- Influence of the accession on flowering parameters of J. curcas during wet season. Parameters Accessions Statistical Table 4:- Effect of accession and season interaction on flowering parameters of J. curcas. Variables Accessions x saisons Statistical parameters CE50 CE95 CE6 CE97 F P Dry season Wet season Dry season Wet season Dry season Wet season Dry season Wet season Number of male flowers per inflorescence 156.83 ab 103.77c 138.83bc 115.37 bc 126.10 bc 187.90a 153.27ab 146.50abc 12.05 <0.001 Number of female flowers per inflorescence 5.18bc 2.18c 5.11bc 5.31bc 1.38c 11.58a 5.45bc 8.31ab 16.71 <0.001 Male to female flower ratio 30.28 47.60 27.17 21.73 91.38 16.23 28.12 17.63 - - (-): no analysis of variance; means followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test. Table 4:- Effect of accession and season interaction on flowering parameters of J. curcas. Variables Accessions x saisons The number of female flowers per inflorescence ranged from 2.18 to 11.58. Highest values were recorded for CE6 during wet season, followed by CE97 during wet season. The range for male to female flower ratio varied from 16.23 to 91.38. Lowest values were observed for CE6 during wet season, followed by CE97 wet season and CE95 wet season. The number of female flowers per inflorescence ranged from 2.18 to 11.58. Highest values were recorded for CE6 during wet season, followed by CE97 during wet season. The range for male to female flower ratio varied from 16.23 to 91.38. Lowest values were observed for CE6 during wet season, followed by CE97 wet season and CE95 wet season. The flowering characteristics of the six accessions in the wet season are presented in Table 5. All accessions produced flowers. The analysis of variance revealed that CE6 had a higher number of male flowers (187.90) compared to the other accessions (table 5). The accession CE50 showed the lowest value of this character (103.77). For the number of female flowers per inflorescence, the highest was seen in CE6 (11.58) while the lowest in CE50 (2.18). Flowering phenology:- These parameters were significantly higher for the accessions CE6 and CE97; consequently the ratio values of these accessions were the lowest. 2141 Int. J. Adv. Res. 5(7), 2138-2148 ISSN: 2320-5407 ISSN: 2320-5407 Table 3:- Influence of the accession on flowering parameters of J. curcas. Parameters Accessions Statistical parameters CE50 CE95 CE6 CE97 F P Number of male flowers per inflorescence 130.30b 127.10b 157.00a 149.88ab 4.33 0.007 Number of female flowers per inflorescence 3.68b 5.21ab 6.48a 6.88a 4.40 0.006 Male to female flower ratio 35.41 24.40 24.23 21.78 - - (-): no analysis of variance; means followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test. The influence of the interaction between accession and season on flowering parameters is summarized in Table 4. The data showed a significant effect (p < 0.05) on all flowering parameters. The number of male flowers per inflorescence varied from 103.77 to 187.90. Maximum value of this parameter was observed for CE6 during wet season, followed by CE50 and CE97 during dry season. Table 4:- Effect of accession and season interaction on flowering parameters of J. curcas. Variables Accessions x saisons Statistical parameters CE50 CE95 CE6 CE97 F P Dry season Wet season Dry season Wet season Dry season Wet season Dry season Wet season Number of male flowers per inflorescence 156.83 ab 103.77c 138.83bc 115.37 bc 126.10 bc 187.90a 153.27ab 146.50abc 12.05 <0.001 Number of female flowers per inflorescence 5.18bc 2.18c 5.11bc 5.31bc 1.38c 11.58a 5.45bc 8.31ab 16.71 <0.001 Male to female flower ratio 30.28 47.60 27.17 21.73 91.38 16.23 28.12 17.63 - - (-): no analysis of variance; means followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test. The number of female flowers per inflorescence ranged from 2.18 to 11.58. Highest values were recorded for CE6 during wet season, followed by CE97 during wet season. The range for male to female flower ratio varied from 16.23 to 91.38. Lowest values were observed for CE6 during wet season, followed by CE97 wet season and CE95 wet season. The flowering characteristics of the six accessions in the wet season are presented in Table 5. All accessions produced flowers. The analysis of variance revealed that CE6 had a higher number of male flowers (187.90) compared to the other accessions (table 5). The accession CE50 showed the lowest value of this character (103.77). Fruiting phenology:- g p gy Figure 2 summarizes the fruits production of accessions from February to November. All accessions showed continuous fruit production during the experiment survey and average fruits production patterns were fairly similar for all accessions. Fruiting patterns is highest concentration between September 15th and October 3rd. Fig. 2:- Dry fruit weight per tree according to the time for six J. curcas accessions. 0 200 400 600 800 1000 1200 1400 1600 27-Feb 13-Mar 28-Mar 12-Apr 27-Apr 12-May 27-May 11-Jun 26-Jun 11-Jul 26-Jul 10-Aug 25-Aug 9-Sep 24-Sep 9-Oct 24-Oct 8-Nov Dry fruit weight per tree (g) Dates CE50 CE95 CE6 CE97 CE98 CE14 Fig. 2:- Dry fruit weight per tree according to the time for six J. curcas accessions. 0 200 400 600 800 1000 1200 1400 1600 27-Feb 13-Mar 28-Mar 12-Apr 27-Apr 12-May 27-May 11-Jun 26-Jun 11-Jul 26-Jul 10-Aug 25-Aug 9-Sep 24-Sep 9-Oct 24-Oct 8-Nov Dry fruit weight per tree (g) Dates CE50 CE95 CE6 CE97 CE98 CE14 Fig. 2:- Dry fruit weight per tree according to the time for six J. curcas accessions. During dry (end of April) CE50, CE95 and CE6 had low fruit production while CE97, CE98 and CE14 had nearly no production. In full production period, CE95 and CE97 produced 66 % and 97 % of total production respectively. For EC50, EC6, CE98 and CE14, average was 73 %, 87 %, 94 % and 95 % of total fruit production. During dry (end of April) CE50, CE95 and CE6 had low fruit production while CE97, CE98 and CE14 had nearly no production. In full production period, CE95 and CE97 produced 66 % and 97 % of total production respectively. For EC50, EC6, CE98 and CE14, average was 73 %, 87 %, 94 % and 95 % of total fruit production. The effect of the season on yield parameters is shown in table 6. The two way analysis of variance revealed that the season had a significant effect (p<0.05) on all the yield parameters except one fruit weight (Table 6). Values recorded for the wet season were significantly higher than those observed in the dry season. Seed yield, noted for the wet season (1282.10 kg ha-1) was 13 times higher than that of the dry season (99.40 kg ha-1). Table 6:- Effect of the season on fruit weight per tree, one fruit weight, seed weight per tree and seed yield. Flowering phenology:- The accessions CE97 and CE98 had intermediate values for this parameter, 8.31 and 6.98 respectively. Male to female flowers ratio recorded was highest for CE6 (16.23) and lowest for CE50 (47.60). Table 5:- Influence of the accession on flowering parameters of J. curcas during wet season. Parameters Accessions Statistical parameters CE50 CE95 CE6 CE97 CE98 CE14 F P Number of male flowers per inflorescence 103.77 c 115.37bc 187.90a 146.50b 137.77bc 122.03bc 9.64 <0.001 Number of female flowers per inflorescence 2.18c 5.31bc 11.58a 8.31ab 6.98b 4.98bc 11.17 <0.001 Male to female flower ratio 47.60 21.73 16.23 17.63 19.74 24.50 - - 2142 Int. J. Adv. Res. 5(7), 2138-2148 ISSN: 2320-5407 (-): no analysis of variance; means followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test. The influence of the accession on yield components is summarized in Table 7. There were significant differences between accessions for yield parameters, out of fruit weight. Fruit weight per tree ranged from 243.20 g (CE98) to 826.35 g (CE6) while seed weight per tree varied from154.38 g (CE98) to 547.90 g (CE6). Seed yield per hectare were between 385.90 (CE98) and 1369.80 (CE6). For all these parameters, the accession CE6 had the highest values, followed by CE97 and CE95. Fruiting phenology:- Parameters Seasons Statistical parameters Dry season Wet season F P Fruit weight per tree (g) 62.25b 788.43a 71.02 <0.001 One fruit weight (g) 1.920a 2.030a 1.46 0.235 Seed weight per tree (g) 39.78b 512.83a 72.60 <0.001 Seed yield (kg ha-1) 99.40b 1282.10a 72.60 <0.001 Means followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test. The influence of the accession on yield components is summarized in Table 7. There were significant differences between accessions for yield parameters, out of fruit weight. Fruit weight per tree ranged from 243.20 g (CE98) to 826.35 g (CE6) while seed weight per tree varied from154.38 g (CE98) to 547.90 g (CE6). Seed yield per hectare were between 385.90 (CE98) and 1369.80 (CE6). For all these parameters, the accession CE6 had the highest values, followed by CE97 and CE95. 2143 Int. J. Adv. Res. 5(7), 2138-2148 ISSN: 2320-5407 Table 7:- Effect of the accession on fruit weight per tree, one fruit weight, seed weight per tree and seed yield. Variables Accessions Statistical parameters CE50 CE95 CE6 CE97 CE98 CE14 F P Fruit weight per tree (g) 272.40b 421.70ab 826.35a 455.12ab 243.20b 333.25b 4.07 0.005 One fruit weight (g) 1.83a 1.86a 1.98a 1.96a 2.07a 2.14a 1.16 0.352 Seed weight per tree (g) 175.50b 265.30ab 547.90a 290.23ab 154.38b 224.51b 4.40 0.003 Seed yield (kg ha-1) 438.7b 663.30ab 1369.80a 725.60ab 385.90b 561.30b 4.40 0.003 Means followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test. The effect of the interaction between accession and season on yield parameters is shown in Table 8. Results indicated that except fruit weight, all yield parameters (fruit weight per tree, seed weight per tree, seed yield) was significantly affected by the interaction between the season and the accession (p <0.05). The highest fruit weight per tree was recorded in CE6 (1552.70 g) and CE97 (893.50 g) during the wet season. The lowest value of this parameter was observed in CE97 (16.70 g) during the dry season. But there was no significant difference between that value and those of CE14, CE98, CE50, CE6 and CE95 during the dry season. Table 8:- Interaction effect of accession and season on fruit weight per tree, one fruit weight, seed weight per tree and seed yield. Fruiting phenology:- Variables Yield parameters Accession Season Fruit weight per tree (g) One fruit weight (g) Seed weight per tree (g) Seed yield (kg ha-1) CE50 Dry Season 80.00c 1.69a 50.80c 127.00c CE50 Wet season 464.80bc 1.96a 300.20bc 750.50bc CE95 Dry Season 139.10c 1.86a 85.40c 213.40c CE95 Wet season 704.30bc 1.85a 445.30bc 1113.10bc CE6 Dry Season 100.00c 1.99a 65.70c 164.30c CE6 Wet season 1552.70a 1.97a 1030.10a 2575.30a CE97 Dry Season 16.70c 1.98a 11.40c 28.4c CE97 Wet season 893.50ab 1.95a 569.10ab 1422.80ab CE98 Dry Season 19.70c 2.01a 12.90c 32.30c CE98 Wet season 466.80bc 2.14a 295.90bc 739.60bc CE14 Dry Season 18.10c 1.96a 12.60c 31.40c CE14 Wet season 648.50bc 2.31a 436.50bc 1091.20bc F 3.50 0.51 3.78 3.78 P 0.012 0.769 0.008 0.008 Means followed by the same letter within columns do not differ significantly at P<0 05 by Tukey’s test Table 8:- Interaction effect of accession and season on fruit weight per tree, one fruit weight, see nd seed yield. tion effect of accession and season on fruit weight per tree, one fruit weight, seed weight per tree s Yield parameters Means followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test. Means followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test Seed weight per tree was significantly higher in CE6 (1030.10 g) and CE97 (569.10 g) during the wet season. It was significantly lower in CE97, CE14, CE98, CE50, CE6 and CE95 in the dry season. The trend is similar for seed yield. ans followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test Discussion:- Discussion: This study has shown significant effect of the studied factors (season, accession) and interaction between them on floral traits of J. curcas. The observations revealed that flowering of the six accessions studied was continuous with two peaks. The first peak occurred in hot dry season while the second, with the highest number of inflorescences, appeared early in the wet season. These results were reported in Asia (Sukarin et al., 1987; Kaur et al., 2011) and Florida (Nietzsche et al., 2014). Full bloom of J. curcas occurred between the beginning of April and the end July. Flowering parameters including the number of female flowers per inflorescence and male to female flower ratio change significantly with the season. Overall, wet season has significantly fostered female flowers development and induced low male to female ratio. Thus, the number of female flowers ranged of 2.47- 11.87 in the wet season with an average of 7.13. In the dry season, this parameter varied from 1.67 to 5.73 with an average of 4.56. This is consistent with observations reported for Florida (Nietzsche et al., 2014). The period of full bloom of the accessions and installation of the second flowering peak coincides with the warmer months with temperatures ranging between 27.5 (June) and 27.7 °C (July). Also, the wet season begins in the study region, between June and July. Therefore, the best performance of flowering in the starting of the wet season (higher peak and highest production of female flowers), established in this study, may be related to climatic conditions particularly the high temperatures and increase of soil moisture with installing of rainfall. These observations suggest that floral initiation and development of female flowers of J. curcas requires high temperatures and moderate humidity. In a study conducted by Nietzsche et al. (2014) in Florida, it has been observed that J. curcas flowering begins with rising temperatures and precipitation. Wu et al. (2011) reported that female sites on inflorescences of J. curcas occur mostly in seasons with high temperature and rainfalls. They also observed that the right amount of rainfall may possibly enhance the number of female flowers for each inflorescence, while too much or too less would not favor the occurrence of female flowers. However, we found in this study, that effect of season on the number of male flowers was not significant. Discussion:- This result suggests that the production of female flowers is more environmentally sensitive than the male flowers that would depend more on genetic factors. It is consistent with the observations of Anandalakshmi et al. (2015) that showed that female flower production is more sensitive than male flowers. In a study conducted in China to understand organization of the flower sexes in J. curcas, Wu et al. (2011) documented that, male and female flowers have similar tissues and organs before female primordia emergence. So, they proposed that there are factors that regulate sex differentiation in this plant, which may selectively affect the action of homeotic genes in one whorl, such as only restraining the initiation of a gynoecium meristem in male J. curcas flowers. This study also showed that the flowering characteristics vary significantly among accessions. In wet season, CE6 had more male (191.60) and female (11.87) flowers per inflorescence with lowest male to female flower ratio (16.40). In contrast, CE50 with lowest number of male (107.47) and female (2.47) flowers per inflorescence had highest male to female ratio (43.00). In the dry season, the highest number of male (154.87), female (5.73) flowers per inflorescence and lowest male to female ratio (22.81) was observed on CE97. These results are within the range of values obtained by previous studies (Raju and Ezradanam 2002; Ghosh and Singh, 2008; Rao et al., 2008; Wijaya et al., 2009; Alam et al., 2011; Kaur et al., 2011; Nietzsche et al., 2014; Mat et al., 2015), for the number of male flowers (25-215), the number of female flowers (0 to 16.6) flowers and male to female flower (7.14-55.4). Wu et al. (2011) explained the lowest number of female flowers on J. curcas inflorescences by the fact that the female and male flowers have remarkable position location features. But, female flowers do not appear at the male sites, while male flowers may develop at the female sites. The present study has shown significant effect of the studied factors (season, accession) and interaction between them on fruiting traits of J. curcas. It showed that the fruiting rhythm of J. curcas in semi-arid area in Senegal is continuous with a short period of full production (September - October). CE14 666.30ab 448.50b 1122.50b F 3.81 3.96 4.27 P 0.012 0.010 0.010 Means followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test. CE14 666.30ab 448.50b 1122.50b F 3.81 3.96 4.27 P 0.012 0.010 0.010 Means followed by the same letter within columns do not differ significantly at P<0.05 by Tukey’s test. Annual yield:- y Cumulative values (from February to November 2012) of yield parameters are shown in table 9. Whatever the parameter considered, highest values were recorded in CE6, CE97 and CE95, while lowest value was observed in CE98. Table 9:- Cumulative values of yield parameters for 6 accessions of J. curcas. Accession Fruit weight per tree (g) Seed weight per tree (g) Seed yield (kg ha-1) CE50 544.50b 350.80b 877.30b CE95 843.00ab 530.00ab 1326.30ab CE6 1652.30a 1095.50a 2739.30a CE97 910.00ab 580.00ab 1451.00ab CE98 486.00b 308.50b 771.50b 2144 Int. J. Adv. Res. 5(7), 2138-2148 ISSN: 2320-5407 ISSN: 2320-5407 ISSN: 2320-5407 ISSN: 2320-5407 Some accessions (CE14, CE97 and CE98) had no fruit set in dry season but their full production ranged from 94 to 97 % of total production. In contrast CE6, CE50 and CE95 had fruit set in dry season and their full production varied from 66 to 87 % of total fruit production. These results indicate that J. curcas harvest should be done in wet and dry season according to accessions. This would reduce the high labor costs of harvesting J. curcas fruit (Everson et al., 2013). This study also noted that the season has a significant influence on yield parameters. Overall, production of fruits and seeds was better in the wet season than in the dry season. Thus, seed weight per tree ranged from 11.40 to 85.40 g in dry season with an average of 39.78 g; in wet season it was between 295.90 and 1030.10 g with an average of 512.83 g. High fruit and seed production of accessions in the wet season are due to better high flowering with a greater number of female flowers (Ferry, 2006). In addition, the values of male to female flowers ratio observed show that there are enough male flowers to ensure good pollination. However, the low number of female flowers is a major constraint of production. Our results can be explained by the fact that in the wet season, the efficiency of flowering (number of fruits / number of female flowers per inflorescence) is higher and humidity conditions are more favorable to fruit growth. Ma et al. (2016) reported that J. curcas have high demand for water during flowering and fruiting period in the dry-hot valley of Chin-sha River China. Anandalakshmi et al. (2015) mentioned that the annual increase in fruiting success of a three year old plantation in Tamil Nadu, India, was due to sufficient rainfall accompanied by availability of nutrients. In South Florida, where summer season is characterized by high average precipitation (1473 mm/yr) and high average temperatures (29 °C), Nietzsche et al. (2014) reported that fruit set average is highest in that season (75.5%) compared to fall, indicating a positive effect of climate on this characteristic. Also, our results showed that there are significant differences among accessions for the cumulative yield (dry and wet seasons). Conclusion:- This work demonstrated that flowering and fruiting of J. curcas were continuous during ten months, with periods of full bloom and full fruit production. The flowering cycles of different accessions showed one to two peaks, one in the dry season and another one early in the rainy season. The characteristics of flowering and fruiting change significantly according to the accessions and the season. The wet season has been more favorable for the production of flowers. Also during this period, J. curcas had between 66 and 97% of total fruit production. Four accessions had highest seed yield (≥1 t ha-1); and could be promising for the production of biofuel. Based on our results, to better understand the reproductive biology and phenology of J. curcas, it is important to determine the role of climate and soil moisture in the flowering and fruiting of this species. ISSN: 2320-5407 In terms of yield, CE6 is the better accession with a cumulative seed yield of 1095.50 g per tree or 2739.3 kg ha-1. CE98 and CE50 with lowest number of flowers in wet season produced 308.50 and 350.80 g per tree as cumulative seed yield, respectively. Previous works (Rao et al., 2008; Wijaya et al., 2009; Wani et al., 2012; Diedhiou et al., 2012b; Shabanimofrad et al., 2013; Singh et al., 2013) in Asia and in South Africa (Everson et al., 2013) demonstrated that seed yield ranged from 32 to 745.72 g per tree for 5-years field of J. curcas. However, the maximum yield obtained in our study is higher than the maximum values found by these authors, due to differences between environmental conditions, tillage and plant material (Chang-wei et al., 2007; Nietzsche et al., 2014). In this study, the highly significant variability of floral and fruiting traits found among accessions, could be associated with the genetic differences between these accessions and the climate conditions, particularly temperature, since plants grown in relatively homogeneous soil conditions. Therefore, it is important to select high yielding accessions to improve the profitability of plantations. In this perspective, as suggested by Singh et al. (2010), the number of female flowers borne by the plant may be given more weightage rather than selecting plants with better male to female ratio and number of inflorescences borne by the plant. However, our results suggest that we can also improve the performance of plantations through water supply at the beginning of flowering and dry season. Discussion:- Considering the average length of fruit set to physiological maturity is 3 to 5 weeks (Silip et al., 2010), that indicates that the majority of fruits are from fecundation that took place during the first ten days of August. 2145 Int. J. Adv. Res. 5(7), 2138-2148 Int. J. Adv. Res. 5(7), 2138-2148 Int. J. Adv. Res. 5(7), 2138-2148 References:- (2011): Floral biology and breeding system of Jatropha curcas in North-Western India. Journal of Tropical Forest Science 23(1): 4–9. 13. Kizito, F., Dragila, M. I., Sène, M., Lufafa, A., Dick, R. P., Diedhiou, I., Dossa, E., Khouma, M., Ndiaye, S. and Badiane, A. (2006): Seasonal soil water variation and root patterns among two semi-arid shrubs coexisting with Pearl millet in Senegal, West Africa. Journal Arid Environments 67: 436–455. 14. Ma S., Zhou L., Pu G., Lei B., Hou L., Dai X. and Yu T. (2016) : Effects of altitude and water on flowering and fruiting of Jatropha curcas L. Pak. J. Bot., 47(2): 537-541. 15. Mat, N. H. C., Bhuiyan, M. A. R., Senan, S., Yaakob, Z., and Ratnam, W. (2015): Selection of high yielding Jatropha curcas L. accessions for elite hybrid seed production. Sains Malaysiana 44(11): 1567–1572. 16 Ni t h S V d W A C J H d P i M C T (2014) A t f d ti 16. Nietsche, S., Vendrame, W. A. Crane, J. H. and Pereira, M. C. T. (2014): Assessm characteristics of Jatropha curcas L. in south Florida. GCB Bioenergy 6: 351–359. 17. Raju, A. J. S. and Ezradanam, V. (2002): Pollination ecology and fruiting behaviour in a mon Jatropha curcas L. (Euphorbiaceae). Current Science 83: 1395-1398. 18. Rao, G., Korwar, G., Shanker, A. K. and Ramakrishna, Y. (2008): Genetic associations, variability and diversity in seed characters, growth, reproductive phenology and yield in Jatropha curcas (L.) accessions. Trees 22: 697– 709. 19. Shabanimofrada, M., Rafiia, M. Y., Megat Wahaba, P. E., Biabania, A. R. and Latif, M. A. (2013): Phenotypic, genotypic and genetic divergence found in 48 newly collected Malaysian accessions of Jatropha curcas L. Industrial Crops and Products 42: 543– 551. p 20. Silip J. J., Tambunan A. H., Hambali H; Sutrisno, S., and Surahman, M. (2011): Life cycle Duration and Maturity Heterogeneity of Jatropha curcas L. Journal of Sustainable Development 3(2): 1-5. 21. Singh A.S., Patel M.P., Patel T.K., Delvadia D.R., Patel D.R., Kumar N., Naraynan S., and Fougat R.S., (2010): Floral biology and flowering phenology of Jatropha curcas. Journal of Forest Science, 26 (2): 95-102. gy g p gy p f 22. Singh, B., Singh, K., Rao, G. R., Chikara, J., Kumar, D., Mishra, D. K., Saikia, S. P., Pathre, U. V., Raghuvanshi, N., Rahi, T. S. and Tuli, R. References:- 1. Ahoton, L E., Quenum, F. (2012): Floral biology and hybridization potential of nine accessions of Physic Nut (Jatropha curcas L.) originating from three continents. Tropicultura 30(4): 193-198. 1. Ahoton, L E., Quenum, F. (2012): Floral biology and hybridization potential of nine accessions of Physic Nut (Jatropha curcas L.) originating from three continents. Tropicultura 30(4): 193-198. 2. Alam, N .C. N., Abdullah, T. L. and Abdullah, N. A. P. (2011): Flowering and Fruit Set Under Malaysian Climate of Jatropha curcas L. American Journal of Agricultural and Biological Sciences 6(1): 142-147. 3. Anandalakshmi, R. Sivakumar, V. Warrier, R. R. and Narmatha Bai, V (2015): Dynamics of flowering and fruiting in Jatropha curcas l. and its implications. Electronic Journal of Plant Breeding 6(3): 813-825. 4. Chang-wei, L. Kun, L. You, C. and Yong-yu, S. (2007): Floral display and breeding system of Jatropha curcas L. Forestry Studies in China 9 (2): 114-119. 5. Diédhiou, I., Diédhiou, P. M., Ndir, K. N., Bayala, R., Ouattara, B., Mbaye, B., Kâne, M., Dia, D., and Wade, I. (2012): Diversity, farming systems, growth and productivity of Jatropha curcas L. Sudano-Sahelian Zone in Senegal, West Africa. In Carels, N; Sujatha, M; Bahadur B (eds) Jatropha, Challenges for a New Energy Crop. Springer Science + Business Media, New York, USA pp. 281-295. st Africa. In Carels, N; Sujatha, M; Bahadur B (eds) Jatropha, Challenges for a New Energy Crop ence + Business Media, New York, USA pp. 281-295. p g pp 6. Everson, C. S., Mengistu, M. G., Gush, and M. B. (2013): A field assessment of the agronomic performance and water use of Jatropha curcas in South Africa. Biomass and Bioenergy 59: 59-69. 7. FAO (1998) World Reference Base for Soil Resources. Rome: Food and Agriculture Organization of the United Nations. 8. Ferry Y. (2006): Menghitung perkiraan produksi Jarak Pagar. Info Tek Jarak Pagar (Jatropha curcas L.) 1(2): 5-8. 9. Fresnedo-Ramirez, J. (2013):The floral biology of Jatropha curcas L. – A review. Tropical Plant Biology 6 (1): 1-15. DOI: 10.1007/s12042-012-9113-x 10. Ghosh, L., and Singh, L. (2008) : Phenological changes in Jatropha curcas in subhumid dry tropical environment. Journal of Basic and Applied Biology 2(1): 1-8. 11. Heller, J. (1996): Physic nut. Jatropha curcas L. Promoting the conservation and use of underutilized and neglected crops. Rome: International Plant Genetic Resources Institute. 12. Kaur, K., Dhillon, G. P. S., and Gill, R. I. S. Acknowledgements:- g The authors are grateful to African Union and to European Union for financial assistance to this study through “10th European Fund for Development” and African Union Research Grant (EU – financed grant contracts for external actions), grant contract N° AURG/094/2012 CRS N° 2012/289-014. The authors are thankful to Moussa NDIONE and Chérif CISSE for help in collection of J. curcas seeds and management of the plantation. 2146 Int. J. Adv. Res. 5(7), 2138-2148 ISSN: 2320-5407 26. Wani, T A., Kitchlu, S., and Ram, G. (2012): Genetic variability studies for morphological and qualitative attributes among Jatropha curcas L. accessions grown under subtropical conditions of North India. South African Journal of Botany 79: 102–105. 28. Wu, J., Liu, Y., Tang, L., Zhang, F. and Chen, F. (2011): A study on structural features in early flower development of Jatropha curcas L. and the classification of its inflorescences. African Journal of Agricultural Research 6 (2): 275-284. 29. Zante, P. 1983. Etude pédologique du domaine de l’Institut National du développement rural (Thiès-Sénégal). Dakar : Ed. ORSTOM. Vidal V. A. (1962) : Oleaginosas do Ultramar Portugês. Mem. Junta Invest. Ultramar 31: 129-145 f f y 27. Wijaya, A., Tidiana, S., Harun, M. U. and Hawalid, H. (2009): Flower characteristics and the yield of Jatropha (Jatropha curcas L.) accessions. HAYATI Journal of Biosciences 16(4): 123-126. References:- (2013): Agro-technology of Jatropha curcas for diverse environmental conditions in India. Biomass and Bioenergy 48: 191-202. gy 23. Sukarin, W., Yamada, Y. and Sakaguchi, S. (1987): Characteristics of physic nut, Jatropha curcas L. a new biomass crop in the tropics. Japan Agricultural Research Quartely 20: 302–303. 24. Traoré, M., Nacro, H. B., Doamba, W. F., Tabo, R. and Nikiema, A. (2015) : Effets de doses variées du tourteau de Jatropha curcas sur la productivité du mil (variété HKP) en condition pluviale en Afrique de l’Ouest. Tropicultura 33(1): 19-25. 2147 ISSN: 2320-5407 Int. J. Adv. Res. 5(7), 2138-2148 Int. J. Adv. Res. 5(7), 2138-2148 Int. J. Adv. Res. 5(7), 2138-2148 ISSN: 2320-5407 26. Wani, T A., Kitchlu, S., and Ram, G. (2012): Genetic variability studies for morphological and qualitative attributes among Jatropha curcas L. accessions grown under subtropical conditions of North India. South African Journal of Botany 79: 102–105. f f y 27. Wijaya, A., Tidiana, S., Harun, M. U. and Hawalid, H. (2009): Flower characteristics and the yield of Jatropha (Jatropha curcas L.) accessions. HAYATI Journal of Biosciences 16(4): 123-126. 28. Wu, J., Liu, Y., Tang, L., Zhang, F. and Chen, F. (2011): A study on structural features in early flower development of Jatropha curcas L. and the classification of its inflorescences. African Journal of Agricultural Research 6 (2): 275-284. 29. Zante, P. 1983. Etude pédologique du domaine de l’Institut National du développement rural (Thiès-Sénégal). Dakar : Ed. ORSTOM. 2148
https://openalex.org/W2801689618	https://europepmc.org/articles/pmc5944396?pdf=render	English	null	Gender Dysphoria in Adults: An Overview and Primer for Psychiatrists	Transgender health	2,018	cc-by	15,734	1Mental Illness Research Education and Clinical Center, James J Peters VA Medical Center, Bronx, New York. 2Department of Psychiatry, Icahn School of Medicine at Mount Sinai and Center for Transgender Medicine and Surgery at Mount Sinai, New York, New York. 3Department of Psychiatry, University of California, San Francisco, San Francisco, California. 4Program in Human Sexuality, Department of Family Medicine and Community Health, University of Minnesota Medical School, Minneapolis, Minnesota. 5Departments of Psychiatry and Family Medicine, University of Vermont College of Medicine, Burlington, Vermont. 6Department of Psychiatry, Division on Substance Use Disorders, College of Physicians and Surgeons of Columbia University, New York, New York. 7Division of Gender, Sexuality, and Health, New York State Psychiatric Institute/Department of Psychiatry, College of Physicians and Surgeons of Columbia University, New York, New York. 8Department of Psychiatry, Division of Child and Adolescent Psychiatry, Hofstra North Shore-LIJ School of Medicine, Albert Einstein College of Medicine, Zucker Hillside Hospital, Ambulatory Care Pavilion, Glen Oaks, New York. 9Department of Psychiatry, Division of Gender, Sexuality and Health, College of Physicians and Surgeons of Columbia University, New York, New York. Address correspondence to: William Byne, MD, PhD, James J. Peters VA Medical Center, Research Bldg, Rm 5F-04B, Bronx, NY 10468, E-mail: william.byne@mssm.edu This report was prepared by the American Psychiatric Association (APA) Workgroup on Gender Dysphoria with oversight by the APA Council on Quality Care. This article has been extracted and revised from a larger document, ‘‘Gender Dysphoria and Gender-variant Patients: A Primer for Psychiatrists,’’ which was approved by the APA Board of Trustees as an APA Resource Document. A course based on this content was also presented as APA Course 315, Transgender and Intersex for the PracticingPsychiatrist, given at the 168th Annual Meeting of the APA, May 16, 2015, Toronto, Canada, and Course 4196 by the same name given at the 169th Annual Meeting of the APA, May 24, 2016, Atlanta, GA. ª William Byne et al. 2018; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Regardless of their area of specialization, adult psychiatrists are likely to encounter gender-variant patients; how- ever, medical school curricula and psychiatric residency training programs devote little attention to their care. This article aims to assist adult psychiatrists who are not gender specialists in the delivery of respectful, clinically competent, and culturally attuned care to gender-variant patients, including those who identify as transgender or transsexual or meet criteria for the diagnosis of Gender Dysphoria (GD) as deﬁned by The Diagnostic and Stat- istical Manual of Mental Disorders (5th edition). The article will also be helpful for other mental health profession- als. The following areas are addressed: evolution of diagnostic nosology, epidemiology, gender development, and mental health assessment, differential diagnosis, treatment, and referral for gender-afﬁrming somatic treat- ments of adults with GD. rds: assessment; gender dysphoria; gender transition; mental health; psychiatry; intersex; transgend history in this area and many are likely to have encoun- tered providers who adhere to outdated stigmatizing the- ories and approaches to treatment.3 Today’s mental health professionals should, therefore, be familiar with the history in this area as it is not unusual for gender- variant patients to have apprehensions about seeking mental healthcare or to raise questions about their providers’ views and approach to treatment consid- ering that history. Gender Dysphoria in Adults: An Overview and Primer for Psychiatrists William Byne,1,2, Dan H. Karasic,3 Eli Coleman,4 A. Evan Eyler,5 Jeremy D. Kidd,6 Heino F.L. Meyer-Bahlburg,7 Richard R. Pleak,8 and Jack Pula9 William Byne,1,2,* Dan H. Karasic,3 Eli Coleman,4 A. Evan Eyler,5 Jeremy D. Kidd,6 Heino F.L. Meyer-Bahlburg,7 Richard R. Pleak,8 and Jack Pula9 Transgender Health Volume 3.1, 2018 DOI: 10.1089/trgh.2017.0053 Transgender Health Volume 3.1, 2018 DOI: 10.1089/trgh.2017.0053 WHITE PAPER ª William Byne et al. 2018; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction Individuals who would likely be considered transgen- der today are evident throughout the historical record.1 The historical and sociocultural conceptualizations of gender variance, and their evolution within mental health professions over the past century and a half are reviewed elsewhere.2 Individuals who would likely be considered transgen- der today are evident throughout the historical record.1 Individuals who would likely be considered transgen- der today are evident throughout the historical record.1 Individuals who would likely be considered transgen- der today are evident throughout the historical record.1 The historical and sociocultural conceptualizations of gender variance, and their evolution within mental health professions over the past century and a half are reviewed elsewhere.2 The historical and sociocultural conceptualizations of gender variance, and their evolution within mental health professions over the past century and a half are reviewed elsewhere.2 Nineteenth and 20th century theories of gender vari- ance and views of appropriate treatment were pathologiz- ing and highly stigmatizing to transgender people.2 While mainstream psychiatry is now more afﬁrming of gender variance, transgender individuals often are aware of the Between 1963 and 1979, over 20 university-based gender identity clinics opened in the United States.2,4 These clinics provided interdisciplinary care that 57 Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 58 network of medical and mental health providers, often af- ﬁliated with the Harry Benjamin International Gender Dysphoria Association (HBIGDA), which was subse- quently renamed the World Professional Association for Transgender Health (WPATH). HBIGDA/WPATH developed and successively revised standards of care (SOC) for gender transition, which are currently in their seventh revision as the WPATH SOC7.7 In the WPATH SOC7, mental health professionals are tasked with determining whether those interested in gender- afﬁrming treatments meet eligibility criteria, have capacity for informed consent, and have adequately anticipated the psychosocial impacts of their transition. included psychiatrists and other mental health profes- sionals and played an important role in the provision of medical services to transgender people and in promot- ing research to improve their care.2,4 The majority of these clinics closed following a 1981 decision of the U.S. On ofﬁcial documents such as birth certiﬁcates, driver’s licenses, and passports, the traditional category ‘‘sex’’ is equivalent to ‘‘gender’’ in current psychological terminology. ‘‘Trans’’ (also ‘‘Trans’’) More recent umbrella terms being increasingly used to avoid distinguishing between transgender and transsexual individuals. DSM, Diagnostic and Statistical Manual of Mental Disorders; GD, Gender Dysphoria; GID, Gender Identity Disorder; ICD, International Classiﬁ- cation of Diseases. Introduction Department of Health and Human Services (HHS) that labeled sex reassignment surgery as experimental,5 a decision what was overturned by HHS in 2014 in a determination that concluded that the 1981 decision was ‘‘unreasonable and contrary to contemporary sci- ence and medical standards of care.’’6 With the closure of the academic gender clinics, trans- gender people in the United States came to rely on a loose Table 1. Glossary t of a gender to an individual. In typically developed newborns, the initial gender assignment (aka ‘‘birth-assigned the basis of the appearance of the external genitalia. Gender binary: a gender-categorization system limited to the two options, male and female. Individuals who identify outside the gender binary may use a variety of gender identity labels, including genderqueer or nonbinary. Gender dysphoria (not capitalized): distress caused by the discrepancy between one’s experienced/expressed gender and one’s assigned gender and/or primary or secondary sex characteristics. y (capitalized): a diagnostic category in DSM-5, with specific diagnoses defined by age group-specific sets of criteria. T y GD in adults. p y y Gender Dysphoria (GD) (capitalized): a diagnostic category in DSM-5, with specific diagnoses defined by age group-sp article addresses only GD in adults. Gender identity: one’s identity as belonging or not belonging to a particular gender, whether male, female, or a nonbinary alternative, aka experienced gender. p g Gender Identity Disorder (GID) a diagnostic category in DSM-III and DSM-IV that was replaced in DSM-5 by GD. Gender incongruence (not capitalized): incongruence between experienced/expressed gender and assigned gender, and/or psychical gender characteristics. y ( ) g g y p y Gender incongruence (not capitalized): incongruence between experienced/expressed gender and assigned gender, and/or psychical gender characteristics. Gender Incongruence (capitalized): a diagnostic category (analogous to GD in DSM-5) proposed for ICD-11. Gender role: cultural/societal definition of the roles of males and females (or of alternative genders). Gender Incongruence (capitalized): a diagnostic category (analogous to GD in DSM-5) proposed for ICD-11. d l l l/ l d f f h l f l d f l ( f l d ) der Incongruence (capitalized): a diagnostic category (analogous to GD in DSM-5) proposed for ICD-11. Gender Incongruence (capitalized): a diagnostic category (analogous to GD in DSM-5) proposed for ICD-11. Gender role: cultural/societal definition of the roles of males and females (or of alternative genders). Gender Incongruence (capitalized): a diagnostic category (analogous to GD in DSM-5) proposed for ICD-1 Gender role: cultural/societal definition of the roles of males and females (or of alternative genders). g p g g y g p p der role: cultural/societal definition of the roles of males and females (or of alternative genders). d h h h h h d d l l h d d h der transition: the process through which individuals alter their gender expression and/or sex characteristics to align w dentity. Table 1. Glossary Gender transition: the process through which individuals alter their gender expression and/or sex characteristics to alig identity. Gender variance: any variation of experienced or expressed gender from socially ascribed norms within the gender b Gendered behavior: behavior in which males and females differ on average. g Genderqueer: an identity label used by some individuals whose experienced and/or expressed gender does/do not conform to the male/female binary or who reject the gender binary. g Genderqueer: an identity label used by some individuals whose experienced and/or expressed gender does/do not conform to the male/female binary or who reject the gender binary. Intersex conditions: a subset of the somatic conditions known as ‘‘disorders of sex development’’ or ‘‘differences of sex development ‘‘in which chromosomal sex is inconsistent with genital sex, or in which the genital or gonadal sex is not classiﬁable as either male or female. Some individuals who report their identity as ‘‘intersex’’ do not have a veriﬁable intersex condition. Intersex conditions: a subset of the somatic conditions known as ‘‘disorders of sex development’’ or ‘‘differences of sex development ‘‘in which chromosomal sex is inconsistent with genital sex, or in which the genital or gonadal sex is not classiﬁable as either male or female. Some individuals who report their identity as ‘‘intersex’’ do not have a veriﬁable intersex condition. Intersex conditions: a subset of the somatic conditions known as disorders of sex development or differences of s chromosomal sex is inconsistent with genital sex, or in which the genital or gonadal sex is not classiﬁable as either ma who report their identity as ‘‘intersex’’ do not have a veriﬁable intersex condition. Sex: a person’s categorization as biologically male or female, usually on the basis of the genitals and reproductive tract.* Sex assigned at birth: the sex or gender first assigned to an individual after birth. Also known as ‘‘natal gender,’’ ‘‘birth-assigned sex,’’ and ‘‘gender assigned at birth.’’ Often queried as ‘‘What sex was listed on your original birth certiﬁcate?’’ Sex: a person’s categorization as biologically male or female, usually on the basis of the genitals and reproductive tract.* Sex assigned at birth: the sex or gender first assigned to an individual after birth. Also known as ‘‘natal gender,’’ ‘‘birth-assigned sex,’’ and ‘‘gender assigned at birth.’’ Often queried as ‘‘What sex was listed on your original birth certiﬁcate?’’ Sex assigned at birth: the sex or gender first assigned to an individual after birth. Table 1. Glossary Also known as ‘‘natal gender,’’ ‘‘birth-assigned sex,’’ and ‘‘gender assigned at birth.’’ Often queried as ‘‘What sex was listed on your original birth certiﬁcate?’’ Sex assigned at birth: the sex or gender first assigned to an individual after birth. Also known as ‘‘natal ge assigned at birth.’’ Often queried as ‘‘What sex was listed on your original birth certiﬁcate?’’ Sexual orientation: a person’s pattern of sexual attraction and physiological arousal to others of the same, other, both, or neither sex. Sexual orientation cannot be inferred from one’s gender identity. As a show of respect, we recommend that the sexual orientation of transgender individuals be expressed in relation to their gender identity rather than their gender assigned at birth; however, all gender scholars do not follow that convention. Ambiguity in charting can be avoided by using terms such as sexually attracted to men, women, both, or neither. Transgender: an umbrella term usually referring to persons whose experienced or expressed gender does not conform to normative social expectations based on the gender they were assigned at birth. p g y g Transsexual: a term often reserved for the subset of transgender individuals who desire to modify, or have modified, their bodies through hormones or surgery to be more congruent with their experienced gender. On ofﬁcial documents such as birth certiﬁcates, driver’s licenses, and passports, the traditional category ‘‘sex’’ is equivalent to ‘‘gender’’ in current psychological terminology. Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 59 The WPATH SOC also provide clinical guidance for health professionals to assist transgender people in their search for psychological well-being in their gen- dered selves. In the absence of other comprehensive English language guidelines, U.S. providers and their professional associations came to rely heavily on the HBIDGA/WPATH SOC.8–10 Similarly, insurance car- riers and tax courts employ WPATH SOC criteria in evaluating the medical necessity of transition treatments for determination of reimbursable and tax-deductible medical expenses.11–14 disruptive behavior disorder, eating disorders, and tic dis- orders. Under this parent category, DSM-IIIR added a new diagnosis, Gender Identity Disorder of Adolescence and Adulthood Nontranssexual Type (GIDAANT). These changes recognized that gender identity disorder (GID) often begins in childhood, may or may not per- sist into adolescence and adulthood, and when it does persist, it may not entail a desire for the primary or sec- ondary sexual characteristics of the other sex. Table 1. Glossary With DSM-IV, the diagnoses of Transsexualism and GIDAANT were discontinued, but GIDC and GIDAA were retained and placed under a new parent category, Sexual and Gender Identity Disorders, a category that also included the unrelated sexual dysfunctions and para- philias.24 Individuals with somatic intersex conditions, who experienced dysphoria attributable to dissatisfaction with their gender assigned at birth, could be diagnosed with Gender Identity Disorder Not Otherwise Speciﬁed. With transition services offered outside of university- based clinics, U.S. medical schools and residency training programs offered little exposure to the provision of tran- sition services, leaving psychiatrists and other physicians poorly prepared for the growth in demand for these ser- vices seen in recent years.15 This article aims to assist adult psychiatrists and other mental health professionals who are not gender specialists in the care of these indi- viduals. Detailed information on the assessment and treatment of gender dysphoria in children and adoles- cents can be found elsewhere.16–19 Retention of the diagnosis by the DSM and its new name, including the word ‘‘disorder,’’ was perceived by many as stigmatizing and contributing to societal dis- crimination against transgender individuals.25 By analogy to homosexuality, much of the distress and functional im- pairment associated with being transgender, and required for the diagnosis of GID, could derive from social stigma- tization rather than from being transgender, per se. On the other hand, removal of a coded diagnosis for medical classiﬁcation and billing purposes would limit access to transition care, deny the full impact of gender dysphoria, and prove harmful to transgender individuals.2,26 A glossary of transgender-related terms is found in Table 1. Providers should be respectful of their patients’ identity labels; however, due to the rapid evolution of gender terminology, they may need to clarify how both their patients and colleagues employ particular terms. Diagnostic and Statistical Manual of Mental In contrast, the ICD is not limited to only mental disorders. In its forthcom- ing eleventh iteration, ICD-11, the diagnosis of Gender Incongruence (GI) (corresponding to GD in DSM-5 ter- minology) will most likely be moved out of the section on mental disorders. Instead, it has been proposed to place it in a separate section tentatively named Conditions Related to Sexual Health or Sexual and Gender Health.29 Placing GI in this section will declassify it as a mental disorder, while maintaining a diagnosis that will facili- tate access to care through third party reimbursement, and could eventually lead to American Psychiatric Asso- ciation (APA) removing GD from the DSM. The prevalence of transgender people receiving gen- der specialty care in the Netherlands has been esti- mated at 0.008% for transgender women and 0.003% for transgender men.32 More recent data for those obtaining surgery in Belgium were similar.33 In Swe- den, point prevalence in 2010 was estimated to be 0.013% for transgender women and 0.008% for trans- gender men.34 A higher percentage, 0.023%, received a diagnosis of GID recorded in the health records of the U.S. Veteran’s Administration.35 Other studies, rather than measuring the propor- tion of a population that received a clinical diagnosis, have reported on those who self-identiﬁed as trans- gender or gender incongruent, and found that mea- suring self-identity yields much higher numbers. In 2016, data from the Center for Disease Control’s Behavioral Risk Factor Surveillance System suggested that 0.6% of U.S. adults identify as transgender, double the estimate utilizing data from the previous decade.36 Importantly, the GD diagnosis does not apply auto- matically to people who identify as transgender but is given only to those who either exhibit clinically signif- icant distress or impairment associated with a perceived incongruence between their experienced/expressed gen- der and their assigned gender or who, after transition, no longer meet full criteria, but require ongoing care (e.g., hormonal replacement therapy). In DSM-5, this lat- ter group is given a ‘‘post-transition’’ speciﬁer. Diagnostic and Statistical Manual of Mental Diagnostic and Statistical Manual of Mental Disorders and Transgender-Related Nosology Disorders and Transgender-Related Nosology The ﬁrst two editions of the Diagnostic and Statistical Manual of Mental Disorders (DSM) published in 1952 and 1968, respectively, did not include any gender diagnosis.20 The diagnosis, ‘‘Transsexualism’’ (sic), ﬁrst appeared in 1975 in the ninth revision of the International Classiﬁcation of Diseases (ICD)-921 and subsequently, in the DSM-III in 1980 under the parent category, Sexual Deviations.22 The deﬁning characteristics of this diagnosis were as follows: (1) discomfort about one’s assigned sex; (2) ‘‘cross-dressing,’’ in reality or fantasy, as the other sex, but not for the purpose of sexual excitement; and (3) the desire to get rid of one’s primary and secondary sex characteristics and to acquire those of the other sex. DSM-III also included ‘‘Gender Identity Disorder of Childhood’’ (GIDC). Ultimately, the diagnosis was retained by DSM-5,27 but its name was changed to Gender Dysphoria (GD), simultaneously removing the stigmatizing ‘‘disorder’’ from its name and shifting the focus to dysphoria as the target symptom for intervention and treatment, rather than gender identity itself.27,28 GD was also moved out of the parent category that included sexual dysfunctions and paraphilias, with which it has nothing in common, and into a separate parent category, also named Gender Dysphoria. Use of the diagnostic label, GD, requires that a person meets the full criteria speciﬁed in DSM-5. This is distinctly differentfrom thehistoricalgenericuseoftheterm,gender dysphoria, which refers to the distress caused by a discrep- ancy between one’s experienced gender and assigned gen- der, whether or not full DSM criteria for GD are met. For clarity here, references to the diagnosis will be capitalized or abbreviated (i.e., Gender Dysphoria or GD) while references to the symptom will not be capitalized or abbre- viated (i.e., gender dysphoria). Both transsexualism and GIDC were carried over into DSM-IIIR, but were no longer categorized as sexual devi- ations. Instead, they were placed within the parent cate- gory, Disorders Usually First Evident in Infancy, Childhood, or Adolescence.23 This category also included Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 60 gender specialty clinics,25 and, therefore, do not reﬂect the prevalence of all individuals with gender dysphoria or who identify as transgender. The DSM is a manual on mental disorders and, there- fore, despite the name change, GD retains its classiﬁ- cation as a mental disorder. Diagnostic and Statistical Manual of Mental In a large Massachusetts population-based phone survey, 0.5% of the population (age 18–64 years) identi- ﬁed as transgender.37 In another large population-based survey in the Netherlands, 1.1% of those assigned male at birth (age 15–70 years) reported an incongruent gender identity (stronger identiﬁcation with a gender other than the one assigned at birth), as did 0.8% of those assigned female at birth.38 Unlike previous versions of the DSM, in DSM-5, gender-dysphoric individuals with somatic intersex conditions, who were previously excluded from the di- agnosis, can now receive the diagnosis with a speciﬁer to indicate the presence of the intersex condition. DSM-5 is also the ﬁrst DSM to recognize the legiti- macy of gender identities outside the gender binary such that individuals with GD are no longer described as identifying simply as ‘‘the other gender,’’ but as ‘‘the other gender (or some alternative gender different from one’s assigned gender).’’ Examples of alternative genders include eunuch, genderqueer, and nonbinary. Recent surveys of youth showed even higher numbers. In New Zealand, 1.2% of high school students surveyed identiﬁed as transgender.39 In a survey of San Francisco middle school students (grades 6–8), 1.3% identiﬁed as transgender.40 More study is needed, but these larger numbers indicate that many transgender people have not been counted in clinical studies, including those with nonbinary identities, those not seeking transition care, those receiving hormones outside of clinics special- izing in transgender care or by self-administration, and others who identify as transgender when surveyed, but do not report gender dysphoria to clinicians. Epidemiology Epidemiological research has employed different mea- sures of transgender populations, resulting in varying es- timates of prevalence.30,31 Some studies assessed the fraction of a population, which had received the DSM- IV diagnosis of GID or the ICD 10 diagnosis of transsex- ualism, both of which were limited to clinical populations who sought binary transition (male-to-female or female- to-male). For example, the prevalences reported in DSM- 5 (0.005–0.014% for birth-assigned males; 0.002–0.003% for birth-assigned females) are based on people who received a diagnosis of GID or transsexualism, and were seeking hormone treatment and surgery from Gender Development Biological considerations p Biological considerations Biological considerations Animal research has established that sex differences in the phenotype of both body and brain as well as behav- iors are the result of multiple, sex-biasing factors. These include hormonal, sex-chromosomal,41 genetic, and epigenetic contributions.42 The sensitivity of brain Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 61 Muchofwhatisknownabouttheroleofearlyhormonal exposureonthedevelopmentofgenderidentityinhumans derives from studies of gender outcomes in individuals with somatic intersex conditions. Biological considerations Androgenization of the brain depends not only upon the level of androgen to which a fetus is exposed but also upon numerous other factors, including the presence of enzymes to convert androgens to the speciﬁc metabolites required by particular brain cells, their steroid receptors, and their postreceptor mechanisms that are involved in the full response to androgens. Receptor structure, which can inﬂuence sensitivity, is genetically deter- mined, while the activity of genes for receptors and post- receptor mediators is subject to epigenetic modulation.54 As the period of genital differentiation largely pre- cedes the sexual differentiation of the brain,55 it is con- ceivable that GD in individuals without somatic intersex conditions could reﬂect a brain-limited intersex condi- tion (i.e., a lack of concordance between the sexually dif- ferentiated state of the brain and body). That hypothesis has been tested in a variety of ways, including searching for features of the brain in individuals with GD that more closely match their experienced gender than their birth-assigned gender.56 Investigations in this regard have included postmortem morphometric and stereo- logical studies,57 as well as in vivo morphometric,58 functional magnetic resonance imaging,59 and diffu- sion tensor imaging studies of the brain,60–62 and ex- amination of otoacoustic emissions.63 Psychosocial factors inﬂuencing gender expression In mammals, and particularly in humans, psychological and social factors have a major additional inﬂuence on be- havioral outcome.65 In humans, these psychosocial pro- cesses include verbal labeling (e.g., ‘‘boy’’ and ‘‘girl’’) and nonverbal gender-cuing (e.g., gender-speciﬁc clothing and haircuts) of children by parents and others in their social environment, as well as the shaping of children’s gendered behavior by positive and negative reinforcement and later by explicit statements of gender-role expecta- tions. Related processes in developing children include gender-selective observational learning/imitation, the formation of gender stereotypes and of related self- concepts, and self-socialization. The effects on gender development have been documented in a vast body of research in developmental psychology.65 As reviewed elsewhere,53,56,64 while some positive ﬁnd- ings in the predicted direction have been reported,56,64 inferences are currently limited. This is because few ﬁndings have been replicated and few studies have ade- quately controlled for potentially confounding variables such as age, sexual orientation, transition status (includ- ing history of gender-afﬁrming hormonal treatment, if any), and hormonal status at the time of study (or of death in the case of postmortem studies).53 The impact of such psychosocial factors, however, is not determinative. Biological considerations Early guidelines for ini- tial gender assignment for such infants relied heavily on the surgical potential to achieve concordance between the gender assigned and the appearance and functional potentialoftheexternalgenitalia,inparticular,thecapac- ity of penile-vaginal intercourse.9 Current guidelines, however, emphasize what is known about the long- term gender outcomes of individuals with intersex conditions on a syndrome by syndrome basis.52 tissues to organizational effects of sex hormones ap- pears to be particularly high at prenatal/perinatal stages of development and gradually declines toward young adulthood.43 The timing of hormonal secretions in the course of development, however, gives the impression of three discrete sensitive periods: (1) pre/perinatal; (2) pubertal44; and (3) for females, the ﬁrst pregnancy.45 tissues to organizational effects of sex hormones ap- pears to be particularly high at prenatal/perinatal stages of development and gradually declines toward young adulthood.43 The timing of hormonal secretions in the course of development, however, gives the impression of three discrete sensitive periods: (1) pre/perinatal; (2) pubertal44; and (3) for females, the ﬁrst pregnancy.45 In humans, statistical sex differences in brain struc- ture are well documented,46 and ﬁndings of sensitive periods for sexual differentiation of the brain appear to parallel those seen in other mammals.47,48 The ev- idence for brain/behavior effects of prenatal andro- genization is particularly strong,49–51 much of which derives from studies of individuals with so- matic intersex conditions and varying degrees of functional androgen exposure.51–53 Overall, these data suggest that regardless of genetic constitution, or gonadal or genital development at birth, individuals prenatally exposed to a full comple- ment of masculinizing hormonal inﬂuences (i.e., an- drogen exposure and the cellular mechanisms for responding fully to androgens as described above) have an increased likelihood of GD when assigned female.51,52 Conversely, most reported 46,XY indi- viduals with complete androgen insensitivity syn- drome (and hence no functional androgenization of the brain) have developed a female gender identity, de- spite having a Y chromosome as well as normally de- veloped and functioning testes.51,52 To date, however, no brain marker of sexual differentiation has been val- idated to guide the initial gender assignment of infants with intersex conditions. Mental Health Assessment and Treatment This section addresses the assessment and treatment of adults with gender identity or expression concerns in the absence of an intersex condition. GD in individuals with intersex conditions is addressed in the Appendix. Treat- ment of GD in prepubescent children, where there is currently less consensus,81 is addressed elsewhere as is treatment of adolescents, including selection of can- didates for pubertal suspension.81,82 The primary roles of the mental health professional in assessing and treating patients with GD are based on expert consensus,7,8,10,20 summarized in Table 2 and described more fully below in the broader context of gender variance. Expert consensus regarding the treatment of adults has been arrived at after many years of clinical experience. Attempts to engage individuals in psychotherapy to Prospective follow-up studies of children, who before puberty had met criteria for the DSM-IV diagnosis of GID, showed that the majority of those diagnosed with GID in early or early middle childhood ‘‘desisted,’’ mean- ingthattheysubsequentlyidentiﬁedastheirbirth-assigned gender and did not meet criteria for GID. As adults, many identiﬁed as lesbian, gay, or bisexual.73–75 Some ‘‘desist- ers,’’ however, subsequently transitioned later in life.73 Biological considerations This is evidenced by individuals in whom gender identity is discordant with the initial gender assignment and gender of rearing, for example, transgender individuals and a higher than expected Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 62 childhood into adolescence are likely to experience an exacerbation of dysphoria with the emergence of (or with the anticipation of) undesired secondary sexual characteristics at puberty, in which case pubertal sus- pension should be considered.10 proportion of individuals with particular intersex con- ditions (i.e., 46,XY individuals with high degrees of so- matic hypomasculinization and 46,XX individuals with high degrees of somatic hypermasculinization66,67). Factors in gender-identity development p Regardless of their initial sexual orientation, during and after transitioning to express their experienced gender, some individuals retain their pretransition sex- ual attraction patterns, while others change.7 In some transgender women, the desire to transition gender is preceded by fantasizing themselves as women, some- times with sexual arousal.78 This phenomenon has been controversially interpreted by some as fetishism.79 Importantly, neither a history of fetishistic arousal nor one’s sexual orientation precludes one from meeting the criteria for the diagnosis of GD27 or eligibility for gender transition services.7,80 Systematic data on gender identity development are much more limited than those on gendered behavior. Yet, the data available, especially for those with intersex conditions, lead to the conclusion that, while early androgenization plays a role, a deﬁnitive biological pre- determination of gender identity seems unlikely. Not a single biological factor, but multiple factors (i.e., bio- logical, psychological, and social) appear to inﬂuence the development of gender identity.50 The need to transition gender is even less understood in individuals without, compared to those with, intersex con- ditions.68 Along with the dramatically increased referrals of gender-variant individuals to specialized clinics in Western Europe and North America over the last two de- cades,69,70 there has been a diversiﬁcation of presenta- tions beyond the original ‘‘transsexual’’ who sought (or was perceived by providers to seek) change to the ‘‘other’’ gender through treatment with gender- afﬁrming hormones and genital surgeries. Currently, many transgender people seek chest, but not genital surgery, or only gender-afﬁrming hormones, or only a social transition without any medical changes. Others may simply desire ﬂexibility in gender expres- sion without transition to ‘‘the other gender,’’ identi- fying, for example, as nonbinary or genderqueer.71,72 Assess and diagnose gender concerns according to current DSM criteria and see that they are addressed. Assess and diagnose any coexisting psychopathology and see that it is addressed. Assess eligibility for hormonal and/or surgical treatments, or refer to professionals capable of making such assessments. Assess capacity to give informed consent for hormonal and surgical treatments. Ensure that eligible individuals are aware of the full range of treatment options and their physical, psychological, and social implications, including risks, beneﬁts, and impact on sexual functioning and reproductive potential. Ensure adequate psychological and social preparation for transition treatments. Refer patients for hormonal or surgical treatments, collaborating with providers as needed. Ensure continuity of mental healthcare as indicated throughout transition and beyond. Table 2. Roles of the Psychiatrist Assess and diagnose gender concerns according to current DSM criteria and see that they are addressed. Assess and diagnose any coexisting psychopathology and see that it is addressed. Assess eligibility for hormonal and/or surgical treatments, or refer to professionals capable of making such assessments. Assess capacity to give informed consent for hormonal and surgical treatments. Ensure that eligible individuals are aware of the full range of treatment options and their physical, psychological, and social implications, including risks, beneﬁts, and impact on sexual functioning and reproductive potential. Ensure adequate psychological and social preparation for transition treatments. Refer patients for hormonal or surgical treatments, collaborating with providers as needed. Ensure continuity of mental healthcare as indicated throughout transition and beyond. Assess and diagnose gender concerns according to current DSM criteria and see that they are addressed. The data available do not allow a clear prediction be- fore puberty of which child will persist and transition permanently, and which child will not.75 With the in- troduction of stricter criteria for the diagnostic cate- gory of Gender Dysphoria in DSM-5, the persistence rate likely will be higher,73 but this needs to be tested by future long-term follow-up studies. For example, the degree of gender nonconformity and whether a child believes they are, as opposed to wishes to be, ‘‘the other’’ gender have been proposed as predictors of persistence.76,77 Those in whom GD persists from Ensure that eligible individuals are aware of the full range of treatment options and their physical, psychological, and social implications, including risks, beneﬁts, and impact on sexual functioning and reproductive potential. Ensure adequate psychological and social preparation for transition treatments. Refer patients for hormonal or surgical treatments, collaborating with providers as needed. Ensure continuity of mental healthcare as indicated throughout transition and beyond. Table 2. Roles of the Psychiatrist Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 63 ities, the transgender population also exhibits marked general health disparities.90 Few of these disparities are linked to sexually transmitted infections or hor- monal or surgical transition treatments,7,10,90 but are instead linked to ﬁnancial barriers to care as well as avoidance of healthcare due to experienced and/or an- ticipated stigma and discrimination in healthcare set- tings, and the widespread belief among transgender individuals that medical professionals are poorly trained to meet their needs,3 a belief that appears to be well- founded.15 Extensive guidance on overcoming these bar- riers to care, including creating a welcoming clinical environment, can be found elsewhere.91 change their gender identity or expression are currently not considered fruitful by the mental health professionals with the most experience working in this area7,9,83 and legal bans of therapies aimed at changing sexual orienta- tion have recently been extended to therapies aimed at changing gender identity or expression in a number of U.S. states and Canadian provinces.84,85 Currently, psy- chotherapeutic involvement with adults with GD is primarily used to assist in clarifying their desire for, and commitment to, changes in gender expression and/or somatic treatments to minimize discordance with their experienced gender, and to ensure that they are aware of and have considered alternatives.7 change their gender identity or expression are currently not considered fruitful by the mental health professionals with the most experience working in this area7,9,83 and legal bans of therapies aimed at changing sexual orienta- tion have recently been extended to therapies aimed at changing gender identity or expression in a number of U.S. states and Canadian provinces.84,85 Currently, psy- chotherapeutic involvement with adults with GD is primarily used to assist in clarifying their desire for, and commitment to, changes in gender expression and/or somatic treatments to minimize discordance with their experienced gender, and to ensure that they are aware of and have considered alternatives.7 Gender questioning, gender-variant, and transgender adults present to mental health services for a variety of reasons. Some presentations may relate explicitly to gen- der. For example, patients may wish to explore their gen- der identity, consider transition options and concerns (e.g., coming out to family or coworkers), or request eval- uation for hormonal or surgical treatments. Assessment of gender concerns Assessment of gender concerns g Treatment should be patient centered and tailored to the needs and individuality of each patient. Patients should be asked what names and pronouns they use and should be addressed by those names and pronouns regardless of their stage of transition. Those who tran- sitioned many years ago and are seeking treatment for another problem typically need much less focus on gender history than those who are questioning their gender identity, just beginning gender transition, or ex- ploring options for gender expression. When gender is not the primary concern, devoting the appropriate amount of attention to gender-related issues is impor- tant, balancing against an overemphasis on gender that can feel inadvertently stigmatizing to the patient or dis- tract from adequate focus on the chief complaint. According to WPATH SOC7, as an alternative to an evaluation by a mental health professional, primary care providers who are competent in the assessment of GD may evaluate patients for hormone therapy, par- ticularly in the absence of signiﬁcant coexisting mental health concerns and when working in the context of a multidisciplinary specialty team.7 While it is important to avoid the assumption that coexisting psychiatric symptoms are due to gender vari- ance, the impact of past and present gender-related stigma should be considered in the biopsychosocial eval- uation. This is particularly important in light of the stress- diathesis model of psychiatric illness and its exacerba- tions.8,92 Suicidality should always be assessed, as should protective factors such as social and family supports.93 Suicidal ideation3,94 and completed suicide90 are dramat- ically increased in this population and GD may be a risk factor for suicidality, independent of other psychiatric conditions.94,95 Up to 47% of transgender adults have considered or attempted suicide.93 Assessment of suicide risk is especially important during periods of heightened vulnerability, such as when transgender identity is dis- closed to family and more broadly.9,83 Patients may also seek couples or family therapy be- fore, during, or after transition to address the impact of the transition on interpersonal or family dynamics. Alternatively, many transgender patients seek or are re- ferred to psychiatric services for reasons that are either unrelated to gender identity or expression (e.g., man- agement of primary psychiatric illnesses), or only par- tially related (e.g., sequela of childhood trauma as a result of minority stress due to gender nonconformity). Table 2. Roles of the Psychiatrist The latter may include requests for referrals for such treatments, in- cluding requests for mental health referral letters as spec- iﬁed by the WPATH SOC7 or required by their providers of transition treatments and/or insurance carriers.7,11–13 Assessment of gender concerns A careful evaluation for a history and psychological sequela of gender-related stigma and abuse, from child- hood on, is crucial given the high rates of violence and bullying experienced by gender-variant individuals, as well as the high rates of discrimination, unemploy- ment, homelessness, sex work, and HIV infection.3,86 High rates of depressive, anxiety, and substance use disorders, as well as suicidal ideation and completed suicide have been linked to such gender minority stress.87–89 In addition to these mental health dispar- Patients may also seek couples or family therapy be- fore, during, or after transition to address the impact of the transition on interpersonal or family dynamics. Alternatively, many transgender patients seek or are re- ferred to psychiatric services for reasons that are either unrelated to gender identity or expression (e.g., man- agement of primary psychiatric illnesses), or only par- tially related (e.g., sequela of childhood trauma as a result of minority stress due to gender nonconformity). A careful evaluation for a history and psychological sequela of gender-related stigma and abuse, from child- hood on, is crucial given the high rates of violence and bullying experienced by gender-variant individuals, as well as the high rates of discrimination, unemploy- ment, homelessness, sex work, and HIV infection.3,86 High rates of depressive, anxiety, and substance use disorders, as well as suicidal ideation and completed suicide have been linked to such gender minority stress.87–89 In addition to these mental health dispar- The gender assessment should include the age and cir- cumstances when the patient ﬁrst became aware of a sense ofdifference from peersofthesamesexassignedatbirthas well as experiences of negative affect or self-perception Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 64 related to that sense of difference.8,20 Any history of peri- pubertal and/or pubertal distress due to the anticipation and/or emergence of unwanted secondary sex character- istics should also be explored, as should past experiences of gender-related stigmatization, discrimination, harass- ment, and violence.8,20 Before masculinizing or feminizing genital reconstruc- tive surgeries, the WPATH SOC7 also recommend 12 continuous months of living in a gender role that is congruent with the patient’s gender identity.7 Diagnosis of gender dysphoria. The DSM-5 diagnostic criteria for GD in adolescents and adults are shown in Table 3. Assessment of gender concerns The current standard of care in major clinics, the WPATH SOC7, and insurance requirements for reim- bursement of services follow a ﬂexible progression of transition steps, which may begin with completely re- versible steps (e.g., change of pronouns, name, and manner of dress), followed by partially reversible changes (e.g., gender-afﬁrming hormones), and then irreversible gender-afﬁrming surgeries.7,10–14,97 There is ﬂexibility in this process given that some people do not pursue all of these interventions or may prefer to do so in a different sequence. For example, transgender men may wish to undergo mastectomy or male breast construction before initiating masculinizing hormones.7 Before gonadectomy, 12 months of continuous hor- mone therapy consistent with the patient’s gender goals are recommended, unless hormones are clinically con- traindicated for the individual. The aim of hormone therapy before gonadectomy is primarily to allow the i di id l t i i d f d fﬁ i Recommendations regarding psychiatric assessment of individuals with GD have focused largely on assessment of eligibility for and decision-making capacity related to medical and surgical gender transition services.7,8,10 Eligi- bility for both gender-afﬁrming hormone therapy and surgeries requires persistent gender dysphoria, a docu- mented diagnosis of GD based on DSM-5 criteria, and the capacity to give informed consent.7 In addition, any signiﬁcant medical or psychiatric concerns must be sufﬁ- ciently controlled so that they do not interfere with the patient’s ability to safely adhere to the treatment regimen. The current standard of care in major clinics, the WPATH SOC7, and insurance requirements for reim- bursement of services follow a ﬂexible progression of transition steps, which may begin with completely re- versible steps (e.g., change of pronouns, name, and manner of dress), followed by partially reversible changes (e.g., gender-afﬁrming hormones), and then irreversible gender-afﬁrming surgeries.7,10–14,97 Th i ﬂ ibilit i thi i th t l The psychiatrist must assess whether some factor other than GD accounts for the expressed desire to transition. If not, coexisting mental illness is not a con- traindication to supporting transition if it is sufﬁciently controlled to not interfere with the patient’s capacity for Table 3. Diagnostic Criteria for Gender Dysphoria in Adolescents and Adults Assessment of gender concerns Diagnosing GD in adults by these criteria is usu- ally straightforward, especially for those with overt mani- festations in childhood, exacerbation of distress with pubertal changes, and persistence into adulthood in the absence of signiﬁcant coexisting mental health concerns.8,9 The patient’s history of coping mechanisms and sup- port systems should also be examined.8,20 Gender ex- pression (e.g., pronoun use, name changes, manner of dress, and bodily modiﬁcations) over time should be explored as well as what has and has not been helpful in improving the sense of well-being. It is important to clarify each patient’s goals and plans for social and/or medical transition, degree of commitment, and expectations.7,96 For those who do not wish to transition, assessing current psychosocial challenges and formulating with the patient how to best address them (e.g., psychotherapy, group therapy, and social support) should not be neglected. Assessment of patients who are seeking transition ser- vices, but do not clearly meet criteria for GD, may require moretimeandexploratorytherapy9 (e.g., a patient desiring hormonal or surgical treatment to transition to another gender, who does not clearly experience incongruence be- tween their experienced gender and their gender assigned at birth). The same is true for those with the onset of gen- der dysphoria in the context of a psychiatric disturbance (e.g., psychosis, dissociative disorder, and autism spectrum disorder) or recent trauma9,98,99; those who are ambivalent about their gender identity or desired sex characteristics; and those who exhibit marked exacerbations and re- missions of dysphoria over time. Recommendations regarding psychiatric assessment of individuals with GD have focused largely on assessment of eligibility for and decision-making capacity related to medical and surgical gender transition services.7,8,10 Eligi- bility for both gender-afﬁrming hormone therapy and surgeries requires persistent gender dysphoria, a docu- mented diagnosis of GD based on DSM-5 criteria, and the capacity to give informed consent.7 In addition, any signiﬁcant medical or psychiatric concerns must be sufﬁ- ciently controlled so that they do not interfere with the patient’s ability to safely adhere to the treatment regimen. Adapted from DSM-5.27 Table 3. Diagnostic Criteria for Gender Dysphoria in Adolescents and Adults A marked incongruence between an individual’s experienced/expressed gender and assigned sex as evidenced by two of the below, which have been present after the onset of puberty for at least 6 months: p p y A marked incongruence between one’s experienced/expressed gender and primary and/or secondary sex characteristics (or the anticipated secondary sex characteristics in young adolescents). There is ﬂexibility in this process given that some people do not pursue all of these interventions or may prefer to do so in a different sequence. For example, transgender men may wish to undergo mastectomy or male breast construction before initiating masculinizing hormones.7 A strong desire to be rid of one’s primary and/or secondary sex characteristics because of a marked incongruence with one’s experienced/expressed gender (or a desire to prevent the development of the anticipated secondary sex characteristics in young adolescents). A strong desire for the primary and/or secondary sex characteristics of another gender. A strong desire to be of a gender different from one’s assigned gender. A strong desire to be treated as a gender different from one’s assigned gender. Before gonadectomy, 12 months of continuous hor- mone therapy consistent with the patient’s gender goals are recommended, unless hormones are clinically con- traindicated for the individual. The aim of hormone therapy before gonadectomy is primarily to allow the individual to experience a period of gender-afﬁrming hormones, before irreversible surgical intervention.7 A strong conviction that one has the typical feelings and reactions of a gender different from one’s assigned gender. The condition is associated with distress or impairment in social, occupational, or other important areas of functioning that are clinically signiﬁcant. Adapted from DSM-5.27 Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 65 decision-making or ability to safely adhere to the de- mands of the desired treatment.7,9,98 decision-making or ability to safely adhere to the de- mands of the desired treatment.7,9,98 Participation in transgender support groups, including peer-led groups, and other interactions with transgender individuals or the transgender community are often use- ful in clarifying the goals of those who experience ambiv- alence about transition. With patients who are otherwise eligible for transition treatments, but express ambivalence about transition, the therapist should maintain a stance of neutrality, creating a safe therapeutic space in which the patient can weigh all options and arrive at a decision in their own time. Table 3. Diagnostic Criteria for Gender Dysphoria in Adolescents and Adults Many transgender adults need some com- bination of hormonal treatment and/or surgical proce- dures for relief of GD, but some experience relief with a change in gender expression without any medical treat- ment.7 Strengthening resilience factors identiﬁed in the transgender population93 should be a focus, particularly, in patients with suicidal ideation. Differential diagnosis. Few conditions can be mis- taken for GD. Simple nonconformity to gender roles can be differentiated from GD based on the degree of associated distress and whether or not the individual identiﬁes as the sex assigned to them at birth. GD can be differentiated from body dysmorphic disorder (BDD), in which an individual may wish a body part to be removed or altered because it is viewed as de- formed.27 In contrast, in GD alterations are sought for anatomical characteristics that are incongruent with one’s gender identity. BDD and GD can, how- ever, coexist and the presence of BDD is not an absolute contraindication for gender-conﬁrming surgery.27 Trans- vestic disorder is characterized by signiﬁcant distress or impairment due to sexual arousal in the context of cross-dressing fantasies, urges, or behavior. It may exist independently or co-occur with GD,27 and is not a contraindication to supporting transition in those who meet criteria for GD.7 Although treatment with exogenous estrogen or tes- tosterone carries a risk for medical side effects,10 both have been associated with improvement with respect to anxiety, mood, and mood stability, as well as overall satisfaction and quality of life for both transgender women and transgender men.101–104 Similarly, review of the available literature9 demonstrates the beneﬁts of surgery in alleviating GD and the rarity of postsurgical regret. Emotional changes may occur with use of either androgen or estrogen supplementation, although these are usually subtle.9 An increase in libido usually occurs with androgen use with female to male transition.10 Although decreased libido due to antiandrogen and/or estrogen treatment in individuals transitioning male to female is common,10 some may experience a stronger interest in sex, perhaps due to the afﬁrming aspects of attaining desired bodily changes. Table 3. Diagnostic Criteria for Gender Dysphoria in Adolescents and Adults Gender-themed delusions have been reported to occur in up to 20% of those with psychotic disorders.100 Such delusions can usually be easily differentiated from GD by their content (i.e., if they do not entail the belief that one’s gender differs from that assigned at birth), as well as by their presence only during psychotic phases of illness, and the absence of other DSM crite- ria required for the diagnosis of GD.98 Importantly, GD and psychotic disorders may coexist and patients with both diagnoses can beneﬁt from gender-afﬁrmative treatment and appropriate hormonal and/or surgical gender interventions.98 Timely diagnosis of GD may be impeded when it is ﬁrst overtly expressed in ado- lescence or early adulthood coincident with, or shortly following, the ﬁrst psychotic episode.98 Safer sex information and instruction in self-protective negotiation in sexual settings should be provided and tai- loredtotheanatomy,needs,andexperiencesoftransgender persons.9 Masculinizing hormones have been associated with a possible destabilization of psychotic and bipolar disorders, especially with supraphysiological blood lev- els of testosterone7 in both cisgender and transgender men.105–106 The likelihood of such episodes can, there- fore, be minimized by careful dosing and monitoring. Mental health treatment Mental health treatment Statements in this section are based on the cited studies supplemented by the authors’ cumulative clinical expe- rience treating patients with GD. Psychotherapy can be useful for patients with GD; however, many success- fully transition or decide against transition with little or no psychotherapy. Psychotherapy may be helpful at different times and for different reasons across the lifespan.7 Many transgender people seek mental health treatment on an intermittent basis, while contemplat- ing gender transition, at key points in the transition pro- cess, or post-transition if symptoms recur or worsen. Detailed information on speciﬁc gender-afﬁrming surgical procedures can be found elsewhere.7,107 Psy- chiatrists should collaborate with other providers (e.g., endocrinologists, surgeons, psychotherapists, primary care providers, social workers, and other mental health professionals) to ensure that patients have the knowl- edge required to adequately evaluate the beneﬁts, risks, and limitations of desired treatments and their Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 66 large online survey, the National Transgender Discrimi- nation Survey88 found that rejection, discrimination, vic- timization, and violence against transgender people occur in a multitude of settings and negatively affect transgen- der people across the life span. Transgender youth are often harassed and assaulted in schools, which is associ- ated with dropping out and subsequent impoverishment. Many transgender people are harassed at work or lose jobs due to their gender identity and expression. Discrim- ination extends to healthcare settings, where patients may be refused care or treated disrespectfully, or do not have access to care.88 alternatives. This is necessary not only for informed consent but also to ensure adequate preparation for surgery and postsurgical needs (e.g., convalescent pe- riod, period of sexual abstinence, and vaginal dilata- tion in the case of vaginoplasty). Helping the patient anticipate and prepare for psy- chosocial impacts of treatment (e.g., impact on social relationships and employment) is also essential. Impor- tantly, transition treatments target GD, not coexisting psychiatric diagnoses, and coexisting diagnoses are likely to require ongoing attention after transition, al- though symptom severity may be ameliorated.98,100,102 U.S. public policy has contributed to the lack of ac- cess to care. A report by the National Center for Health Care Technology of the HHS Public Health Service issued in 1981, titled ‘‘Evaluation of Transsexual Sur- gery,’’ deemed these procedures ‘‘experimental,’’ and recommended that Medicare not cover transition- related care. Referrals for hormones and surgery Whether the initial evaluation for hormones is done by the hormone prescriber or by a mental health profes- sional, criteria for starting hormones are the same: the presence of persistent GD, the ability to give in- formed consent, and relative mental health stability.7 Insurance carriers and surgeons require mental health evaluation before transition-related surgeries to assess and document eligibility, readiness, and medical necessity of the requested procedure.7,10–14 The speciﬁc requested content of referral letters varies among surgical providers and insurance plans. To avoid unnecessary delays in treatment, letter writ- ers should be aware of such differences and ensure that their letters meet the requirements of all relevant parties. The content requested by most providers and insurance carriers is similar to that outlined in the WPATH SOC7. Genital and gonadal surgeries usually require documentation from two licensed mental health professionals, while chest surgeries generally require just one evaluation and referral.7,108 Although not requirements of WPATH SOC7, some insurers re- quire one letter from a psychiatrist or other doctoral level mental health provider, or may specify a minimal duration of mental healthcare.13 Such requirements vary by health system, insurance carrier, and state, and raise challenges for those without access to reim- bursement for mental healthcare. Many transgender health insurance exclusions have been removed recently. This trend started with increas- ing numbers of employers in the last 15 years adding transition care to health coverage. Starting in 2013, some states have ruled that transgender healthcare ex- clusions are discriminatory and have banned them from state-regulated health insurance plans. In 2014, the 1981 Medicare policy was reversed, removing cate- gorical exclusions for transgender care.6 In 2015, the HHS moved to end categorical exclusions for transgen- der care from all insurance and care providers who ac- cept federal funding or reimbursement;109 and since 2016, insurers in the Federal Employees Health Bene- ﬁts Program must include transition-related coverage for transgender federal employees.110 During this same period, executive orders and other guidance from the Obama administration conferred in- creased protection against discrimination to transgender individuals in workplace and educational settings,111 the ban on open military service of transgender individuals was lifted,112 and changes at the HHS and the National Institutes of Health (NIH) facilitated research to better deﬁne and address the health needs of transgender indi- viduals.111 Much work remains, however, to fully Mental health treatment This was formalized in a 1989 Health Care Financing Administration National Coverage Determination.5 Exclusion of transgender healthcare in private insurance as well as Medicaid and Medicare was near universal in the decades to come. A lack of funding for clinical care and research led to the closing of transgender care programs at academic institutions in the years following the 1981 report. Referrals for hormones and surgery Current Social Issues: Stigmatization and Access to Care Transgender health advocates have worked to address so- cietal discrimination against transgender people, includ- ing stigmatization of identity, discrimination in schools, workplaces, and healthcare, and to improve access to care. Increasingly, this advocacy has been embraced by major institutional and governmental agencies. One Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 67 of the reasons these individuals may seek psychiatric care and, regardless of their area of specialization, psychi- atrists should be adept at conducting respectful, culturally sensitive, and afﬁrming gender assessments without placing an undue emphasis on gender when it is not the patient’s presenting concern. Mental health profes- sionals must fully appreciate that the focus of treatment for GD is on the dysphoria, not the gender identity. At the same time, they must appreciate the role of minority stress in gender minority mental health disparities, screen for related manifestations, including anxiety dis- orders, depression, and suicidality, and consider resil- ience factors in treatment planning. actualize these policy changes. In addition, progress has been slowed on the federal level by the change in presi- dential administrations and legal actions.113 WPATH SOC77 has attempted to improve access to care by including the informed consent model for hor- mone administration. In multidisciplinary clinics pro- viding transgender care, primary care providers can assess for and diagnose longstanding GD that might beneﬁt from treatment with hormones and administer hormones without referral from a mental health pro- fessional. However, patients with cooccurring mental health conditions should be referred to mental health providers when appropriate. WPATH has advocated for the depathologization of transgender identity, the medical necessity of transgender care, and improved access to legal gender change.7 Psychiatrists should also be competent in the provi- sion of routine psychiatric care that is gender afﬁrming to gender variant patients with serious mental illnesses without assuming that the gender variance is a mani- festation of the illness. They should not expect coexist- ing serious mental illness, especially in the context of strong genetic loading, to fully resolve with successful treatment of GD and should assist the patient in for- mulating realistic expectations. The APA has also attempted to reduce stigma and im- prove access to care. As discussed previously, the DSM-IV diagnosis of GID, regarded as stigmatizing by many transgender health and advocacy groups, was replaced with GD in DSM-5.114 In addition, the APA approved position articles on discrimination and access to care. Current Social Issues: Stigmatization and Access to Care Its statement on discrimination against transgender and gender-variant individuals115 opposes all private and pub- lic discrimination against transgender individuals, and its statement on access to care for transgender and gender- variant individuals116 urged the removal of all categorical healthcare exclusions for transgender people and advo- cated for the expansion of access to care. If not included in their residency or fellowship train- ing,orsupervisedclinicalexperience,psychiatristsshould familiarize themselves with the standards of care for gen- dertransitionasdescribedintheWPATHSOC7andout- lined in this article, as well as the roles and minimal competencies of mental health professionals working with adults with GD.7 In addition to the minimal com- petencies, WAPTH SOC7 recommends that health professionals take steps to sustain or augment their cul- tural competency to work with transgender and other gender minority patients by participating in continu- ing education and becoming knowledgeable about community, advocacy, and public policy issues that affect transgender individuals and their families.7 Increased access to care must be accompanied by culturally competent research in transgender health, recommended by the Institute of Medicine86 and out- lined in the NIH’s Strategic Plan to Advance Research on the Health and Well-being of Sexual and Gender Minorities.117 Expanded and improved education of healthcare providers is necessary, and the American Association of Medical Colleges has produced guide- lines for curricular and climate change to improve transgender health.118 Principles of culturally competent care for transgender and nonbinary patients should be included in residency training as well, including psychi- atric residency programs. g All providers should work within their sphere of competency and refer patients when necessary. 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Cite this article as: Byne W, Karasic DH, Coleman E, Eyler AE, Kidd JD, Meyer-Bahlburg HFL, Pleak RR, Pula J (2018) Gender dysphoria in adults: an overview and primer for psychiatrists, Transgender Health 3:1, 57–A3, DOI: 10.1089/trgh.2017.0053. Cite this article as: Byne W, Karasic DH, Coleman E, Eyler AE, Kidd JD, Meyer-Bahlburg HFL, Pleak RR, Pula J (2018) Gender dysphoria in adults: an overview and primer for psychiatrists, Transgender Health 3:1, 57–A3, DOI: 10.1089/trgh.2017.0053. 106. Moore E, Wisniewski A, Dobs A. Endocrine treatment of transsexual people: a review of treatment regimens, outcomes, and adverse effects. J Endocrinol Metab. 2003;88:3467–3473. 107. Ettner R, Monstrey S, Coleman E (eds). Principles of Transgender Medi- cine and Surgery. New York, NY: Routledge, 2016. 108. Karasic DH. Mental Health Care and Assessment of Transgender Adults. 2015. Available at www.lgbthealtheducation.org/topic/transgender- health. Accessed February 1, 2018. Abbreviations Used Abbreviations Used APA ¼ American Psychiatric Association BDD ¼ Body Dysmorphic Disorder DSM ¼ Diagnostic and Statistical Manual of Mental Disorders GD ¼ Gender Dysphoria GID ¼ Gender Identity Disorder GIDAANT ¼ Gender Identity Disorder of Adolescence and Adulthood Nontranssexual Type GIDC ¼ Gender Identity Disorder of Childhood HBIGDA ¼ Harry Benjamin International Gender Dysphoria Association HHS ¼ U.S. Department of Health and Human Services ICD ¼ International Classiﬁcation of Diseases NIH ¼ National Institutes of Health SOC ¼ Standards of Care WPATH ¼ World Professional Association for Transgender Health APA ¼ American Psychiatric Association BDD ¼ Body Dysmorphic Disorder DSM ¼ Diagnostic and Statistical Manual of Mental Disorders GD ¼ Gender Dysphoria GID ¼ Gender Identity Disorder GIDAANT ¼ Gender Identity Disorder of Adolescence and Adulthood Nontranssexual Type GIDC ¼ Gender Identity Disorder of Childhood HBIGDA ¼ Harry Benjamin International Gender Dysphoria Association HHS ¼ U.S. Department of Health and Human Services ICD ¼ International Classiﬁcation of Diseases NIH ¼ National Institutes of Health SOC ¼ Standards of Care WPATH ¼ World Professional Association for Transgender Health 109. U.S. Department of Health and Human Services. Non-discrimination in Health Programs and Activities Proposed Rule. Available at www.hhs .gov/civil-rights/for-individuals/section-1557/nondiscrimination-health- programs-and-activities-proposed-rule/index.html. Accessed March 28, 2018. 110. FEHB Program Carrier Letter 2015-12. U.S. Ofﬁce of Personnel Manage- ment. Available at www.opm.gov/healthcare-insurance/healthcare/ carriers/2015/2015-12.pdf. Accessed March 28, 2018. 111. Byne W. Sustaining progress toward LGBT health equity: a time for vigilance, advocacy, and scientiﬁc inquiry. LGBT Health. 2017;4:1–3. 112. Rosenberg M. Transgender people will be allowed to serve openly in military. New York Times, 2016. Available at www.nytimes.com/2016/07/ 01/us/transgender-military.html. Accessed February 11, 2018. 113. Byne W. A year into the Trump administration: LGBT health equity ﬁghting to stand ground. LGBT Health. 2018;5:1–5. Publish in Transgender Health -Immediate, unrestricted online access -Rigorous peer review -Compliance with open access mandates -Authors retain copyright -Highly indexed -Targeted email marketing liebertpub.com/trgh (Appendix follows /) A1 Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 Gender evaluation h Gender evaluation The questionnaires and interview schedules developed for the assessment of gender development in transgender individuals who do not have an intersex conditionA15,A16 apply to those with intersex conditions as well, but need to be complemented by detailed medical, surgi- cal, and related psychosocial histories, including the histories of disclosure to the patient of her/his medical condition, efforts made to reinforce the initial gender assignment, and responses by parents and providers to behaviors perceived as atypical with respect to the gender assignment. Mental health providers should also assess the patient’s knowledge of their surgical history, their understanding of the implications with respect to fertility and gender-afﬁrming hormonal and surgical procedures, and any history of shaming or other stigma due to their condition, or perceived gender atypicality with respect to their gender assigned at birth. g g Persons with the combination of GD and intersex condition encounter fewer barriers to legal gender reas- signment, and the barriers to hormonal and surgical treatments are much lower.A1 This is because, depend- ing on the particular condition, individuals with an in- tersex condition may have been gonadectomized (often due to concern about risk of malignancy) before pu- berty so that administration of exogenous hormones is required as part of routine care to induce puberty. In addition, infertility is quite common whether due to the condition itself or to gonadectomy, and genital surgery has often been done in infancy or childhood with the intent of afﬁrming, both to the patient and the parents, the gender to which the individual was assigned. Furthermore, such early procedures may have been followed by additional surgical modiﬁcations in adolescence or young adulthood. Gender Dysphoria in Patients with Intersex Conditions Gender Dysphoria in Patients with Intersex Conditions sentations of the various intersex conditions. Thus, to be fully effective, the mental health provider needs to be informed about the medical and surgical history of the individual,A6,A7 the available data on long-term gen- der development (e.g., contentment vs. dysphoria in the assigned gender), and other psychological outcomes of patients on a syndrome by syndrome basis.A5,A8 More- over, intersex conditions are frequently associated with stigma, even in medical settings, which may result in shame and maladaptive coping mechanisms on the part of the patients as well as their parents.A9–A12 As reviewed elsewhere,A1 Gender Dysphoria (GD) and patient-initiated gender transition occur with increased frequency in individuals with intersex conditions. Because Diagnostic and Statistical Manual of Mental Disorders-5 now allows gender-dysphoric individuals with somatic intersex conditions to receive the diagno- sis of GD, psychiatrists need to be aware of assessment- and treatment-relevant characteristics of such individuals that differ from gender-dysphoric individuals without somatic intersexuality.A2 Providers need to be aware of the many ways in which some individuals with intersex conditions report having been stigmatized by their treatment by clinicians and parents (e.g., failure of age-appropriate disclosure of their condition, attempts to modify their gender ex- pression, and repeated genital examinationsA9,A13). Efforts are under way to develop decision-making tools and clinical checklists to ensure that parents and affected children are adequately assessed and in- formed as active participants in decision-making pro- cesses and that the intersex condition and its ramiﬁcations are disclosed to the affected individual in an age-appropriate manner.A14 Intersex conditions are a subset of conditions relatively recently designated as ‘‘disorders of sex development’’A3 or ‘‘differences of sex development’’ (DSD).A4 We use the term ‘‘intersex’’ in this document as our focus is on that subset of individuals with DSDs who were born with atypical external genitalia or lack of concordance among various sex characteristics such as sex chromo- somes, gonads, or external genitalia so that questions often arise as to which gender should be assigned at birth. GD may develop from late preschool age through late adulthood with a range from 0% to 70% depending on the speciﬁc intersex syndrome, its severity (degree of androgen insensitivity, degree of 21-hydroxylase deﬁ- ciency, degree of genital atypicality, etc.), the gender orig- inally assigned, and the postnatal history of exposure to both endogenous and exogenous sex hormones.A5 Decisions regarding gender transition Thus, the view- points of patients (and caregivers) within their cultural contexts should be explored in detail and taken into con- sideration when these individuals are provided with psy- choeducation about gender and other issues related to their intersex conditions.A19 p As is often seen in many individuals with uncommon medical conditions, many people with intersex condi- tions experience varying degrees of isolation and lone- liness.A1 Therefore, linking them to existing intersex support groups by internet or face-to-face meetings can be very beneﬁcial. Despite the emotional relief that support groups can provide, such contacts may sometimes cause additional concerns. For instance, the composition of the group (e.g., the syndromes rep- resented within the group, the personalities of some group members, or the goals of the group) may not meet the individual’s expectations, and the information provided may not always be accurate. Thus, some mon- itoring of the patient’s experience with the chosen group is recommended. As with other transgender patients, when working with patients with an intersex condition and GD, clini- cians should engage the patient in a detailed discussion of their expectations from the gender transition: the so- cial effects of public gender change as well as the med- ical and social effects of the attendant change in hormone treatment and, if desired, of genital or chest surgery. Some of their expectations may be unrealistic, and after detailed discussion, some patients may mod- ify the hormonal and/or surgical treatments they desire or decide against medical treatments or legal gender change, and pursue other ways of ﬁnding authenticity in their gender expression. Patients may be happy with their gender-atypical bodies and/or adapt a nonbi- nary gender identity such as ‘‘intersex.’’ Mental health providers should not assume that patients would ben- eﬁt from conforming to ﬁt within a gender binary, physically or with respect to gender identity. Hormonal and surgical treatments As reviewed elsewhere,A1 many individuals with both an intersex condition and GD will be agonadal in later adolescence or adulthood, either because they were born that way (e.g., in syndromes involving gonadal dysgenesis) or due to surgery, for instance, for the pre- vention of gonadal malignancy. In those with intact gonads (especially 46,XX congenital adrenal hyperpla- sia raised female), loss of fertility may be another issue of concern. Persons who are agonadal are usually on hormone replacement therapy by the time of late ado- lescence. Decisions regarding gender transition Decisions regarding gender transition For individuals with intersex conditions, GD usually raises the question of transition to a different gender, For individuals with intersex conditions, GD usually raises the question of transition to a different gender, Decisions regarding hormonal and surgical proce- dures are complicated by the highly variable somatic pre- Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 A2 tersex status in adolescence or adulthood, and may have been stigmatized for gender nonconformity or ho- mosexuality, or subjected to irreversible hormonal or surgical treatments consistent with their assigned rather than their experienced gender. Upon discovery of their biological status, such patients may feel betrayed by their parents and physicians, feeling they colluded to keep them in ignorance of their medical condition, damaged their bodies, or punished or stig- matized them for their gendered behaviors. Such pa- tients need empathic validation of their feelings. Assurance that parents and providers had their best in- tentions at heart, while usually true, is likely to be expe- rienced as an empathic failure and negatively impact the formation of a therapeutic alliance. and all issues of relevance to transgender persons with- out these conditions should also be considered here. Yet, the situation is often more complex than in GD in the absence of an intersex condition. Factors con- tributing to the desire to transition may include the awareness of the discrepancy between assigned gender and genetic factors such as the karyotype, anatomic factors such as the type of gonads, and secondary sex characteristics like breast development in men or hirsut- ism and masculine habitus in women. Related psychoso- cial inﬂuences may derive from being misidentiﬁed as the ‘‘other’’ gender or from frank stigmatization due to gender-atypical physical features. Different cultures and even subcultures within a given countrymaydifferintheroles(includingrights)associated withone’sgender,andinthesalienceandweightofcriteria used in decision-making on gender reassignment.A17,A18 When discussing gender options, clinicians need to con- sider the legal regulations of the country in which they work as well as the religious and other ideologies that can inﬂuence the gender perspectives of patients (and of caregivers for minors). These considerations are also very important when doing clinical work with visitors or immigrants from foreign countries. Decisions regarding gender transition Cessation of that treatment, change to treat- ment with hormones congruent with their gender identity, patient education for informed consent, and the monitoring of treatment effects are tasks of the en- docrinologist. Also, the technical aspects of genital surgery are more complex than in patients receiving gender- conﬁrming genital surgeries, who do not have intersex Empathic listening is especially important in work- ing with intersex individuals, perhaps particularly with those who have inadvertently discovered their in- Byne, et al.; Transgender Health 2018, 3.1 http://online.liebertpub.com/doi/10.1089/trgh.2017.0053 A3 A4. Johnson EK, Rosoklija I, Finlayson C, et al. Attitudes towards ‘‘disorders of sex development’’ nomenclature among affected individuals. J Pediatr Urol. 2017;13:402–413. conditions. Both the external genitalia and the internal reproductive tract in intersex conditions typically dif- fer from what most surgeons are familiar with in transgender patients without these conditions. In ad- dition, many patients with intersex conditions have al- ready undergone one or more genital surgeries by late adolescence. The resulting postsurgical anatomy con- stitutes an additional challenge for the surgeon per- forming gender-conﬁrming surgery, and a good sex- functional outcome may be more difﬁcult to achieve. A5. Meyer-Bahlburg HFL. Introduction: gender dysphoria and gender change in persons with intersexuality. Arch Sex Behav. 2005;34:371–373. A6. Rey RA, Josso N. Diagnosis and treatment of disorders of sexual devel- opment. In: Endocrinology: Adult and Pediatric, 7th ed. Vol II. (Jameson JL, De Groot LJ; eds). Philadelphia, PA: Elsevier/Saunders, 2016, pp. 2086–2118. A7. New MI, Lekarev O, Parsa A, et al. (eds). Genetic Steroid Disorders. London, UK: Academic Press/Elsevier, 2014. A8. Meyer-Bahlburg HFL. Psychoendocrinology of congenital adrenal hy- perplasia. In: Genetic Steroid Disorders (New MI, Lekarev O, Parsa A, et al.; eds). London, UK: Academic Press/Elsevier, 2014, pp. 285–300. A9. Consortium on the Management of Disorders of Sex Development. Handbook for Parents. Rohnert Park, CA: Intersex Society of North America, 2006. Decisions regarding gender transition Mental health providers should also be aware that not all individuals who identify their gender or gender identity as intersex have a somatic intersex condition, and should ensure that those who do have an intersex condition are receiving adequate medical care, includ- ing hormones (to prevent osteoporosis) and cancer screenings, as appropriate to their particular condi- tion.A3 Without challenging a patient’s identity label, this distinction can usually be made by inquiring about the name of the patient’s condition, when and how they learned of it, and any history of related sur- geries, hormonal replacement, or ongoing follow-up evaluations. If there is any doubt, appropriate referrals should be made to ensure that the patient is receiving adequate follow-up and treatment. A10. Meyer-Bahlburg HFL, Khuri J, Reyes-Portillo J, et al. Stigma associated with classical congenital adrenal hyperplasia in women’s sexual lives. Arch Sex Behav. 2018;47:943–951. A11. Meyer-Bahlburg HF, Reyes-Portillo JA, Khuri J, et al. Syndrome-related stigma in the general social environment as reported by women with classical congenital adrenal hyperplasia. Arch Sex Behav. 2017;46: 341–351. A12. Meyer-Bahlburg HFL, Khuri J, Reyes-Portillo J, New MI. Stigma in medical settings as reported retrospectively by women with congenital adrenal hyperplasia (CAH) for their childhood and adolescence. J Pediatr Psy- chol. 2017;42:496–503. A13. Consortium on the Management of Disorders of Sex Development. Clinical Guidelines for the Management of Disorders of Sex Develop- ment. Rohnert Park, CA: Intersex Society of North America, 2006. A14. Siminoff LA, Sandberg DE. Promoting shared decision making in dis- orders of sex development (DSD): decision aids and support tools. Horm Metabol Res. 2015;47:335–339. A15. Zucker KJ. Measurement of psychosexual differentiation. Arch Sex Behav. 2005;34:375–388. A16. Meyer-Bahlburg HFL. Gender monitoring and gender reassignment of children and adolescents with a somatic disorder of sex development. Child Adolesc Psychiatr Clin N Am 2011;20:639–649. A3. Hughes IA, Houk C, Ahmed SF, et al. Consensus statement on man- agement of intersex disorders. J Pediatr Urol. 2006;2:148–162. A2. Meyer-Bahlburg HFL. Variants of gender differentiation in somatic dis- orders of sex development: recommendations for Version 7 of the World Professional Association for Transgender Health’s Standards of Care. Int J Transgend. 2009;11:226–237. A1. Byne W, Bradley SJ, Coleman E, et al. Report of the American Psychiatric Association task force on treatment of gender identity disorder. Arch Sex Behav. 2012;41:759–796. Appendix References A1. Byne W, Bradley SJ, Coleman E, et al. Report of the American Psychiatric Association task force on treatment of gender identity disorder. Arch Sex Behav. 2012;41:759–796. A2. Meyer-Bahlburg HFL. Variants of gender differentiation in somatic dis- orders of sex development: recommendations for Version 7 of the World Professional Association for Transgender Health’s Standards of Care. Int J Transgend. 2009;11:226–237. A3. Hughes IA, Houk C, Ahmed SF, et al. Consensus statement on man- agement of intersex disorders. J Pediatr Urol. 2006;2:148–162. Appendix References y A17. Lang C, Kuhnle U. Intersexuality and alternative gender categories in non-Western cultures. Horm Res. 2008;69:240–250. A18. Meyer-Bahlburg HF. Introduction to the special section on culture and variants of sex/gender: gias and stigma. Arch Sex Behav. 2017;46: 337–339. A19. Meyer-Bahlburg HF, Baratz Dalke K, Berenbaum SA, et al. Gender as- signment, reassignment and outcome in disorders of sex development: update of the 2005 Consensus Conference. Horm Res Paediatr. 2016;85: 112–118.
https://openalex.org/W4232679240	https://peerj.com/articles/9082v0.3/submission	English	null	Peer Review #3 of "On the importance of spatial scales on beta diversity of coral assemblages: a study from Venezuelan coral reefs (v0.1)"	null	2,020	cc-by	11,111	Emy Miyazawa Corresp., 1 , Luis M Montilla 1, 2 , Esteban Alejandro Agudo-Adriani 1, 3 , Alfredo Ascanio 1, 4 , Gloria Mariño- Briceño 1 , Aldo Croquer Corresp. 1, 5 1 Laboratorio de Ecología Experimental, Universidad Simón Bolivar, Caracas, Venezuela 2 Integrative Marine Ecology Department, Stazione Zoologica Anton Dohrn, Naples, Italy 3 Department of Biology, University of North Carolina at Chapel Hill, North Carolina, United States 4 Department of Biology, Miami University of Ohio, Oxford, Ohio, United States 5 Centro de Innovación Marina, The Nature Conservancy, Punta Cana, Dominican Republic Corresponding Authors: Emy Miyazawa, Aldo Croquer Email address: 12-11222@usb.ve, acroquer@usb.ve 1 Laboratorio de Ecología Experimental, Universidad Simón Bolivar, Caracas, Venezuela 2 Integrative Marine Ecology Department, Stazione Zoologica Anton Dohrn, Naples, Italy 3 Department of Biology, University of North Carolina at Chapel Hill, North Carolina, United States 4 Department of Biology, Miami University of Ohio, Oxford, Ohio, United States 5 Centro de Innovación Marina, The Nature Conservancy, Punta Cana, Dominican Republic Corresponding Authors: Emy Miyazawa, Aldo Croquer Email address: 12-11222@usb.ve, acroquer@usb.ve Estimating variability across spatial scales has been a major issue in ecology because describing patterns in space is extremely valuable to propose speciﬁc hypotheses aim to unveil key processes behind these patterns. This paper aims to estimate the variability of the coral assemblage structure at diﬀerent spatial scales in order to determine which scales explain the greatest variability onβ-diversity. For this, a fully-nested design including a series of hierarchical-random factors encompassing three spatial scales:(1) regions, (2) localities and (3) reefs sites across the Venezuelan territory. The variability among spatial scales were tested with a permutation analysis of variance (Permanova) based on Bray-Curtis index. Dispersion in species presence/absence across scales (i.e., β- diversity) was tested with a PermDisp analysis based on Jaccard's index. We found the highest variability in the coral assemblage structure between sites within locality (Pseudo- F = 5.34; p-value = 0.001, CV = 35.10%), We also found that longitude (Canonical corr = 0.867, p = 0.001) is a better predictor of the coral assemblage structure in Venezuela, rather than latitude (Canonical corr = 0.552, p = 0.021). Largest changes inβ-diversity of corals occurred within sites (F = 2.764, df1= 35, df2 = 107, p = 0.045) and within localities (F = 4.438, df1= 6, df2 = 29, p = 0.026). Our results suggest that processes operating at spatial scales of hundreds of meters and hundreds of kilometers might both be critical to shape coral assemblage structure in Venezuela, whereas smaller scales (i.e., hundreds of meters) showed to be highly- important for the species turnover component of β-diversity. Manuscript to be reviewed On the importance of spatial scales on beta 1 diversity of coral assemblages: a study 2 from Venezuelan coral reefs 3 my Miyazawa1, Luis M. Montilla1, 2, Esteban Alejandro Agudo-Adriani1, 3 f d A i 1 4 Gl i M i˜ B i ˜ 1 d Ald C ´ 1 5 Miyazawa1, Luis M. Montilla1, 2, Esteban Alejandro Agudo-Adriani1, 3, edo Ascanio1, 4, Gloria Mari˜no-Brice˜no1, and Aldo Cr´oquer1, 5 Corresponding author: 11 Emy Miyazawa, Aldo Cr´oquer1 12 Email address: 12-11222@usb.ve, acroquer@usb.ve 13 Email address: 12-11222@usb.ve, acroquer@usb.ve 13 Emy Miyazawa Corresp., 1 , Luis M Montilla 1, 2 , Esteban Alejandro Agudo-Adriani 1, 3 , Alfredo Ascanio 1, 4 , Gloria Mariño- Briceño 1 , Aldo Croquer Corresp. 1, 5 This result highlights the importance of creating scale-adapted management actions in Venezuela and likely across the Caribbean region. PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) Manuscript to be reviewed On the importance of spatial scales on beta 1 diversity of coral assemblages: a study 2 from Venezuelan coral reefs 3 Emy Miyazawa1, Luis M. Montilla1, 2, Esteban Alejandro Agudo-Adriani1, 3, 4 Alfredo Ascanio1, 4, Gloria Mari˜no-Brice˜no1, and Aldo Cr´oquer1, 5 5 1Laboratorio de Ecolog´ıa Experimental, Universidad Sim´on Bol´ıvar 6 2Integrative Marine Ecology Department, Stazione Zoologica Anton Dohrn 7 3Department of Biology, University of North Carolina at Chapel Hill 8 4Department of Biology, Miami University of Ohio 9 5Centro de Innovaci´on Marina, The Nature Conservancy 10 Corresponding author: 11 Emy Miyazawa, Aldo Cr´oquer1 12 Email address: 12-11222@usb.ve, acroquer@usb.ve 13 ABSTRACT 14 Estimating variability across spatial scales has been a major issue in ecology because describing patterns in space is extremely valuable to propose speciﬁc hypotheses aim to unveil key processes behind these patterns. This paper aims to estimate the variability of the coral assemblage structure at different spatial scales in order to determine which scales explain the greatest variability on β-diversity. For this, a fully-nested design including a series of hierarchical-random factors encompassing three spatial scales: (1) regions, (2) localities and (3) reefs sites across the Venezuelan territory. The variability among spatial scales were tested with a permutation analysis of variance (Permanova) based on Bray-Curtis index. Dispersion in species presence/absence across scales (i.e., β-diversity) was tested with a test of homogeneity of dispersions (PermDisp) based on Jaccard's index. We found the highest variability in the coral assemblage structure between sites within locality (Pseudo-F = 5.34; p-value = 0.001, CV = 35.10%). We also found that longitude (Canonical corr = 0.867, p = 0.001) is a better predictor of the coral assemblage structure in Venezuela, rather than latitude (Canonical corr = 0.552, p = 0.021). Largest changes in β-diversity of corals occurred within sites (F = 2.764, df1= 35, df2 = 107, p = 0.045) and within localities (F = 4.438, df1= 6, df2 = 29, p = 0.026). Our results suggest that processes operating at spatial scales of hundreds of meters and hundreds of kilometers might both be critical to shape coral assemblage structure in Venezuela, whereas smaller scales (i.e., hundreds of meters) showed to be highly- important for the species turnover component of β-diversity. This result highlights the importance of creating scale-adapted management actions in Venezuela and likely across the Caribbean region. Manuscript to be reviewed In addition, oceanographic processes 61 which partly deﬁne the environmental setting of a reef are also extremely variable within habitats, across 62 sites, reef systems, and regions (Chollett et al., 2010; Eidens et al., 2015). Furthermore, biological and 63 environmental factors may interact with each other to produce different patterns in species distribution 64 across several spatial scales. In consequence, understanding the underlying factors controlling the coral 65 species richness in a reef is not a simple task (Edmunds, 2013; Eidens et al., 2015; Karlson and Cornell, 66 1999) for it is a multi-scale problem (Komyakova et al., 2018). 67 Total species richness of a region, frequently named gamma diversity (γ), can be partitioned in two 68 components: (1) α-diversity (i.e., the number of species by site), and (2) β-diversity (i.e., the variation 69 in the species identities from site to site, sensu Whittaker (1960, 1972)). For decades, ecologist have 70 debated ways to estimate and interpret α and β-diversity; but in recent years, the study of β-diversity has 71 gained a lot of interest for it is what actually makes assemblages of species more or less similar to one 72 another at different places and times (Anderson et al., 2011; Vellend, 2010). Many different measures of 73 β-diversity have been introduced, but there is no overall consensus about which ones are most appropriate 74 for addressing particular ecological questions (Jurasinski et al., 2009; Tuomisto, 2010a,b). Anderson et al. 75 (2011) distinguished two types of β-diversity: (a) turn-over and (b) variation. Turn-over refers to changes 76 in community structure among sampling units distributed along well-deﬁned environmental gradients, 77 whereas variation portrays variability in species composition among sample units within a given spatial or 78 temporal extent, or within a given category of a factor (such as a habitat type or experimental treatment). 79 On the other hand, Baselga (2010) partitioned the total β-diversity into two components: (1) nestedness, 80 i.e. when the species composition of sample units with low richness represent a subset of the species 81 found in the richest sample units, and (2) species replacement, i.e. a turn-over of species (Gaston et al., 82 2007; Leibold et al., 2004; Svenning et al., 2011). Manuscript to be reviewed depend on four important processes: (1) climate, (2) species interactions, (3) habitat structure and (4) 46 dispersal capabilities, each one operating with different strength at a range of spatial scales (McGill, 47 2010). Generally, the presence or absence of organisms within a community may depend on rare or 48 large-scale (region-speciﬁc) dispersal and colonization events, while local abundance is more a function 49 of frequent, ﬁne-spatial scale processes such as biotic interactions and habitat heterogeneity, e.g. Ricklefs 50 (1987). This implies that communities are structured by both abiotic and biotic factors nested along 51 different spatial scales which often occur along environmental gradients (Johnson and Goedkoop, 2002; 52 Whittaker and Heegaard, 2003). Concomitantly, the species richness of a community is also expected to 53 be highly dependent on spatial scales evaluated (Barton et al., 2013; Field et al., 2009; Melchior et al., 54 2017; Whittaker et al., 2001). 55 depend on four important processes: (1) climate, (2) species interactions, (3) habitat structure and (4) 46 dispersal capabilities, each one operating with different strength at a range of spatial scales (McGill, 47 2010). Generally, the presence or absence of organisms within a community may depend on rare or 48 large-scale (region-speciﬁc) dispersal and colonization events, while local abundance is more a function 49 of frequent, ﬁne-spatial scale processes such as biotic interactions and habitat heterogeneity, e.g. Ricklefs 50 (1987). This implies that communities are structured by both abiotic and biotic factors nested along 51 different spatial scales which often occur along environmental gradients (Johnson and Goedkoop, 2002; 52 Whittaker and Heegaard, 2003). Concomitantly, the species richness of a community is also expected to 53 be highly dependent on spatial scales evaluated (Barton et al., 2013; Field et al., 2009; Melchior et al., 54 2017; Whittaker et al., 2001). 55 Coral reefs are one of the most complex and diverse ecosystems of the planet. Reef species diversity 56 has been estimated at 600,000 to more than 9 million species worldwide (Plaisance et al., 2011; Reaka- 57 Kudla, 1997). The habitat and shelter for the majority of these species is largely provided by scleractinian 58 corals (Alvarez-ﬁlip et al., 2009). There is compiling evidence indicating that ecological processes 59 controlling the structure of coral assemblages (e.g. substrate availability, recruitment, competition, and 60 herbivory) are strongly dependent on spatial scales (Pandolﬁ, 2002). INTRODUCTION 33 The importance of scales in ecology has been largely acknowledged for decades (Schneider, 2001; Mac 34 Nally and Quinn, 1998; Wiens, 1989). MacArthur (1972) and Levin (1992) assays deeply inﬂuenced 35 modern ecologists by promoting the view that ecological processes act at a variety of spatial and temporal 36 scales, and they generate patterns that may differ from those at which processes act (Chave, 2013). Today 37 it is known that ecological dynamics tends to be stochastic at small scales, but variability is conditional 38 on the resolution of description (Chave, 2013; Levin, 1992). Furthermore, there has been an increased 39 recognition that the problem of scale at which ecological processes act, should be considered as critical if 40 it is wanted to produce general predictions about patterns in space and time (Chave, 2013). Thus, modern 41 ecological thinking agrees that to understand a system (e.g. a community), it is important to study it at the 42 appropriate scale (Chave, 2013). 43 It is clear that increasing consideration of scale is helping to address a key issue in ecology: the question 44 of what inﬂuences the distribution and abundance of organisms (McGill, 2010). Species distributions 45 PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) Manuscript to be reviewed Regardless the point of view, the study of each of 83 these components is relevant to understand processes that control ecological communities and a range of 84 ecosystem functions (Arias-Gonz´alez et al., 2008; Harborne et al., 2006). 85 While spatial patterns of γ and α-diversity of coral assemblages have been studied extensively; only 86 few studies have focused on measuring β-diversity, e.g. Arias-Gonz´alez et al. (2008); Connell et al. 87 (2004); Harborne et al. (2006). This is the case of Venezuela, where most of the papers published to date 88 have only been focused on site descriptions based on species composition and abundance, whereas the 89 inﬂuence of spatial variation across different scales on coral assemblages remains poorly explored. The 90 Venezuelan coast is highly heterogeneous with clear longitudinal environmental gradients (Chollett et al., 91 2010; Miloslavich et al., 2003) which are deeply inﬂuenced by up-welling regimes that play an important 92 role for the distribution of marine biodiversity (Miloslavich et al., 2010). In fact, algal communities in 93 rocky shores (Cruz-Motta, 2007) and sessile organisms associated to mangrove roots have been found to 94 vary at different spatial scales along the Venezuelan coast (Guerra-Castro et al., 2011). Thus, it should not 95 be surprising to ﬁnd coral assemblages to be extremely variable across spatial scales in Venezuela. We 96 expected that greater changes in community structure and β-diversity of coral assemblages will occur 97 at scales of thousand of kilometers (i.e., between the eastern and western regions) and within sites (i.e., 98 hundreds of meters). This is because of existing contrasting environmental settings driven by upwelling 99 spots that have been described along the Venezuelan coast line (Chollett et al., 2010; Miloslavich et al., 100 2/15 PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) Manuscript to be reviewed Figure 1. Map of the Venezuelan coast with the seven locations used in this study. Wester region, represented in blue, include: MOR= Morrocoy National Park and OCU= Ocumare de la Costa. Central region, represented in red, include: ROQ= Archipielago Los Roques National Park and CHI= Chichiriviche de la Costa. Eastern region, represented in green, included MOC= Mochima National Park, CUB= Cubagua and FRA= Los Frailes. Figure 1. Map of the Venezuelan coast with the seven locations used in this study. Wester region, represented in blue, include: MOR= Morrocoy National Park and OCU= Ocumare de la Costa. Study area 105 y We conducted a multi-scale sampling design comprising coastal areas as well as continental and oceanic 106 islands (Figure 1). Speciﬁcally, seven locations were sampled along the Venezuelan territory encompassing 107 three contrasting regions (Figure 1). The western region, included two localities: (1) Morrocoy National 108 Park (MNP) and (2) Ocumare de la Costa. The former is a continental reef system formed by a group of 109 keys and lagoons surrounded by fringing and patch reefs located nearby mangroves and seagrass beds 110 (Weil, 2003); whereas the latter is a small bay protected by reef barriers with seagrass and mangroves 111 dominating the inner and shallower habitats. Likewise, the central region, entailed two localities: (3) Los 112 Roques National Park (LRNP) which is an oceanic archipelago with a central lagoon, characterized by 113 extensive reef banks/patches and two large coralline barriers located south and east of the archipelago 114 (Weil, 2003); and (4) Chichirivivhe de la Costa, a location of rocky reefs with steep slopes and scattered 115 coral assemblages. Finally, in the eastern region three localities were included: (5) Mochima National 116 Park (MoNP), (6) Los Frailes and (7) Cubagua. In Mochima, seagrass beds and mangroves border a rocky 117 coastline with steep slopes and fringing reef communities. Los Frailes and Cubagua are islands lying at 118 the continental shelf and dominated by small patch reefs with scattered coral assemblages bordering their 119 coastlines. The whole eastern coast of Venezuela and its continental islands are subjected to seasonal 120 upwelling due to its connection with the Cariaco trench (Weil, 2003). The selection of these locations 121 aimed to cover the vast majority of reef habitats described for Venezuela (Weil, 2003). Permit for taking 122 pictures at marine protected areas was given by Ministerio del Poder Popular para el Ecosocialismo y 123 Aguas - Direcci´on General de Diversidad Biol´ogica under the ofﬁce n° 0033. 124 Manuscript to be reviewed Central region, represented in red, include: ROQ= Archipielago Los Roques National Park and CHI= Chichiriviche de la Costa. Eastern region, represented in green, included MOC= Mochima National Park, CUB= Cubagua and FRA= Los Frailes. 2003).The goal of this study was two-fold: (1) quantify spatial variation of coral assemblages from 101 hundreds meters to hundreds of kilometers, and (2) to determine if there are patterns of β-diversity across 102 these scales. 103 Data analysis 150 A Log(x + 1) transformation was applied to the data and the Bray-Curtis index was used to build the 151 similarity matrices. Hypothesis test were performed with a permutation-based analysis of variance 152 (PERMANOVA). A test of homogeneity of dispersions (PermDisp) was performed before running the 153 PERMANOVA to verify dispersion of data across different spatial scales. For the majority of the spatial 154 scales the PermDisp conﬁrmed homogeneity in dispersion except for sites (Supp. 2). However the 155 PERMANOVA test has been shown to be robust to identify dispersion versus location effects for balanced 156 designs like the one presented here (Anderson and Walsh, 2013). 157 With the PERMANOVA, we tested the spatial variability of coral assemblage structure (i.e., absolute 158 abundance of each species) from meters to hundreds of kilometers. The variance components were 159 estimated depending on each source of variation in the analysis following the procedures outlined by 160 Clarke and Gorley (2006); Anderson et al. (2008). Spatial patterns (if any) were visualized by using an 161 Analysis of Principal Coordinates (PCO) aimed to represent the position of centroids of each site in a 162 Bray-Curtis space. Also, a Similarity Percentage Analysis (SIMPER) was carried out at each spatial 163 scale to identify and estimate the species that contribute the most to the patterns observed. SIMPER 164 results were represented as heat maps to show the relative contribution of each coral specie to the average 165 Bray-Curtis similarity. Additionally, a Canonical Analysis of Principal Coordinates (CAP) based on 166 Bray-Curtis similarity index was carried out. We used the scale that most contributed to the variation 167 in the dissimilarity of coral species to determine the correlation between the coverage of scleractinian 168 corals and their latitudinal and longitudinal position. For coral cover we used the same variables as in the 169 PERMANOVA, and the latitude and longitude were understood as proxies of distance respect to the coast 170 and position along the coast, respectively. All analyses were carried out using Primer 6 - Permanova + 171 (Clarke and Gorley, 2006; Anderson et al., 2008). 172 Differences on β-diversity were tested across different spatial scales using a test of homogeneity of 173 dispersions (PermDisp) (Anderson, 2006) based on presence/absence Jaccard index. Benthic surveys 132 At each reef site, benthic surveys were conducted during 2017 and 2018, following the guidelines 133 outlined by the Global Coral Reef Monitoring Network-Caribbean (GCRMN) (GCRMN, 2014) with 134 slight modiﬁcations. In order to increase the number of sampled sites, we surveyed four instead of 135 ﬁve 30m-long transects parallel to the shoreline following the bottom contour between 8-10 m depth. 136 Transects were set randomly, with the ﬁrst transect being always layout at the ﬁrst spot of diving. From 137 that point, each transect was moved up or down from the ﬁrst transect. Distance among transects varied 138 from 5 to 6 m, so each operational unit was inter-spaced across the sampled reef habitat. For each transect, 139 15 80x90-cm photos were taken every other meter to determine the benthic community structure (N = 60 140 photos per site). A reference frame was used in the ﬁeld to calibrate each photograph in the laboratory for 141 further analysis of benthic cover. 142 Manuscript to be reviewed and β-diversity from hundreds of meters (sites) to hundreds of kilometers (region). The factor region 128 encompassed three levels (West, Center, and East); nested within region there were two/three localities, 129 four to seven reef sites within each locality and four 30m-long transects within each site, understood as 130 the operational unit. 131 and β-diversity from hundreds of meters (sites) to hundreds of kilometers (region). The factor region 128 encompassed three levels (West, Center, and East); nested within region there were two/three localities, 129 four to seven reef sites within each locality and four 30m-long transects within each site, understood as 130 the operational unit. 131 Analysis of photo-quadrats 143 The photo quadrat analysis was performed using PhotoQuad (Trygonis and Sini, 2012). For this, every 144 coral was identiﬁed to species level and the percentage cover was estimated from 25 points randomly set 145 in an area of approximately 7,200 cm2. From the analysis of photo quadrats, we obtained two matrices: 146 (1) absolute cover of coral species and (2) coral species presence/absence. Data cleaning and quality 147 control were performed using R (R Core Team, 2019). Thus, coral cover estimates were done from a 148 randomly-selected sample composed of 375 points per transect (15 photos x 25 points = 375). 149 Experimental design 125 p g A fully-nested design encompassing three hierarchical-random factors (i.e., site, locality and region) was 126 used to determine spatial variation on coral assemblage structure (i.e., absolute cover of coral species) 127 A fully-nested design encompassing three hierarchical-random factors (i.e., site, locality and region) was 126 used to determine spatial variation on coral assemblage structure (i.e., absolute cover of coral species) 127 used to determine spatial variation on coral assemblage structure (i.e., absolute cover of coral species) 127 3/15 PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) Manuscript to be reviewed et al., 2014). To verify this, we estimated sampling coverage (i.e. the relationship of interpolation and 181 extrapolation of the effective number of species in the community) (Hsieh et al., 2016; Chao et al., 2014). 182 This methodological approach allows statistical inferences without the bias of underestimated rare species 183 in assemblies and/or assigning them the same weight as abundant species (Chao et al., 2014). The 184 sampling coverage was calculated using the iNEXT package in the R software (Hsieh et al., 2016). We 185 found that 80% of the sites had a sample coverage greater than 0.7 (Table 1, Supp. 3), which is considered 186 a proper sampling effort to describe species assemblages Hsieh et al. (2016). 187 et al., 2014). To verify this, we estimated sampling coverage (i.e. the relationship of interpolation and 181 extrapolation of the effective number of species in the community) (Hsieh et al., 2016; Chao et al., 2014). 182 This methodological approach allows statistical inferences without the bias of underestimated rare species 183 in assemblies and/or assigning them the same weight as abundant species (Chao et al., 2014). The 184 sampling coverage was calculated using the iNEXT package in the R software (Hsieh et al., 2016). We 185 found that 80% of the sites had a sample coverage greater than 0.7 (Table 1, Supp. 3), which is considered 186 a proper sampling effort to describe species assemblages Hsieh et al. (2016). 187 Patterns of coral assemblage structure 189 The results show that species composition and abundance of corals in Venezuela varied across different 190 spatial scales. The greatest variability was found at the scale of sites (Pseudo-F = 5.34, p-value = 0.001) 191 which accounted for 35.1% of the total variance (table 2). The scale of hundreds of kilometers was the 192 second most important source of variation in the analysis (Pseudo-F = 3.35, p-value = 0.01, CV = 21.35% 193 table 2). This result indicates that coral assemblages in Venezuela only vary by 21.35% at the scale of 194 region. Also, we found statistical signiﬁcance at the scale of locations within regions, explaining 11.42% 195 of the total variance (table 2). Thus, our results indicate that coral assemblages are much variable at small 196 to medium scales (i.e., from hundreds of meters to tens of kilometers) rather than hundreds of kilometers 197 (i.e., regions) alone. 198 With over 70% of the total variance explained by the ﬁrst three PCO axes, localities within regions 199 ordinate according to changes in cover of coral species (Figure 2). Our results show that the eastern 200 and central regions are much more similar to each other concerning the western region. The SIMPER 201 analysis showed clear patterns deﬁning regions and locations across Venezuela, however, sites showed 202 variable species composition and cover (Figure 3). Overall, Western region was largely composed of 203 Orbicella faveolata reefs, whereas the eastern reefs were dominated by Pseudodiploria strigosa (Figure 204 3). On the other hand, across the Central regions which included Oceanic and Coastal Reefs, mixed coral 205 communities were found. These species accounted for more than 75% of dissimilarities across localities, 206 sites and regions (Supp. 4-6). 207 Longitude (correlation = 0.867, p = 0.001) was highly correlated with observed spatial patterns in 208 contradiction to latitude (correlation = 0.552, p = 0.021). This result indicates that the relative position 209 of each site along the Venezuelan coast (i.e., longitudinal variation), is an important factor to determine 210 the features of coral assemblages in Venezuela, instead of the proximity to the coast (i.e., latitudinal 211 variation). 212 Data analysis 150 To maintain the 174 nested design we used transects to test dispersion across sites, the composition of sites to test dispersion 175 across localities, and the composition of localities to test dispersion across regions. The Jaccard index 176 was then split into the components of nestedness and turn-over using the BetaPart package in R (Baselga 177 and Orme, 2012). 178 Due to the life history strategies of scleractinian corals (i.e. large colonies of a single species may 179 often occupy an entire quadrat), rare species in the assemblages might have been underestimated (Chao 180 4/15 PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) Patterns of beta diversity 213 When assessing β-diversity, we found the highest variation in species presence/absence occurring between 214 transect of the same site (F = 2.764, p = 0.045) and between sites of the same locality (F = 4.438, p = 0.026). 215 On the other hand, at larger scales, we found no signiﬁcant dispersion in species composition between 216 localities of the same region (table 3, Supp. 7-9). In addition, site and locality were the spatial scales 217 with the largest Jaccard dissimilarity, with turn-over component as the main contributor. Furthermore, at 218 larger scales, Jaccard dissimilarity decreased and the contribution of turn-over and nestedness became 219 evener (Figure 4). Thus, our result clearly shows that in Venezuela it is more likely to ﬁnd changes in 220 coral species composition at small to medium scales (i.e., hundreds of meters to tens of kilometers) than 221 at larger scales (i.e., hundreds of kilometers). Finally, the results indicate that coral species found between 222 the western, central and eastern region of Venezuela can result either from species replacement or from 223 species loss, which is interpreted as a subset of a total pool of species. 224 DISCUSSION 225 While coral assemblages have been extensively studied in Venezuela, this is the ﬁrst multi-scale assessment 226 to show the importance of spatial scales in determining the structure of these communities. Overall, 227 we found that coral assemblages in Venezuela are variable from hundreds of meters and hundreds of 228 kilometers. Additionally, the largest changes in the composition of coral species occurred at a small scale 229 with a clear predominance of species turn-overs. Also, longitude and latitude are a good predictors of 230 5/15 PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) Manuscript to be reviewed Table 1. Sampling coverage for each reef site. p g g Locality Site Replicates Incidence Richness Sampling coverage Morrocoy Boca Seca 4 12 6 0.900 Bajo Caim´an 4 9 4 0.952 Medio 4 13 7 0.736 Mero 4 16 10 0.796 Norte 4 20 7 0.979 Sombrero 4 24 11 0.836 Sur 4 18 8 0.885 Ocumare Ci´enaga este 4 15 8 0.706 Ci´enaga interna 4 24 11 0.779 Ci´enaga oeste 4 25 10 0.917 Guabinitas 4 16 7 0.925 Los Roques Cayo Agua 4 25 11 0.893 Boca de Cote 4 33 12 0.937 Dos Mosquises sur 4 21 10 0.790 Madrisqui 4 17 7 0.912 Pelona de Rabusqui 4 14 6 0.839 Salinas 4 23 10 0.851 La Venada 4 7 4 0.679 Chichiriviche de la costa La Pared 4 21 9 0.901 Media Legua 4 15 9 0.640 Punta de Media Legua 4 17 9 0.741 Petaquire 3 11 7 0.636 Playa Tibur´on 4 20 7 0.979 Punta Mono 4 19 8 0.828 Mochima San Agust´ın 4 1 1 1.000 Blanca 4 23 9 0.893 Carabela 4 2 2 0.400 Punta Cruz 4 14 6 0.893 Gabarra 4 11 5 0.864 Garrapata 4 20 7 0.940 Cubagua Charagato 4 6 4 0.625 Punta Conejo 4 6 4 0.625 La Muerta 4 6 4 0.625 Los Frailes Cominoto 4 6 2 1.000 La Pecha 4 1 1 1.000 Puerto Real 4 8 2 1.000 Table 2. Three-way permutation-based analysis of variance (PERMANOVA) based on Bray-Curtis Similarity to test differences in coral assemblage structure. Source df SS MS Pseudo-F P(perm) Unique perms %CV Region 2 7.11x104 35529 3.35 0.01 998 21.35 Locality 4 4.09x104 10219 2.28 0.002 998 11.42 Site 29 1.30x105 4483 5.34 0.001 994 35.10 Residuals 107 8.99x104 840.17 32.14 Total 142 3.36x105 6/15 PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) Manuscript to be reviewed Figure 2. Principal coordinates ordination plot (PCO) showing the variation of coral assemblages at the site level nested into locality. The distance between samples are interpreted as % of similarity. Panels A, B, and C represent the different combined pairs of the ﬁrst three principal coordinates axes. Figure 2. Principal coordinates ordination plot (PCO) showing the variation of coral assemblages at the site level nested into locality. The distance between samples are interpreted as % of similarity. Manuscript to be reviewed Panels A, B, and C represent the different combined pairs of the ﬁrst three principal coordinates axes. Figure 2. Principal coordinates ordination plot (PCO) showing the variation of coral assemblages at the site level nested into locality. The distance between samples are interpreted as % of similarity. Panels A, B, and C represent the different combined pairs of the ﬁrst three principal coordinates axes. Table 3. Test of homogeneity of dispersions (PermDisp) to compare the distances from each factor to centroids as a test for similarity in β diversity among factors. Df F N.Perm P(perm) Site 35 2.764 999 0.045 107 Locality 6 4.438 999 0.026 29 Region 2 8.216 999 0.283 4 7/15 PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) Manuscript to be reviewed Figure 3. Heat map showing the species contribution to similarity (Cut off for low contributions = 75 Panels A, B, and C represent the three scales considered in the study. Figure 3. Heat map showing the species contribution to similarity (Cut off for low contributions = 75%). Panels A, B, and C represent the three scales considered in the study. 8/15 PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Figure 4. Total dissimilarity as Jaccard index and their respective turn-over and nestedness components for each spatial scale. Figure 4. Total dissimilarity as Jaccard index and their respective turn-over and nestedness components for each spatial scale. coral assemblage structure (i.e., species composition and abundance) further indicating that large-scale 231 processes are also important to determine the structure of these communities. 232 coral assemblage structure (i.e., species composition and abundance) further indicating that large-scale 231 processes are also important to determine the structure of these communities. 232 Manuscript to be reviewed at broad spatial scales (hundreds of kilometers). However similar to our study, they found a higher degree 256 of variability within sites highlighting the relevance of local ecological drivers (e.g. rugosity and wave 257 exposure) in structuring coral assemblages. Other studies have also taken into account the importance of 258 spatial scales for coral assemblages, but must of then have focused on total live coral cover (Murdoch and 259 Aronson, 1999) and total abundance of colonies (Hughes et al., 1999). 260 It is widely acknowledged that behind patterns are ecological processes that shape communities (Barton 261 et al., 2013). In coral reef ecosystems, processes such as predation (e.g. herbivory) and competition 262 have profound impacts on species abundance and composition at scales of a few meters. For example, 263 very high densities or the absence of Diadema antillarum can determine the composition of corals in 264 patches of few m2 (Sammarco, 1980). Also, reef ﬁsh can preferentially prey on certain coral species, thus 265 decreasing their abundance or making them less competitive than other neighboring corals. In addition, 266 the presence of vermetids could potentially modify the survival rates of coral species (Lenihan et al., 2011). 267 Furthermore, factors such as structural complexity (e.g. rugosity and micro scale habitat heterogeneity) 268 may inﬂuence competition and survival of colonies depending on their sizes (Almany, 2004; Zilberberg 269 and Edmunds, 2001). Coral reefs in Venezuela are known to be highly variable within and between 270 sites but the processes responsible for these patterns have not been ﬁrmly established. However, spatial 271 variation of coral assemblages in Los Roques has been associated to changes in reef slopes (Cr´oquer and 272 Villamizar, 1998). 273 Various anthropogenic stressors can alter biological interactions thereby affecting the structure of 274 coral assemblages. For example, over ﬁshing often leads to the dominance of macroalgae which escape 275 to herbivory control (Hughes, 1994). The selective extraction of species of carnivorous ﬁsh can lead 276 to an increment in the abundance of echinoderms, which may also steer to increasing erosion, loss of 277 topographic complexity and live coral cover (Roberts, 1995; Mcclanahan and Muthiga, 1988). Manuscript to be reviewed Also, high 278 intensity of recreational activities represents an important disturbance to marine communities (Milazzo 279 et al., 2002), for example, coral cover and the proportion of massive corals is being found to be lower 280 in places with high recreational diving intensity (Tratalos and Austin, 2001). Thus, spatial variability 281 recorded within sites of Morrocoy and Los Roques National Parks could be explained by their differences 282 in touristic use for not all sites within these MPAs are exposed to the same human pressures. 283 In addition, experimental evidence shows that some coral species differ in resistance to environmental 284 stressors such as sedimentation (McClanahan and Obura, 1997; Rogers, 1983) which could explain the 285 patterns observed within sites and between locations showing higher sedimentary regimes and river 286 presence (e.g. coastal versus oceanic reef sites) (Dikou and van Woesik, 2006). Furthermore, local 287 oceanographic events can generate mortality which leads to changes in the structure of coral assemblages. 288 Differences between Playa Caim´an, Cayo Norte and Sombrero (Morrocoy National Park) represents a 289 example of how abnormal oceanographic conditions can alter benthic communities by killing dominant 290 species in speciﬁc sites while promoting stable alternative states which hampers recovery (Bastidas et al., 291 2006). Our results seem to support that each site/location in Venezuela possess different communities 292 because they may have been affected/unaffected by different stressors and/or mortality events in different 293 times. Thus, high spatial variability on coral assemblages in Venezuela could be related to the differences 294 in the disturbance regime and local history as noticed in other studies (Pisapia et al., 2019). 295 Coral bleaching mortality events may be patchy (West and Salm, 2003) and could potentially affect 296 coral assemblages at different spatial scales (e.g. within sites, localities and regions). For example, in 297 2010 an increase in seawater temperature in Los Roques National Park affected 72% of the colonies 298 at the study sites, showing bleaching and prevalence of diseases such as black band and white plague. 299 Extensive mortality caused changes in the community structure a year later (Bastidas et al., 2012). Patterns of coral assemblage structure 233 Previous studies have acknowledged the importance of spatial scales on coral assemblages in the Caribbean 234 and in Venezuela (Weil, 2003). Particularly, the effect of upwelling and other related oceanographic 235 processes has been pinpointed as strong factors that shape coral assemblage structure along the Venezuelan 236 coast where at least 12 upwelling points have been targeted (Miloslavich et al., 2003; Chollett et al., 2012). 237 Our study shows that coral assemblages in Venezuela are much variable within and between localities than 238 we originally expected. We found two fold higher variability at small to medium scales when compared to 239 regions. However, coral assemblages between the western, central and eastern regions differed by 21.35%, 240 further indicating that differences at scale of region cannot be neglected. Moreover, about 32% of the 241 total variance in coral species composition and abundance was associated to the residual which indicates 242 that other variables like levels of anthropogenic disturbance, oceanic inﬂuence or other intermediate 243 scales between those taken into consideration might also be relevant to determine the structure of these 244 assemblages. 245 In Venezuela, encrusting communities associated with mangrove roots have been studied following 246 a spatial hierarchical design (Guerra-castro et al., 2016). Similar to our results, higher variation for 247 this assemblages were found at the smallest and biggest scales (Guerra-castro et al., 2016). Moreover, 248 algal assemblages associated rocky platforms have previously been found to be highly variable at tens of 249 kilometers and not between localities or regions, further illustrating the importance of local processes in 250 providing structure to different assemblages of sessile organisms in the country (Cruz-Motta, 2007). 251 Only a few numbers of studies encompassing multiple hierarchical spatial scales have been conducted 252 in the Caribbean. For example, in a multi-scale study Williams et al. (2015) surveyed a series of reef sites 253 across locations and different bio-regions in the Caribbean for three major coral taxonomic groups: corals, 254 sponges and octocorals. They concluded these faunas exhibited considerable biogeographical variability 255 9/15 PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) Patterns of beta diversity 322 We found that differences in coral species composition occurred at spatial scales of hundreds of meters 323 to tens of kilometers. Although it is known that β-diversity depends on the spatial scale at which it is 324 measured, opposite to our results, in most studies β-diversity is assumed to be homogeneous at small 325 spatial scales (Hewitt et al., 2005; Whittaker, 1975). Changes in species composition at scales of tens 326 of meters often occurs in highly-heterogeneous habitats sampled with enough resolution to detect these 327 changes (Hewitt et al., 2005). In our study, we found more likely to have different species composition 328 within transects of a single site than between localities belonging to different regions. Thus, our results 329 clearly support that coral habitats in Venezuela are extremely variable at local scales, suggesting signiﬁcant 330 environmental heterogeneity within reef habitats, with coral species probably forming mosaics or patches 331 within a single habitat. However, it is not clear what are the conditions favoring this heterogeneity within 332 the reef sites. This variation at the smallest scale could mean that Venezuelan coral assemblies are in 333 good condition, as well as there are patches of mortality, with more or fewer species, reﬂected in a high 334 turnover rate. 335 At larger scales (i.e., between the eastern, central and western Venezuelan coast) we found quite 336 similar and homogeneous coral species composition, which may be partly due to the reduced pool of 337 species that exist in the Caribbean when compared with the Indo Paciﬁc region. In regions with larger 338 species pools such as the Indo Paciﬁc, β-diversity tends to be higher at larger spatial scales because 339 species represent a subset of a total species pool, e.g. Zvuloni et al. (2010). In Venezuela, coral reefs 340 located at the oceanic sites and the western coast are dominated by Orbicellas , whereas in the eastern 341 coast and the majority of sites located at the central coast Pseudodiplorias and Colpophyllias become 342 more important. 343 Our results indicate that at scales of tens of kilometers species nestedness (loss) becomes as important 344 as species turnover (replacement). These two components arise from different ecological phenomena 345 (Baselga, 2010). Manuscript to be reviewed suggested that during these periods the assemblages of macroalgae become more dominant (Diaz-Pulido 311 and Garz´on-Ferreira, 2002) which could modify the coral assemblage structure through competitive 312 processes. 313 suggested that during these periods the assemblages of macroalgae become more dominant (Diaz-Pulido 311 and Garz´on-Ferreira, 2002) which could modify the coral assemblage structure through competitive 312 processes. 313 The Venezuelan coast is characterized by an upwelling period that occurs between January and June, 314 in particular, the eastern region of the country is characterized by a large area of upwelling (Castellanos 315 et al., 2002). However, studies such as those of Jim´enez and Cort´es (2003) and Rodr´ıguez et al. (2009) did 316 not ﬁnd an effect of the upwelling on factors such as the reproductive behavior of spawn corals and the 317 growth rates of colonies. On the other hand, the dynamic of black band disease, one of the most important 318 factors producing rapid coral mortality in Cubagua has been shown to be deeply inﬂuenced by upwelling 319 events (Rodriguez and Croquer, 2008). Thus, our results indicate that upwelling alone is not sufﬁcient to 320 explain the extremely variable nature of coral assemblage. 321 Manuscript to be reviewed Other 300 bleaching events recorded in Venezuela since 1998 primarily affected reefs in several oceanic islands and 301 the western and central coast of Venezuela, but these events did not produce extensive mortality events 302 like the one reported in 2010 (Rodr´ıguez et al., 2010; Bastidas et al., 2012; M´onaco et al., 2012). 303 It is likely that the eastern coast of Venezuela remained less affected by bleaching events because of 304 seasonal upwelling and unique environmental conditions. In fact, Nakamura and van Woesik (2001) found 305 differences in coral mortality rates during bleaching events, according to local environmental settings 306 (e.g. light intensity, penetration, temperature and currents). However, Chollett et al. (2010) argued that 307 upwelling does not necessarily guarantee a refuge for corals. Thus, in Venezuela differences between 308 geographical regions could be strongly inﬂuenced by factors such as nutrient input and temperature 309 decrease associated with the upwelling season (Birkeland, 1988; Weil, 2003). In fact, it has been 310 10/15 PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) REFERENCES 377 Agardy, T., Di Sciara, G. N., and Christie, P. (2011). Mind the gap: addressing the shortcomings of marine 378 protected areas through large scale marine spatial planning. Marine Policy, 35(2):226–232. 379 Almany, G. R. (2004). Does increased habitat complexity reduce predation and competition in coral reef 380 ﬁsh assemblages? 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Navigating the multiple meanings of beta-diversity: a roadmap for the practicing ecologist. 389 Ecology Letters, 14:19–28. 390 Anderson, M. J., Gorley, R. N., and Clarke, K. R. (2008). PERMANOVA+ for PRIMER: Guide to Software 391 and Statistical Methods. PRIMER-E, Plymouth. 392 Anderson, M. J. and Walsh, D. C. (2013). Permanova, anosim, and the mantel test in the face of 393 heterogeneous dispersions: what null hypothesis are you testing? Ecological monographs, 83(4):557– 394 574. 395 Arias-Gonz´alez, J. E., Legendre, P., and Rodr´ıguez-zaragoza, F. A. (2008). Journal of Experimental Marine 396 Biology and Ecology Scaling up beta diversity on Caribbean coral reefs. Journal of Experimental 397 Marine Biology and Ecology, 366(1-2):28–36. 398 Barton, P. S., Cunningham, S. A., Manning, A. D., Gibb, H., Lindenmayer, D. B., and Didham 99 (2013). The spatial scaling of beta diversity. Global Ecology and Biogeography, 22(6):639–6 00 Baselga, A. (2010). Partitioning the turnover and nestedness components of beta diversity. Global 401 Ecology and Biogeography, 19(1):134–143. 402 Baselga, A. and Orme, C. D. L. (2012). Betapart: An R package for the study of beta diversity. Methods 403 in Ecology and Evolution, 3(5):808–812. Manuscript to be reviewed whereas oceanographic/coastal processes could explain latitudinal variability. Regarding β-diversity, 365 coral assemblages are fairly homogeneous across the Venezuelan coast, while increasing spatial resolution 366 shows greater heterogeneity, with smaller scales revealing a greater change in species composition. In 367 addition, the replacement of species is a relevant phenomenon to explain these diversity patterns. Results 368 from this study highlights the importance of taking into account local variability during the design and 369 implementation of speciﬁc conservation efforts. 370 whereas oceanographic/coastal processes could explain latitudinal variability. Regarding β-diversity, 365 coral assemblages are fairly homogeneous across the Venezuelan coast, while increasing spatial resolution 366 shows greater heterogeneity, with smaller scales revealing a greater change in species composition. In 367 addition, the replacement of species is a relevant phenomenon to explain these diversity patterns. Results 368 from this study highlights the importance of taking into account local variability during the design and 369 implementation of speciﬁc conservation efforts. 370 ACKNOWLEDGMENTS 371 This research was funded by the Waitt Foundation through the Rapid Ocean Conservation (ROC) Grant 372 Program. We thank the support provided by Fundaci´on para la Defensa de la Naturaleza (FUDENA), 373 Instituto de Estudios Avanzados (IDEA) and the Escuela de Ciencias Aplicadas del Mar (ECAM), as well 374 as Jos´e Cappelletto, Zlatka Rebolledo and Alejandra Verde who helped with the ﬁeld work. We also thank 375 Rita Peachey and all the 39th AMLC meeting committee for their help to attend the conference. 376 This research was funded by the Waitt Foundation through the Rapid Ocean Conservation (ROC) Grant 372 This research was funded by the Waitt Foundation through the Rapid Ocean Conservation (ROC) Grant 372 Program. We thank the support provided by Fundaci´on para la Defensa de la Naturaleza (FUDENA), 373 Instituto de Estudios Avanzados (IDEA) and the Escuela de Ciencias Aplicadas del Mar (ECAM), as well 374 as Jos´e Cappelletto, Zlatka Rebolledo and Alejandra Verde who helped with the ﬁeld work. We also thank 375 Rita Peachey and all the 39th AMLC meeting committee for their help to attend the conference. 376 g pp p y p ( ) Instituto de Estudios Avanzados (IDEA) and the Escuela de Ciencias Aplicadas del Mar (ECAM), as well 374 as Jos´e Cappelletto, Zlatka Rebolledo and Alejandra Verde who helped with the ﬁeld work. We also thank 375 Rita Peachey and all the 39th AMLC meeting committee for their help to attend the conference 376 Instituto de Estudios Avanzados (IDEA) and the Escuela de Ciencias Aplicadas del Mar (ECAM), as well 374 as Jos´e Cappelletto Zlatka Rebolledo and Alejandra Verde who helped with the ﬁeld work We also thank Instituto de Estudios Avanzados (IDEA) and the Escuela de Ciencias Aplicadas del Mar (ECAM), as well 374 as Jos´e Cappelletto, Zlatka Rebolledo and Alejandra Verde who helped with the ﬁeld work. We also thank 375 Rita Peachey and all the 39th AMLC meeting committee for their help to attend the conference. 376 Rita Peachey and all the 39th AMLC meeting committee for their help to attend the conference. 376 Patterns of beta diversity 322 Species nestedness occurs when the biotas of sites with smaller numbers of species are 346 subsets of the biotas at richer sites, reﬂecting a non-random process of species loss as a consequence 347 of any factor that promotes the orderly disaggregation of assemblages (Baselga, 2010). On the other 348 hand, species turn-over (replacement) occurs as a consequence of environmental sorting and spatial 349 and historical constraints (i.e., stochastic process) (Qian et al., 2004). For example, processes such as 350 settlement selectivity of coral larvae could explain species turn overs at tens of meters and kilometers. 351 Coral larvae are known to select certain characteristics in the habitat to settle down, e.g. presence of 352 certain species of coralline algae (Ritson-Williams et al., 2010) or sounds of the reef (Vermeij et al., 353 2010).Our results therefore indicate that coral assemblage structure in Venezuela is probably regulated by 354 a series of interconnected processes acting alone and/or in combination at various spatial scales. This 355 result highlights the importance of creating scale-adapted management actions in Venezuela since the 356 smallest scales reﬂect the greatest variability. However, very small MPAs are often ineffective in achieving 357 their conservation goals, so they must necessarily be chained into a large-scale strategy (Agardy et al., 358 2011). 359 In summary, coral assemblage structure in Venezuela is highly variable at different spatial scales but 360 within locality variability seem to be very important. The processes that could underlie these patterns are 361 diverse and complex and little experimental efforts to untangle the speciﬁc contribution of each factor 362 have been conducted. Longitude is a good predictor of coral assemblages in Venezuela. Upwelling-related 363 processes could be targeted as potential candidates to explain longitudinal variation of coral assemblages, 364 11/15 PeerJ reviewing PDF \| (2019:08:40572:2:0:NEW 1 Apr 2020) Manuscript to be reviewed and van Woesik, R. (2006). Survival under chronic stress from sediment load: Spatial 434 patterns of hard coral communities in the southern islands of Singapore. 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https://openalex.org/W2793405560	http://urlib.net/sid.inpe.br/mtc-m21c/2018/05.04.19.08	English	null	Sensitivity of Numerical Weather Prediction to the Choice of Variable for Atmospheric Moisture Analysis into the Brazilian Global Model Data Assimilation System	Atmosphere	2,018	cc-by	7,047	Article Sensitivity of Numerical Weather Prediction to the Choice of Variable for Atmospheric Moisture Analysis into the Brazilian Global Model Data Assimilation System Thamiris B. Campos 1,, Luiz F. Sapucci 1, Wagner Lima 2 and Douglas Silva Ferreira 3 ID 1 National Institute for Space Research, Center for Weather Forecasting and Climate Research, Cachoeira Paulista, SP 12227-010, Brazil; luiz.sapucci@inpe.br 2 Climatempo, São José dos Campos, SP 12247-016, Brazil; wﬂauber@gmail.com 3 Instituto Tecnológico Vale, Belém, PA 66055-090, Brazil; douglas.silva.ferreira@itv.org Correspondence: thamiris.campos@cptec.inpe.br Received: 9 February 2018; Accepted: 13 March 2018; Published: 23 March 2018 Received: 9 February 2018; Accepted: 13 March 2018; Published: 23 March 2018 Received: 9 February 2018; Accepted: 13 March 2018; Published: 23 March 2018 Abstract: Due to the high spatial and temporal variability of atmospheric water vapor associated with the deﬁcient methodologies used in its quantiﬁcation and the imperfect physics parameterizations incorporated in the models, there are signiﬁcant uncertainties in characterizing the moisture ﬁeld. The process responsible for incorporating the information provided by observation into the numerical weather prediction is denominated data assimilation. The best result in atmospheric moisture depend on the correct choice of the moisture control variable. Normalized relative humidity and pseudo-relative humidity are the variables usually used by the main weather prediction centers. The objective of this study is to assess the sensibility of the Center for Weather Forecast and Climate Studies to choose moisture control variable in the data assimilation scheme. Experiments using these variables are carried out. The results show that the pseudo-relative humidity improves the variables that depend on temperature values but damage the moisture ﬁeld. The opposite results show when the simulation used the normalized relative humidity. These experiments suggest that the pseudo-relative humidity should be used in the cyclical process of data assimilation and the normalized relative humidity should be used in non-cyclic process (e.g., nowcasting application in high resolution). Keywords: atmospheric water vapor; numerical weather prediction; variational data assimilation; moisture control variable; pseudo-relative humidity; normalized relative humidity Atmosphere 2018, 9, 123; doi:10.3390/atmos9040123 1. Introduction The atmospheric moisture ﬁeld presents variation of high and low frequencies. These frequencies depend directly on the heating of the terrestrial surface and indirectly on some variability patterns of low frequency by teleconnection [1,2]. In addition, the space ﬁeld presents variability associated with wind, topography and surfaces types. Wind changes moisture by advectives or recycling processes. Topography is responsible for large amount of water vapor moving windward of the mountain, and small amount moving downwind. The plane and coastal surfaces present a larger storage of water vapor than continental areas [3,4]. The numerical representation of the atmospheric moisture ﬁeld involving these characteristics still presents a challenge in modeling. Although the models of Numerical Weather Prediction (NWP) are quite useful to spatially characterize the behavior of the atmospheric moisture, they are deﬁcient at physically representing all the involved processes. Due to temporal and spatial discretization Atmosphere 2018, 9, 123; doi:10.3390/atmos9040123 www.mdpi.com/journal/atmosphere 2 of 12 Atmosphere 2018, 9, 123 applied in this model, several of these processes require a parameterization that has been developed for simulations of the atmosphere. A portion of the imprecision of these simulations is from the uncertainties contained in the observations [5,6]. Statistical combination of atmospheric moisture observations with the predicted ﬁelds using a numerical model in a optimization methodology is denominated data assimilation. This strategy can be the best solution to obtain a more realistic space representation of atmospheric moisture. The observations are introduced in the cycles of numerical prediction to minimize the increase of error during the model integration in a feedback process. This generates initial conditions that are inﬂuenced by observation to better represent the physical reality, considering the deﬁciency of the used model. This cyclical procedure is one of the most important characteristics of the data assimilation process because it allows the concatenation of the contribution of the observations in different steps in the process of integration of the model [7]. This minimizes the deﬁciency in the collected data and the atmospheric modeling. The efﬁciency of the data assimilation process in the moisture ﬁelds is directly related to the choice of the atmospheric moisture control variable employed in the process of uncertainties minimization. The moisture control variable is correlated with other variables, which promote modiﬁcation by moisture observation not only in the moisture ﬁeld, but also in the ﬁelds of the correlated variable. 1. Introduction The suitable choice of this control variable permits us to minimize the contamination of the moisture observation uncertainty to other variables measured and predicted with larger precision, e.g., temperature. Furthermore, the moisture control variable has an important impact on the total precipitable water predicted by the model. This has implications for the quality of the precipitation forecast, which is the most important NWP product for human activities. Several previous studies have investigated the impact of a suitable choice of moisture control variable in the data assimilation process [8–10]. Using a Physical-Space Statistical Analysis System (PSAS), Dee et al. [9] observed the impact on the moisture initial conditions when different variables (such as mixing ratio, specific moisture, logarithm of specific moisture, relative moisture and pseudo-relative humidity) were chosen as moisture control variable of the atmospheric moisture fields. Lorenc et al. [11] showed that the preservation of the relative humidity when humidity observations are not available can be advantageous to global data assimilation system at the UK Met Ofﬁce, because the cloud parameterization in the model is directly associated with relative humidity. However, if the model has cold bias in the stratosphere, the increase in the values of the observed temperature causes spurious humidity accumulation, which is condensed in the stratosphere by the model. To contain the increase this stratospheric humidity, it is possible to introduce an artiﬁcial variable or to use pseudo-relative humidity as moisture control variable. Pseudo-relative humidity is dependent on the background temperature and is consequently related to all of the processes of correction of the model trajectory inside of the cycle of data assimilation [9]. The correct choice of the moisture moisture control variable is very important. It is necessary to consider the skill of the model, the availability of humidity observation systems and the data assimilation method. Different centers use different atmospheric moisture control variables; the Center for Weather Forecast and Climate Studies (CPTEC) from National Institute for Space Research (INPE) uses the normalized relative humidity. However, the current data assimilation system (GSI, Gridpoint Statistical Interpolation) permits us to explore the pseudo-relative humidity as moisture control variable [9,12]. The objective of this work is to evaluate the sensitivity of the initial conditions and forecasts in the basic state and atmospheric moisture ﬁelds as a function of the selected moisture control variable in the data assimilation process. 2. Materials and Methods The model used in this study was Brazilian Global Model from CPTEC/INPE, T299L64, with a horizontal resolution of approximately 44 km near the equator. The equations are written in spectral form and the equations of horizontal motion are transformed into vorticity and divergence equations. The initial condition undergoes an initialization process using the normal modes of the linearized model of a basic state at rest and considering temperature only as a vertical function [13]. The Brazilian Global Model uses the Simple Biosphere Model (SSiB) to represent the terrestrial surface [14]. Dynamic processes and physical parameterizations involve the Kuo scheme for deep convection [15], the Tiedtke for shallow convection, Mellhor and Yamada closure scheme for the vertical diffusion in the planetary boundary layer, and the biharmonic-type diffusion for the horizontal diffusion. The surface variables (soil surface temperature, soil moisture, surface albedo and snow thickness) are introduced at the beginning of integration with the climatological values and adjusted throughout the integration. More information about the conﬁguration of the Brazilian Global Model can be found in Cavalcanti et al. [16]. The Grid point Statistical Interpolation (GSI, [17]) system implemented at CPTEC/INPE uses the previous 6-h forecast from Brazilian Global Model as background. Then, a new estimate of the atmospheric state (analysis) is required every 6 h to initialize the Brazilian Global Model that covers the 6-h data window centered on the analysis time. The analyses are used as the initial conditions for subsequent forecasts and the cycle continues. The GSI system is a three-dimensional (3-D) variational data assimilation (3DVAR) method. The solution of 3D variational data assimilation is sought as the minimum of the following cost function [17,18]. J(x) = 1 2 Jb(x) + 1 2 J0(x) = 1 2(xb −x)TB−1(xb −x) + 1 2[y0 −H(x)]TR−1[xb −H(x)]. (1 (1) where x is the state vector composed of the model variables at every grid point, xb is the background state vector, y0 is the vector of observations, and H is the non-linear observation operator, which provides a map from the gridded model variables to the observation locations. The J0 term contains R, the observational error covariance matrix. The Jb term contains B, the background error covariance matrix. By deﬁnition, exact values of R and B would require the knowledge of the true state of the atmosphere at all times and everywhere on the model computational grid. 1. Introduction To do this, it is necessary to accomplish simulations in cyclical experiments of assimilation using GSI coupled in the CPTEC/INPE model. In this case, the normalized relative humidity and pseudo-relative humidity are used as moisture control variables in the data assimilation. In Section 2, the methodology is presented. An emphasis is placed on the GSI and CPTEC/INPE model, the experiment designer and the evaluation strategy employed here. We also prioritize a 3 of 12 Atmosphere 2018, 9, 123 discussion about which moisture control variables are available in the process of data assimilation implemented in GSI. In Section 3, the results are presented and described. In Section 4, the conclusions and ﬁnal comments are discussed. 2. Materials and Methods However, the matrix is too large to calculate explicitly and to store in present-day computer memories. As a result, the B matrix needs to be modeled [19]. Therefore, we need to deﬁne the analysis control variable that will be used to represent the stream function, velocity potential, temperature, surface pressure, ozone and moisture, etc. In this study, we used the same B matrix in both experiments; we only changed the analyzed moisture. To do this, there are two choices from GSI (3DVAR) normalized relative moisture (Equation (2)) and pseudo-relative humidity (Equation (3)). normalized −RH = r rs(T, P). (2) (2) where r is the mixing ratio and rs is the mixing ratio of a volume of air that is saturated with water vapor, which are affected by temperature T and pressure P. pseudo −RH = r rbs(Tb, P). (3) (3) Atmosphere 2018, 9, 123 4 of 12 where r is the mixing ratio and rb s is the mixing ratio of a volume of air that is saturated with water vapor from background, which are affected by temperature from background Tb and atmospheric pressure P. where r is the mixing ratio and rb s is the mixing ratio of a volume of air that is saturated with water vapor from background, which are affected by temperature from background Tb and atmospheric pressure P. Thus, if the atmospheric moisture control variable is the normalized RH, temperature or moisture observations can affect both the analyzed temperature and speciﬁc moisture during the cycle process. For example, in the absence of moisture observations, a single temperature observation is enough to change the temperature ﬁeld from analysis to the background. Thus, because the mixing ratio depends on temperature, the mixing ratio of the analysis will be different to the mixing ratio of the background but the normalized RH from background stays the same [9]. If the atmospheric moisture control variable is the pseudo-RH, the background pseudo-RH and relative humidity ﬁelds are identical. However, the observed pseudo-RH is not equal to the observed relative humidity. More information can be found in Dee et al. [9]. The normalized RH and pseudo-RH only differ with respect to temperature, because the normalized RH depends on temperature observations while the pseudo-RH depends on temperature forecast. Therefore, the choice between normalized RH and pseudo-RH depends on three tools: the numeric model, observations, and data assimilation. 2. Materials and Methods The normalized RH depends mainly on whether the temperature measurements are of good quality and the pseudo-RH depends mainly on whether the numerical model is skilled at forecasting temperature. To achieve the objectives of this work, two experiments were carried out in the period 1–31 August 2014, in which pseudo-RH and normalized RH were used as variables controlling atmospheric moisture in the CPTEC/INPE data assimilation system. The normalized RH experiment was considered as a control experiment because it is the variable that is used operationally by the CPTEC/INPE data assimilation system. To evaluate the sensitivity of numerical weather prediction to choice the atmospheric moisture variable control, the values of the mean ﬁeld, Mean Square Error, anomaly coefﬁcient and bias in the state variable and moisture ﬁelds were used. The analyses were carried out in the ﬁelds of initial conditions and forecasts generated by Brazilian Global Model Data Assimilation System for the domains Global, Southern Hemisphere, Northern Hemisphere, South America and Equator. The mean ﬁeld values of the initial conditions were obtained through the means of the initial conditions of 00:00, 06:00, 12:00 and 18:00 UTC from 1 to 31 August 2014. The reference values for RMS when calculating the forecast ﬁelds provided us with the initial conditions. 3. Results and Discussion The sensibility of initial conditions for selecting moisture control variables in the assimilation was veriﬁed through the initial conditions of the mean ﬁelds of zonal wind at 200 hPa (U200 hPa), 500 hPa geopotential height (Z500 hPa), mean sea level pressure (MSLP), and precipitable water (PW). The mean ﬁelds were obtained to the experiments that utilized pseudo-RH, normalized RH and the difference between pseudo-RH and normalized RH in August (Figure 1). Greater differences were noted between normalized RH (NRH) and pseudo-RH (PRH) experiments in the Antarctic and region of the polar jet stream in the Southern Hemisphere when compared with the U200 hPa, Z500 hPa and MSLP ﬁelds (Figure 1, right side). For the experiment utilizing PRH (Figure 1, left side), the U200 hPa varied from 0 to 15 m/s, the Z500 hPa was 4700 m, and MSLP was between 600 hPa and 700 hPa at the Antarctic. In the region of polar jet stream in the Southern Hemisphere, U200 hPa ranged from 15 to 35 m/s, the Z500 hPa was 5450 m, and MSLP was 950 hPa. The U200 hPa results is consistent with the ﬁndings of a previous study [20], which identiﬁed a zonal mean climatology wind of 10 m/s in 200 hPa at latitude of 80◦S (Antarctic’s latitude) and a wind between 20 and 30 m/s at the region of polar jet stream. 5 of 12 5 of 12 Atmosphere 2018, 9, 123 Figure 1. Mean ﬁelds of the initial conditions of zonal wind at 200 hPa (U200 hPa), 500 hPa geopotential height (Z500 hPa), mean sea level pressure (MSLP), and water precipitable (PW) compared with experiments that utilized pseudo-RH (PRH) and normalized RH (NRH) in August. Figure 1. Mean ﬁelds of the initial conditions of zonal wind at 200 hPa (U200 hPa), 500 hPa geopotential height (Z500 hPa), mean sea level pressure (MSLP), and water precipitable (PW) compared with experiments that utilized pseudo-RH (PRH) and normalized RH (NRH) in August. The experiment using NRH (Figure 1, center) achieved incoherent results with an average behavior of the atmosphere on the Antarctic and region of the polar jet stream in the Southern Hemisphere. This control experiment (NRH) showed similar rates to those presented in Cavalcanti et al. [16]. The performance of Brazilian Global Model from CPTEC/INPE (without the coupling between GSI) was compared with a reanalysis of National Center for Atmospheric Research (NCAR-NCEP) by Cavalcanti et al. 3. Results and Discussion [16] and revealed that the mean of U200 hPa is overestimated in 30◦S and 60◦S, achieving values between 30 and 40 m/s. These results are higher than rates obtained by reanalyzing NCEP-NCAR, i.e., between 25 and 35 m/s in austral winter (June, July and August). Cavalcanti et al. [16] observed the Southern Hemisphere at subtropical latitudes. They showed that the wavenumber 1 observed in the reanalysis of NCEP-NCAR is reproduced in the Brazilian Global Model model from CPTEC/INPE, but there are some differences in the intensity and position of zonal anomaly centers. The anomalous centers at mid- and high latitudes are weaker in the model than in the reanalysis, representing the weaker amplitude of the stationary wave in the model. Here, the PRH experiment was intenser than the NRH experiment in Z500 hPa. The MSLP was underestimated in the Antarctic in the austral winter when evaluated with the reanalysis of NCEP-NCAR [16]. Furthermore, the PRH experiment reached higher rates in comparison with the experiment using NRH (Figure 1), i.e., PRH adjusts the bias identified in the NRH experiment. The data assimilation using the results obtained in the NRH experiment could not solve all the issues pointed out by Cavalcanti et al. [16]. This is because the temperature observations on the Antarctic and region of the polar jet stream were not good enough to improve the ﬁelds. Nevertheless, the PRH experiment produced better results for these regions because they depend on the temperature forecast. Cavalcanti et al. [16] identiﬁed that the Brazilian Global Model presented a negative bias for temperatures between 1000 and 800 hPa in latitudes between 75◦S and 90◦S, as well as in levels over 700 hPa in latitudes around 60◦S. An accurate result may be obtained if an appropriate monitoring system is installed on these locations. Nevertheless, Sapucci et al. [21] demonstrated Atmosphere 2018, 9, 123 6 of 12 that AIRS-TPW was the only sensor to provide observations of air temperature on 15 June 2009. Therefore, the tools available in this study present a negative bias for air temperatures and a lack of observations. Dee et al. [9] showed that the pseudo-relative humidity predicts relative humidity fairly well, depending on the accuracy of the background temperature estimates. The precipitable water ﬁeld produced the highest difference between the experiments. 3. Results and Discussion The highest variations were detected in several regions, such as the Kalahari Desert, the Northeast, Center and Northwest of South America, the Indian Ocean, West and East Paciﬁc Oceans. The NRH experiment showed rates between 10 and 15 mm in the Kalahari Desert of Africa, while the PUR experiment showed rates between 15 and 20 mm (Figure 1). Howarth [22] found that this desert has one of the driest territories in the South Hemisphere. Therefore, the NRH experiment is consistent with Howarth [22]. This is because the experiment utilizing NRH indicated that the descending vertical movement in 500 hPA is slightly higher in the desert and more intensive over the southwest coast of Africa when contrasted with the experiment using PRH (Figure 2). Figure 2. The mean ﬁelds of the initial condition of divergence ﬁelds in 200 hPa, vertical movement in 500 hPa, and humidity ﬂow divergence in 925 hPa during the experiments that utilized PRH and NRH in August. Figure 2. The mean ﬁelds of the initial condition of divergence ﬁelds in 200 hPa, vertical movement in 500 hPa, and humidity ﬂow divergence in 925 hPa during the experiments that utilized PRH and NRH in August. In the tropics of the Indian Ocean, and the West and East Paciﬁc Oceans, a divergence of mass, an ascendant vertical movement in 500 hPa, and humidity convergence ﬂow was observed (Figure 2). Altogether, these environmental variables make a proper analysis of rainfall. The PRH experiment showed minor intense values of these variables (Figure 2). Howarth [22] identified values of 50 mm of rainfall at tropics of the Indian Ocean and East Pacific Ocean, while the West Pacific was drier than the East Pacific. Howarth [22] results were consistent with the RH experiment. South America reached higher values of precipitable water on at northwest and center (Figure 1). Rao et al. [1] measured the rainfall in South America and found a higher volume at the Brazilian Northwest in July. The bigger difference between both experiments is over the center of South America Atmosphere 2018, 9, 123 7 of 12 because the humidity convergence at lower levels is stronger in the PRH experiment than in the NRH experiment. In a statistical analysis, differences in the root mean square (RMS) among the experiments have been evaluated. 3. Results and Discussion This result indicates 8 of 12 Atmosphere 2018, 9, 123 that Brazilian Global Model present deﬁciency in the suitable characterization of the humidity ﬁelds forecast considering the analyzed ﬁelds obtained when PRH is used as moisture control variable. The sensibility of the initial conditions (Figure 1) and of the forecasts (Figure 3) in the choice of the moisture control variable is different over several parts of the globe. This difference is not constant along the evaluated period. To assess the temporal behavior of this difference, Figure 4 shows the temporal series of the anomaly correlation coefﬁcient and RMS values of the forecast for 48 h with the height geopotential at 500 hPa as a function of the integration time for each experiment in the global domains, Southern Hemisphere, Equator and Northern Hemisphere in the analyzed period. This variable was chosen because it represents the basic state and has a relevant inﬂuence on the weather forecast in the extratropical areas, indicating the approach of frontal systems and high-pressure centers. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 20 40 60 80 100 GL Anomaly correlations of the 500 hPa geopotencial height NRH PRH 00 24 48 72 96 120 0 40 80 120 160 200 240 RMS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 20 40 60 80 100 SH 00 24 48 72 96 120 0 40 80 120 160 200 240 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 20 40 60 80 100 EQ 00 24 48 72 96 120 0 40 80 120 160 200 240 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Days 20 40 60 80 100 NH 00 24 48 72 96 120 Hours 0 40 80 120 160 200 240 Figure 4. 3. Results and Discussion These experiments utilized the PRH and NRH variables in the forecasting of 24, 72, and 120 h to zonal wind ﬁelds at 250 hPa (U250 hPa), geopotential heights at 500 hPa (Z500 hPa), mean sea level pressure (MSLP), and precipitable water (PW) (Figure 3). Then, positive values mean a greater difference between the 24, 72 and 120 h of forecast and the initial conditions to the PRH experiment, and negative values show a greater difference between the 24, 72 and 120 h of forecast and the initial conditions to the NRH experiment. Figure 3. Differences in the root mean square (RMS) between PRH and NRH experiments for 24, 72, and 120 h of forecast. Fields: zonal wind of 250 hPa (U250 hPa), geopotential heights of 500 hPa (Z500 hPa), mean sea level pressure (MSLP), and precipitable water (PW). Figure 3. Differences in the root mean square (RMS) between PRH and NRH experiments for 24, 72, and 120 h of forecast. Fields: zonal wind of 250 hPa (U250 hPa), geopotential heights of 500 hPa (Z500 hPa), mean sea level pressure (MSLP), and precipitable water (PW). These results show that Brazilian Global Model tends to approximate the forecasts of 24, 72 and 120 h of the initial conditions in the ﬁelds of the basic state when PRH is used as a moisture control variable. There are subtle differences between the forecast and initial conditions in 30◦S–90◦S to MSLP and U250 hPa. These differences increase to Z500 hPa. However, in the region of the polar jet stream in the Southern Hemisphere and in the Antarctic, greater differences are found for all variables of the basic state during the NRH experiment. Figure 1 shows that the initial conditions of the ﬁelds of the basic state (U250 hPa, Z500 hPa, and MSLP) were more sensitive to experiment with PRH and Figure 3 shows smaller RMS values in the forecasts of 24, 72 and 120 h over 30◦S and 90◦S region in this experiment. On the other hand, although the water precipitable ﬁelds were more sensitive over Kalahari Desert, South America and tropics, the Indian Ocean, and the West and East Paciﬁc Oceans in the PRH experiment, Figure 3 shows larger RMS values for the forecasts of 24, 72 and 120 h in these areas. Atmosphere 2018, 9, 123 Atmosphere 2018, 9, 123 9 of 12 Atmosphere 2018, 9, 123 These results align with the analysis of Figure 1, which showed that the PRH experiment is closer of the observed values in the reanalysis of NCEP-NCAR. On the other hand, the anomaly correlation values were degraded in the PRH experiment in Equatorial region, particularly after 20 days. In physical terms, the uncertainty is expected to be large where the atmosphere has a high capacity for water vapor, i.e., at low levels and high temperatures [9]. Figure 5 presents a summary of RMS, bias and anomaly correlation coefﬁcient values among the initial conditions of 00:00, 06:00, 12:00 and 18:00 UTC and the forecasts of 24, 48, 72, 96 and 120 h of all the prognostic variables of the Brazilian Global Model. The variables are: zonal and meridional wind component (V and U); the geopotential height (Z) at 250, 500 and 850 hPa; water precipitable; speciﬁc humidity (q) at 500, 850 and 925 hPa; pressure at the mean sea level pressure (MSLP); air temperature (T); and virtual temperature (Tv) at 500, 850 and 925 hPa. The black triangles indicate that the PRH experiment was better than the NRH experiment and the inverted white triangle represents the opposite. The triangle sizes indicate the statistical signiﬁcance of the metrics, small is less signiﬁcant and the larger represents the opposite. The ash squares show that the values between the initial conditions and the forecasts are not statistically signiﬁcant. Figure 5. In this study we show the summary impact of RMS, bias and anomaly correlation coefﬁcient values between PRH and NRH experiments and the global domain in all variables. The black triangles indicate that the PRH experiment was better than the NRH experiment and a white triangle represents the opposite. The triangle sizes indicate statistical signiﬁcance of the metrics (small is less signiﬁcant, and vice versa). The ash squares show that the values between the initial conditions and the forecasts are not statistically signiﬁcant. Figure 5. In this study we show the summary impact of RMS, bias and anomaly correlation coefﬁcient values between PRH and NRH experiments and the global domain in all variables. The black triangles indicate that the PRH experiment was better than the NRH experiment and a white triangle represents the opposite. The triangle sizes indicate statistical signiﬁcance of the metrics (small is less signiﬁcant, and vice versa). 3. Results and Discussion Temporal series of the anomaly correlation coefﬁcient and RMS values of the 48-h forecast of the height geopotential at 500 hPa as a function of the integration time for PRH and NRH experiments in the global domains, Southern Hemisphere, Equator and Northern Hemisphere. The correlations present a statistical signiﬁcance of 99.95% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 20 40 60 80 100 GL Anomaly correlations of the 500 hPa geopotencial height NRH PRH 00 24 48 72 96 120 0 40 80 120 160 200 240 RMS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 20 40 60 80 100 SH 00 24 48 72 96 120 0 40 80 120 160 200 240 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 20 40 60 80 100 EQ 00 24 48 72 96 120 0 40 80 120 160 200 240 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Days 20 40 60 80 100 NH 00 24 48 72 96 120 Hours 0 40 80 120 160 200 240 Figure 4 Temporal series of the anomaly correlation coefﬁcient and RMS values of the 48 h for Anomaly correlations of the 500 hPa geopotencial heigh 2 Figure 4. Temporal series of the anomaly correlation coefﬁcient and RMS values of the 48-h forecast of the height geopotential at 500 hPa as a function of the integration time for PRH and NRH experiments in the global domains, Southern Hemisphere, Equator and Northern Hemisphere. The correlations present a statistical signiﬁcance of 99.95% Figure 3 shows that there was little difference between the forecast and initial condition in the Southern Hemisphere during the PRH experiment and in the Equator during the NRH experiment. In Figure 4, we can see the same behavior. The RMS value is lower for the PRH experiment than the NRH experiment associated with the Southern Hemisphere. The RMS value is equal between the PRH and NRH experiments over the Equator. Atmosphere 2018, 9, 123 The ash squares show that the values between the initial conditions and the forecasts are not statistically signiﬁcant. Figure 5 indicates the gain in the variable temperature dependence for the PRH experiment. This is represented as zonal and meridional wind components, geopotential height, the mean sea level pressure, the air temperature and virtual temperature. The basic state ﬁelds presented an anomaly correlation with statistical signiﬁcance from 48 h of the forecast. The NRH experiment improved 10 of 12 Atmosphere 2018, 9, 123 the humidity fields compared with PRH experiment, which presented smaller RMS values for water precipitation and specific humidity in all evaluated levels. The values of the anomaly correlation showed statistical significance from 48 h just in the field of the water precipitable. The bias values indicate that the NRH experiment generated a better forecast for the humidity fields. In this case, the forecast was systematically closer to the initial condition, and the results are better for larger model integration times. y y g g In a synthesis of those results, the PRH should be a choice for moisture control variable when the model state needs to be maintained at a stable state during the assimilation cycle. However, this option punishes the forecast quality of the atmospheric humidity ﬁelds. On the other hand, when the data assimilation system is used to generate more appropriate humidity ﬁelds, the best choice is NRH. However, in that case, the basic state in the model is punished. In non-cyclical applications of the data assimilation and those that require high-quality precipitation forecasts, the NRH variable is the best option. The previous results showed that the forecast ﬁelds generated by the model are distinct among different areas of the globe. This can be expressed as a function of the choice of the humidity variable control in the data assimilation system at CPTEC/INPE. To assess these results, a sensibility analysis of the forecast ﬁelds in different domains was carried out on differences of anomaly correlation coefﬁcients obtained for PRH and NRH experiments. Figure 6 shows the results as a function of the model integration time for the zonal wind component at 250 hPa, 500 hPa geopotential height, mean sea level pressure and water precipitable for the domains global, Southern Hemisphere, Northern Hemisphere, Equator region and South America. Atmosphere 2018, 9, 123 Note that positive difference indicates that the PRH experiment generated larger values of the anomaly correlation than the NRH experiment, and vice versa. 0 24 48 72 96 120 Forecast (h) -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 Anomaly Correlation U 250 hPa (PRH-NRH) 0 24 48 72 96 120 Forecast (h) -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 Anomaly Correlation Z 500 hPa (PRH-NRH) 0 24 48 72 96 120 Forecast (h) -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 Anomaly Correlation U 250 hPa (PRH-NRH) 0 24 48 72 96 120 Forecast (h) -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 Anomaly Correlation Z 500 hPa (PRH-NRH) 0 24 48 72 96 120 Forecast (h) -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 Anomaly Correlation MSLP (PRH-NRH) 0 24 48 72 96 120 Forecast (h) -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 Anomaly Correlation PW (PRH-NRH) GL SA SH NH EQ Figure 6. Differences in the anomaly correlation as a function of the model integration time for the zonal wind component at 250 hPa, 500 hPa geopotential height, mean sea level pressure and water precipitable for global domains, Southern Hemisphere, Northern Hemisphere, Equator region and South America. 0 24 48 72 96 120 Forecast (h) -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 Anomaly Correlation MSLP (PRH-NRH) 0 24 48 72 96 120 Forecast (h) -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 Anomaly Correlation PW (PRH-NRH) GL SA SH NH EQ Figure 6. Differences in the anomaly correlation as a function of the model integration time for the zonal wind component at 250 hPa, 500 hPa geopotential height, mean sea level pressure and water precipitable for global domains, Southern Hemisphere, Northern Hemisphere, Equator region and South America. In Figure 5, the PRH experiment presented better results than NRH experiment for the temperature-dependent variable of the global domain. Figure 6 shows that domains such as the Southern Hemisphere and South America also presented superior values of anomaly correlation in 11 of 12 Atmosphere 2018, 9, 123 the PRH experiment compared with the NRH experiment with the temperature-dependent variable (U250 hPa, Z500 hPa and MSLP). The NRH experiment shows superior values of anomaly correlation over the Equator than the PRH experiment in PW, U250 hPa, Z500 hPa and MSLP. 4. Summary and Conclusions The performance of the initial conditions and forecasts for the moisture and basic state variables was evaluated by sensitivity tests with the normalized-RH and the pseudo-RH as control moisture variables in the Brazilian Global Model Data Assimilation System. The sensitivity of numerical weather prediction when choosing the atmospheric moisture control variable should consider three aspects: numeric model, observations, and data assimilation. For the Brazilian Global Model Data Assimilation System, these three aspects show that the fields of forecasts and initial condition of the basic state improve in the pseudo-RH experiment, mainly between 30◦S and 90◦S, but damage the atmospheric moisture field. The normalized-RH experiment improves the fields of forecasts and initial condition of the moisture, mainly in the regions across the Equator, but damages the field of the basic state in the initial condition and forecasts. These patterns can be explained because the fields of forecasts and initial condition of the basic state are benefited by balancing the mixing ratio and temperature from the background to pseudo-RH experiment. However, the pseudo-RH harmed the atmospheric moisture field because it is too different to the observed relative humidity. In the Equator, the fields of forecasts and initial condition of the moisture are improved because of the temperature observations. However, the basic state is harmed because of the direct relation between mixing ratio and temperature observations. Therefore, we suggest that the pseudo-RH variable is used in a cyclic data assimilation system, as it substantially improves the physics of the Brazilian Global Model. Moreover, the normalized-RH variable should be assimilated in the non-cyclic system because it will not propagate the errors generated in the basic state and it will help to improve the predicted precipitation. This study is the first performed for the Brazilian Global Model Data Assimilation System and the reported results call attention to choice of moisture control variable in data assimilation process. Acknowledgments: The authors thank the CAPES (Coordination of Improvement of Higher Level Personnel) and CNPQ (National Council for Scientiﬁc and Technological Development) for the scholarship to carry out this research. Additionally, we wish to thank the SCAMTEC “Community System for Evaluation of Weather and Climate Model” team for allowing us to use the statistical tool, and the National Institute For Space Research and Center for Weather Forecasting and Climate Research for the computational infrastructure used in this study. Author Contributions: Thamiris B. Campos and Luiz F. Atmosphere 2018, 9, 123 The biggest anomaly correlation for the NRH experiment was the Z500 hPa ﬁeld at the Equator. In Figure 4, the NRH experiment showed larger anomaly correlation after 20 August. The precipitable water was also a ﬁeld with high anomaly correlation for the NRH experiment at the Equator. These results show that the choice of the NRH is the best option to use as a moisture control variable in the assimilation of data over the Tropical region. 4. Summary and Conclusions Sapucci conceived of and designed the experiments. Thamiris B. Campos performed the experiments. Thamiris B. Campos and Luiz F. Sapucci analyzed the data. Wagner Lima and Douglas Silva Ferreira provided valuable comments and suggestions. Conﬂicts of Interest: The authors declare no conﬂict of interest. s of Interest: The authors declare no conﬂict of interes 1. Rao, V.B.; Cavalcanti, I.F.A.; Hada, K. Annual variation of rainfall over Brazil and water vapor characteristics over South America. J. Geophys. Res. Atmos. 1996, 101, 2156–2202. p y 2. Labraga, J.C.; Frumento, O.; López, M. The atmospheric water vapor cycle in South America and the tropospheric circulation. J. Clim. 2000, 13, 1899–1915. 4. Nóbrega, R.S.; Cavalcanti, E.P.; Souza, E.P. Reciclagem de vapor d’água sobre a América do Sul utilizando reanálises do NCEP-NCAR. Rev. Bras. Meteorol. 2005, 20, 253–262. p p J 3. Sato, T.; Kimura, F. A two-dimensional numerical study on diurnal cycle of mountain lee precipitation. J. Atmos. Sci. 2003, 16, 1992–2003. References 1. Rao, V.B.; Cavalcanti, I.F.A.; Hada, K. 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Sapucci, L.F.; Bastarz, C.F.; Cerqueira, F.; Avanço, L.A.; Herdies, D.L. Impacto de perﬁs de rádio ocultação GNSS na qualidade das previsões de tempode do CPTEC/INPE. Rev. Bras. Meteorol. 2014, 29, 551–567. 22. Howarth, D. A. Seasonal Variations in the Vertically Integrated Water Vapor Transport Fields over the Southern Hemisphere. Mon. Weather Rev. 1983, 111, 1259–1272. c⃝2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4230115797	https://www.researchsquare.com/article/rs-10538/latest.pdf	English	null	Distinct Actions of the Fermented Beverage Kefir on Host Behaviour, Immunity and Microbiome Gut-Brain Modules in the Mouse	Research Square (Research Square)	2,020	cc-by	17,082	nct Actions of the Fermented Beverage Kefir on Host viour, Immunity and Microbiome Gut-Brain Modules in the se Marcel van de Wouw University College Cork National University of Ireland Aaron M. Walsh Teagasc Food Research Centre Moorepark Fiona Crispie Teagasc Food Research Centre Moorepark Lucas van Leuven University College Cork National University of Ireland Joshua M. Lyte University College Cork National University of Ireland Marcus Boehme University College Cork National University of Ireland Gerard Clarke University College Cork National University of Ireland Timothy G. Dinan University College Cork National University of Ireland Paul D. Cotter Teagasc Food Research Centre Moorepark John Cryan (  j.cryan@ucc.ie ) University College Cork National University of Ireland h Joshua M. Lyte University College Cork National University of Ireland Marcus Boehme University College Cork National University of Ireland Marcus Boehme University College Cork National University of Ireland Gerard Clarke University College Cork National University of Ireland Timothy G. Dinan University College Cork National University of Ireland Research DOI: https://doi.org/10.21203/rs.2.19926/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Microbiome on May 18th, 2020. See the published version at https://doi.org/10.1186/s40168-020-00846-5. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 1/28 Page 1/28 Abstract Background: Mounting evidence suggests a role for the gut microbiota in modulating brain physiology and behaviour through bi- directional communication along the gut-brain axis. As such, the gut microbiota represents a potential therapeutic target for influencing centrally-mediated events and host behaviour. It is thus notable that the fermented milk beverage kefir has recently been shown to modulate the composition of the gut microbiota in mice. It is unclear whether kefirs have differential effects on microbiota-gut-brain axis and whether they can modulate host behaviour per se. Methods: To address this, two distinct kefirs (Fr1 and UK4) or unfermented milk control were administered to mice that underwent a battery of tests to characterise their behavioural phenotype. In addition, shotgun metagenomic sequencing of ileal, cecal and faecal matter was performed, as was faecal metabolome analysis. Finally, systemic immunity measures and gut serotonin levels were assessed. Statistical analysis was performed by ANOVA followed by Dunnett's post hoc test or Kruskal-Wallis test followed by Mann-Whitney U test. Results: Fr1 ameliorated the stress-induced decrease in serotonergic signalling in the colon and reward-seeking behaviour in the saccharin preference test. On the other hand, UK4 decreased repetitive behaviour and ameliorated stress-induced deficits in reward-seeking behaviour. Furthermore, UK4 increased fear-dependent contextual memory, yet decreased milk gavage-induced improvements long-term spatial learning. In the peripheral immune system, UK4 increased the prevalence of Treg cells and interleukin 10 levels, whereas Fr1 ameliorated the milk gavage stress-induced elevation in neutrophil levels and CXCL1 levels. Analysis of the gut microbiota revealed that both kefirs significantly changed the composition and functional capacity of the host microbiota, where specific bacterial species were changed in a kefir-dependent manner. Furthermore, both kefirs increased the capacity of the gut microbiota to produce GABA, which was linked to an increased prevalence in Lactobacillus reuteri. Conclusions: Altogether, these data show that kefir can signal through the microbiota-gut-immune-brain axis and modulate host behaviour. In addition, different kefirs may direct the microbiota toward distinct immunological and behavioural modulatory effects. These results indicate that kefir can positively modulate specific aspects of the microbiota-gut-brain axis and support the broadening of the definition of psychobiotic to include kefir fermented foods. Introduction Mounting evidence suggests that the gastrointestinal microbiota influences host behaviour via bi-directional communication through what has been coined the microbiota-gut-brain axis (Rhee et al. 2009, Collins et al. 2012, Cryan and Dinan 2012, Mayer et al. 2014, Foster et al. 2017, Cryan et al. 2019, Sherwin et al. 2019). Various nutritional interventions have already been demonstrated to influence this axis, with host-indigestible dietary fibres (prebiotics) and live bacterial strains that confer health benefits (probiotics) receiving particular attention (Kao et al. 2016, Cryan et al. 2019, long-Smith et al. 2019). Such interventions that modulate mood through manipulation of the microbiota have been coined psychobiotics (Dinan et al. 2013). It is becoming apparent that fermented foods may also confer beneficial effects on aspects of mood, as fermented food intake is associated with decreased social anxiety (Hilimire et al. 2015) and gestational depression in humans (Miyake et al. 2014). In addition, a fermented milk product, which was produced using known probiotics, has been demonstrated to modulate brain activity in healthy women (Tillisch et al. 2013). Such findings merit an investigation into the mechanisms by which different fermented foods might affect the microbiota-gut-brain axis. One such fermented foods are kefir, a traditional fermented milk beverage originating from the Caucasus mountains that is produced by adding a kefir grain to milk. These grains consist of exopolysaccharide matrices harbouring symbiotic microbial communities, including bacteria and yeasts, which together are responsible for fermentation (Bourrie et al. 2016). Notably, the word kefir is derived from the Turkish keyif, which translates as “good feeling”. Indeed, numerous health benefits have been ascribed to kefir (Rosa et al. 2017, Slattery et al. 2019), such as anti-inflammatory effects in animal models (Rodrigues et al. 2005, Liu et al. 2006, Lee et al. 2007), reduced obesity symptomatology in high fat diet-induced obese mice (Kim et al. 2017, Bourrie et al. 2018, Gao et al. 2019), and reduced hypertension in spontaneously hypertensive rats (Silva-Cutini et al. 2019). Furthermore, kefir administration has been shown to reduce physical fatigue and improve exercise performance in mice (Hsu et al. 2018). A recent randomized, controlled trial has even shown that kefir can reduce bloating and improve mood in patients with inflammatory Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 2/28 Page 2/28 bowel disease (Yilmaz et al. 2019). Finally, kefir has been shown to modulate the composition of the gastrointestinal microbiota in rodents (Kim et al. 2017, Bourrie et al. Kefir microbiota was relatively stable over time The milk kefir used in this study was generated in a manner to represent traditional kefir production (i.e., repeated fermentation of milk by a kefir grain). Considering that kefir contains a complex microbiota community composed of a variety of strains (Bourrie et al. 2016), we aimed to determine if this community remained stable over time. Shotgun metagenomics was used to determine the species-level composition of the two kefirs, Fr1 and UK4, at 6 time-points at intervals of 2 weeks throughout the in vivo study. Overall, the populations were generally temporally stable, with both kefirs being dominated by Lactococcus lactis, while also consistently containing L. kefiranofaciens (Figure S1). Several other species were identified at specific time-points at >1% relative abundance in both kefirs, such as Bifidobacterium breve and Pseudomonas species. Notably, L. kefiranofaciens was more abundant in kefir Fr1 at some time points. Kefir did not affect measures of gastrointestinal physiology and motility Assessment of gastrointestinal motility by carmine red administration showed that kefir did not induce any changes in gastrointestinal propulsion (Figure S3). In line with these findings was the absence of differences in faecal pellet weight and faecal water content (Figure S3). Finally, no differences in cecum weight and colon length were detected at the end of the study (Figure S3). Overall, these data indicate that changes in the gut microbiota are likely independent of host gastrointestinal physiology and motility. Introduction 2018, Hsu et al. 2018, Gao et al. 2019). Overall, current evidence indicates that the traditionally fermented milk drink kefir merits investigation to determine its ability to modulate the microbiota-gut-brain axis and affect the mood of the host. It is not clear if kefir’s exert differential influence across the microbiota-gut-brain axis due to their compositional differences (Bourrie et al. 2016). As such, we aimed to investigate if two different kefirs could affect the microbiota of ileal, cecal and faecal contents, the faecal metabolome, gastrointestinal function, host adaptive and innate immunity, and behaviour in mice. The fermented milk drink kefir is well-tolerated Kefir administration did not affect body weight, body composition, food intake and drinking water intake (Figure S2). In addition, no differences were found in basal body temperature, as detected in the stress-induced hyperthermia test, as well as the locomotor activity assessed in the open field test (Figure S2). Overall, this indicates that the fermented milk drink kefir was well-tolerated by mice. Kefir does not affect sociability All groups exhibited normal social preference and recognition in the 3-chamber social interaction test, indicating that kefir did not affect sociability (Figure S5). All groups exhibited normal social preference and recognition in the 3-chamber social interaction test, indicating that kefir did not affect sociability (Figure S5). Kefirs differentially impact the peripheral immune system There was an increase in circulating LY6Chigh monocytes in mice receiving milk, compared to undisturbed mice, indicating an activation of the innate immune system (Gururajan et al. 2019, van de Wouw et al. 2019). Furthermore, there were increased levels of various inflammatory cytokines in the peripheral circulation (Figure S6). In line with this finding, was an increase in neutrophil levels induced by milk administration (t(22) = −3.583, p = 0.002) (Figure 3A), which was ameliorate by Fr1 administration (F(2,34) = 5.412, p = 0.009) (Figure 3A). Similarly, Fr1 ameliorated the increased CXCL1 levels observed in mice chronically stressed by milk gavage (t(21) = −2.589, p = 0.017; F(2,32) = 7.006, p = 0.003) (Figure 3B), which is one of the major chemoattractants for neutrophils (Silva et al. 2017). UK4 increased the prevalence of T regulatory cells (Treg) cells in mesenteric lymph nodes (MLNs) (F(2,34) = 8.709, p < 0.001) (Figure 3C), an anti-inflammatory T helper cell subset known to be induced by gut microbial metabolites (Tanoue et al. 2016). These cells did not express the Helios transcription factor (F(2,34) = 7.548, p = 0.002) (Figure 3D). This indicated that they were induced in the periphery (pTreg) rather than in the thymus (Shevach and Thornton 2014), suggesting that gut microbial-derived metabolites could have driven this increase in Treg cells. We subsequently investigated the prevalence of MLN CD103+ dendritic cells, which are known to induce Treg cell differentiation (Tan et al. 2016). No corresponding differences were found, as an increased prevalence of MLN CD103+ dendritic cells was observed in the milk and kefir treatment groups, which is in line with the increased levels of inflammatory cytokines in the peripheral circulation (Figure S7). The effects of UK4 also reached the peripheral circulation, where there was an increased prevalence of Treg cells (F(2,31) = 3.420, p = 0.046) (Figure 3E). Similarly, we observed increased plasma IL-10 levels (F(2,32) = 6.205, p = 0.006) (Figure 3F), one of the primary cytokines secreted by Treg cells (Sabat et al. 2010). Kefir modulates repetitive behaviour and reward-seeking behaviour In the marble burying test, we found that administration of UK4 decreased the number of marbles buried indicative of reduced repetitive behaviour (F(2,35) = 5.464, p = 0.009) (Figure 1A). No changes were observed in tests assessing anxiety-like behaviours such as the elevated plus maze, open field test and stress-induced hyperthermia test (Figure S4), as well as depressive-like behaviour in the forced swim test and tail-suspension test (Figure S4). Notably, repeated stress of milk gavage increased the corticosterone response to an acute stressor, which remained unaffected by kefir (Figure S4) Page 3/28 In the female urine sniffing test of reward-seeking, mice receiving milk spent less time interacting with the cotton bulb containing water compared to undisturbed mice ( 2(1) = 6.367, p = 0.012), which was ameliorated by both Fr1 and UK4 ( 2(2) = 13.238, p < 0.001) (Figure 1B). In addition, mice receiving UK4 spent more time interacting with the cotton bulb containing the urine from a female mouse in esterus ( 2(2) = 6.280, p = 0.043) (Figure 1B), even though no differences were observed in the preference index (Figure 1C). Finally, Fr1 administration increased saccharin preference in the saccharin preference test ( 2(2) = 12.826, p = 0.002), which is often used as a measure of reward-seeking behaviour (Figure 1D, E).fi Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 3/28 Page 3/28 Kefir – UK4 modulates contextual learning and memory No differences were observed in the fear conditioning test in phase 1 – acquisition, as determined by the time mice spent frozen during the presentation of the cue, as well as in-between the cues (Figure 2A, B). In addition, no differences were seen during phase 2, when cued-dependent fear memory was assessed (Figure 2C). However, mice receiving UK4 showed a trend towards increased freezing behaviour in phase 3 – contextual memory (F(2,34) = 3.181, p = 0.055) (Figure 2D). Conversely, mice receiving UK4 made more errors in the reverse learning phase of the appetitive Y-maze as seen by the percentage correct choices (treatment effect: F(2,33) = 3.870, p = 0.031) (Figure 2E), and the amount of entries mice needed to reach the food reward compared to milk control (treatment effect: F(2,33) = 3.387, p = 0.046) (Figure 2F). Notably, a similar difference was found on day 10 in the percentage correct choices made between the undisturbed control and Milk control group (t(22) = -2.303, p = 0.031), where the mice receiving milk control performed superior to the undisturbed control (Figure 2E). Both kefirs affect gut microbiota composition, at both the species- and strain-levels We subsequently investigated if kefir administration could affect the composition of the ileal, cecal and faecal microbiota. Alpha diversity (Shannon) was not significantly altered by kefir Fr1 administration (Fr1; Ileum: p = 0.11; Cecum: p = 0.19; Faeces: p = 0.16), whereas kefir UK4 increased alpha diversity in the cecum (p = 0.017), but not ileum and faeces (p = 0.44; p = 0.24) (Figure 5A). Analysis of beta diversity revealed a trend towards significant separation induced by the administration of Fr1 (Ileum: p = 0.088, R2 = 0.111; Cecum: p = 0.087, R2 = 0.087; Faeces: p = 0.077, R2 = 0.114) and UK4 (Ileum: p = 0.058, R2 = 0.092; Cecum: p = 0.1, R2 = 0.092; Faeces: p = 0.073, R2 = 0.09) (Figure 5B). It is notable that, the kefir treatment overall did influence beta diversity (p-value ranged from 0.05 < p ≤ 0.10 for all regions). Notably, no significant differences were found between the administration of Fr1 and UK4 in alpha diversity (Ileum: p = 0.37; Cecum: p = 0.34; Faeces: p = 0.8) and beta diversity (Ileum: p = 0.316, R2 = 0.05; Cecum: p = 0.14, R2 = 0.07; Faeces: p = 0.2, R2 = 0.062). A total of 15 bacterial species were identified as being differentially abundant between at least one pair of groups in at least one region of the gut (Figure 5C). Both kefirs increase the abundance in or more region in the gut of Lactobacillus reuteri (Fr1, cecum: LDA=4.36, UK4, cecum: LDA=4.02, UK4, faeces: LDA=4.07), Eubacterium plexicaudatum (Fr1, faeces: LDA=3.77, UK4, cecum: LDA=4.22, UK4, faeces: LDA=3.67), Bifidobacterium pseudolongum (Fr1, ileum: LDA=4.93, UK4, cecum: LDA=4.7). Both kefirs induced a decrease in the prevalence of Lachnospiraceae bacterium 3_1_46FAA (Fr1, cecum: LDA=4.25, UK4: cecum: LDA=4.28), Propionibacterium acnes (Fr1, faeces: LDA=3.25, UK4, faeces, LDA=4.04), and Bacillus amyloliquefaciens (Fr1, faeces: LDA=3.04, UK4, faeces: LDA=3.58). Only Fr1 increased the prevalence of Parabacteroides goldsteinii (cecum: LDA=3.99), Bacteroides intestinalis (faeces: LDA=3.49), Anaerotruncus unclassified (faeces: LDA=3.75), and Parabacteroides goldsteinii (faeces: LDA=4.02). Conversely, only UK4 increased the prevalence of Alistipes unclassified (cecum: LDA=4.45) and decreased Candidatus Arthromitus unclassified (ileum: LDA=4.45). We subsequently correlated significantly altered behavioural and immunological parameters with bacterial species present throughout the gastrointestinal microbiota (Figure 6). Most notable was the correlation between ileal C. Arthromitus unclassified abundances and circulating Treg cell levels (p = 0.004, R = -0.49), and ileal B. Both kefirs affect gut microbiota composition, at both the species- and strain-levels pseudolongum abundances and circulating neutrophil levels (p = 0.001, R = -0.52). PanPhlAn was used alongside StrainPhlAn to characterise differentially abundant species to the strain-level. Both tools indicated that the detected B. pseudolongum strain was closely related to B. pseudolongum UMB-MBP-01 (Figure S8). Similarly, PanPhlAn indicated that the detected L. reuteri strain was closely related to L. reuteri TD1. No other differentially abundant species could be characterised to the strain-level. Finally, neither PanPhlAn nor StrainPhlAn identified any kefir-derived strains in the gut microbiota of mice receiving kefir, establishing that the microbiota of the administered kefir did not colonise to high levels. Kefir – Fr1 selectively increases colonic serotonergic activity Serotonergic signaling is well-known to play a key-role in microbiota-host cross-talk (Sudo et al. 2004, O'Mahony et al. 2015), which is why we quantified gut serotonin (5-HT) levels. We found that mice receiving milk showed decreased ileal 5-HT levels compared to undisturbed mice (t(21) = 2.650, p = 0.015) (Figure 4B). This resulted in an increased 5HIAA/5-HT ratio (t(22) = 2.650, p < 0.001) (Figure 4C), indicating an increased serotonin turnover and serotonergic activity. The opposite was observed in the colon, where the milk induced a trend towards increased 5-HT levels (t(22) = -1.937, p = 0.066) (Figure 4E), whilst decreasing the 5HIAA/5-HT ratio (t(22) = 2.907, p = 0.008) (Figure 4F). This phenotype in the colon, but not ileum, was ameliorated by Fr1 (for 5- HT; F(2,35) = 6.387, p = 0.005, for 5HIAA/5-HT ratio; F(2,35) = 9.026, p < 0.001) (Figure 4E, F).fi Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 4/28 Page 4/28 Kefir increases the prevalence of a Lactobacillus reuteri strain with the potential to produce GABA reuteri was potentially converting 2-oxoglutarate to glutamate, which was subsequently converted into GABA, resulting in the production of succinate as a by-product. Subsequently, we employed metabolic modelling using FVA of L. reuteri to explore this hypothesis in depth. The results indicated that L. reuteri had the ability to consume 2- oxoglutarate and glutamate, while it also could secrete GABA and succinate (Figure 8B). In line with the predicted capacity of L. reuteri to produce GABA, we observed a negative correlation between L. reuteri and faecal levels of 2-oxoglutarate (R=-0.474, p=0.047) and glutamate (R=-0.420, p=0.083), as detected by GC-MS (Figure 8A). Furthermore, there was a trend towards a positive correlation between L. reuteri and succinate levels (R=0.60, p=0.100) (Figure 8A). Furthermore, faecal succinate levels were increased in mice receiving kefir (Fr1: unadjusted p = 0.037; UK4: unadjusted p = 0.030). The pattern of correlation suggests that L reuteri was potentially converting 2-oxoglutarate to glutamate which was subsequently In line with the predicted capacity of L. reuteri to produce GABA, we observed a negative correlation between L. reuteri and faecal levels of 2-oxoglutarate (R=-0.474, p=0.047) and glutamate (R=-0.420, p=0.083), as detected by GC-MS (Figure 8A). Furthermore, there was a trend towards a positive correlation between L. reuteri and succinate levels (R=0.60, p=0.100) (Figure 8A). Furthermore, faecal succinate levels were increased in mice receiving kefir (Fr1: unadjusted p = 0.037; UK4: unadjusted p = 0.030). The pattern of correlation suggests that L. reuteri was potentially converting 2-oxoglutarate to glutamate, which was subsequently converted into GABA, resulting in the production of succinate as a by-product. Subsequently, we employed metabolic modelling using FVA of L. reuteri to explore this hypothesis in depth. The results indicated that L. reuteri had the ability to consume 2- oxoglutarate and glutamate, while it also could secrete GABA and succinate (Figure 8B). Kefir increases the prevalence of a Lactobacillus reuteri strain with the potential to produce GABA Subsequently, changes in the microbiome were explored in the context of the gut-brain axis by examining the abundances of gut- brain modules (GBMs) (Figure 7C), which are groups of KEGG Orthogroups (KOs) that are associated with the production of neuroactive compounds (Valles-Colomer et al. 2019). In mice receiving Fr1, cecal “p-Cresol biosynthesis” was decreased, while “Quinolinic acid synthesis” was decreased in the faeces (LDA=4.43) compared to milk control. In mice receiving UK4, there was an increase in ileal "Inositol synthesis" (LDA=4.77) and "GABA degradation" (LDA=4.70). Finally, the GBM "GABA synthesis III" was significantly higher in mice receiving either kefir (Fr1: (LDA=4.39; UK4: LDA=4.21) in the faeces. These increases in "GABA synthesis III" were attributed to L. reuteri, which showed a significantly higher prevalence of this GBM compared to milk controls (Fr1: LDA=4.20, UK4: LDA=4.11). Similarly, the GBM "S-Adenosylmethionine synthesis" from P. goldsteinii was significantly higher in the faeces of mice receiving Fr1 (LDA=4.21) and UK4 (LDA=4.08). Furthermore, the GBM "GABA degradation" from L. reuteri was significantly higher in the ileum of mice receiving UK4 (LDA=4.16). While "Glutamate synthesis I" from B. pseudolongum was higher in the faeces of Fr1 mice (LDA=4.64). To corroborate the results from HUMAnN2, PanPhlAn gene-family matrices were examined to identify genes associated with the production of neurotransmitters in the detected strains. The detected B. pseudolongum strain encoded two enzymes involved in the production of glutamate: glutamate synthase and glutamine--fructose-6-phosphate transaminase (isomerising). Furthermore, this strain also encoded a glutamate/GABA antiporter, which may be involved in exporting glutamate from the cell. Similarly, the detected L. reuteri strain was also found to encode two enzymes associated with the production of glutamate from glutamine: glutaminase and glutamine--fructose-6-phosphate transaminase (isomerising). Importantly, this strain encoded glutamate decarboxylase, which produces GABA by the decarboxylation of glutamate, along with a glutamate/GABA antiporter. In line with the predicted capacity of L. reuteri to produce GABA, we observed a negative correlation between L. reuteri and faecal levels of 2-oxoglutarate (R=-0.474, p=0.047) and glutamate (R=-0.420, p=0.083), as detected by GC-MS (Figure 8A). Furthermore, there was a trend towards a positive correlation between L. reuteri and succinate levels (R=0.60, p=0.100) (Figure 8A). Furthermore, faecal succinate levels were increased in mice receiving kefir (Fr1: unadjusted p = 0.037; UK4: unadjusted p = 0.030). The pattern of correlation suggests that L. Kefir induces shifts in the functional potential of the gut microbiome We subsequently investigated if kefir administration could affect the functional potential of the microbiome in mice. Fr1 induced a significant functional separation in the microbiome in the ileum (p = 0.052, R2 = 0.099) and the cecum (p = 0.019, R2 = 0.079), but not in the faeces (p = 0.108, R2 = 0.068) (Figure 7A). Similarly, UK4 induced a significant functional separation in the cecum (p = 0.018, R2 = 0.092) and the faeces (p = 0.010, R2 = 0.09), but not in the ileum (p = 0.212, R2 = 0.092) (Figure 7A). No significant functional separations were identified between any other pair of groups in any other regions. The faecal metabolome was assessed using GC-MS from a subset of animals from each group (n = 6) to validate any changes observed in the predicted functional potential of the gut microbiome (sTable 3). Analysis of the beta diversity of the faecal metabolome revealed a predominant effect of kefir Fr1 on the measured metabolites (p = 0.045, R2 = 0.196). Notably, no significant differences in the concentrations of any compound between any pair of groups were identified following p-value adjustment, which included short- chain fatty acid (SCFA) levels (Figure S9). A total of 59 level-4 enzyme commission (EC) categories were differentially abundant between at least one pair of groups in at least one region of the gut. Notably, there were significant differences in several EC categories involved in the production of neuroactives (Figure 7B). Specifically, ileal glutamine--fructose-6-phosphate transaminase (isomerising) (EC 2.6.1.16) levels, which Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 5/28 produces glutamate, was elevated by the administration of both Fr1 (p = 0.002) and UK4 (p = 0.021). In addition, glutamate-- ammonia ligase (EC 6.3.1.2), which produces glutamine, was higher in the ileum of mice receiving Fr1 (p = 0.024). UK4 increased the prevalence of predicted cecal and faecal glutamate--cysteine ligase (EC 6.3.2.2) levels (p = 0.038; p = 0.011 respectively), while cecal tryptophan synthase (EC 4.2.1.20) was decreased (p = 0.028). Discussion In the present study, we demonstrate that two different traditionally fermented kefirs differentially affect host behaviour and immunity. For instance, Kefir Fr1 increased reward-seeking behaviour and ameliorated stress-induced increases in circulating neutrophil and CXCL1 levels. Furthermore, UK4 decreased repetitive behaviour, increased circulating Treg cells and IL-10 levels, and ameliorated deficits in reward-seeking behaviour induced by chronic oral gavage stress. In addition, UK4 increased fear- dependent contextual memory, yet decreased milk gavage-induced improvements long-term spatial learning. Both kefirs modulated the composition and functional capacity of the microbiota, which was associated with an increased capacity to produce GABA. This function was linked to an increased prevalence of L. reuteri. We observed some changes in the gut microbiota that were specific to kefir Fr1, such as an increase in P. goldsteinii, B. intestinalis, Anaerotruncus unclassified and P. goldsteinii. Analysis of GBMs revealed that Fr1 decreased the GBM “p-Cresol biosynthesis” and “Inositol synthesis”, while UK4 increased “Inositol synthesis”. The increase in P. goldsteinii abundances were linked to an Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 6/28 Page 6/28 increased in the GBM "S-Adenosylmethionine synthesis". S-Adenosylmethionine supplementation has been studied in numerous randomized, controlled trials involving depressed adults (Joffe et al. 2010, Sharma et al. 2017). Notably, one of the key features of depression is decreased reward-seeking behaviour (i.e. anhedonia) and that Fr1 increased reward-seeking behaviour in the saccharin preference test. As such, the increase in predicted S-Adenosylmethionine synthesis could indicate increased levels of S- Adenosylmethionine, which in turn might have contributed to the Fr1-induced increase in reward-seeking behaviour. It is important to note, however, that no changes were observed in other measures of depressive-like behaviour. Our results also reveal that Fr1 ameliorated stress-induced deficits in colonic serotonergic signalling, even though it is important to note that no corresponding serotonin-related changes were observed in the gut microbiome and metabolome and that the determined effects were weak. Furthermore, Fr1 increase circulating neutrophil and CXCL1 levels. Notably, p-Cresol, of which the predicted biosynthesis was decreased by Fr1, alters neutrophil function in dogs (Bosco et al. 2016). Furthermore, Fr1 increased B. pseudolongum abundances in the ileum, whereas UK4 increased B. pseudolongum abundances in the cecum. In tandem, ileal B. pseudolongum abundances correlated with circulating neutrophil levels, indicating that B. pseudolongum abundances in the ileum specifically, might contribute to the decrease in neutrophil levels observed in mice receiving Fr1. Discussion Some of the other changes in the composition of the microbiota were specific to UK4, such as an increased prevalence of Alistipes unclassified and decreased C.Arthromitus unclassified. In tandem, UK4 decreased repetitive behaviour, and modestly increased reward-seeking behaviour in the female urine sniffing test, also often used as a measure of depression-related anhedonia. It is important to note, however, that no changes were observed in other measures of depressive-like behaviour. UK4 also decreased milk gavage-induced improvements long-term spatial learning, yet increased fear-dependent contextual memory, indicating that not all behavioural effects induced by kefir are positive. Finally, UK4 increased circulating Treg cells and IL-10 levels. In tandem, ileal C. Arthromitus unclassified abundances correlated with circulating Treg cell levels, indicating that this bacterial species is likely contributing to the increase in Treg cell levels induced by UK4. Notably, supplementation of L. kefiri CIDCA 8348 results in increased gene expression of IL-10 in the ileum and MLNs in mice (Carasi et al. 2015). In addition, specific bacterial species that were increased by kefir administration, such as B. pseudolongum and L. reuteri, have previously been associated with increased levels of the anti-inflammatory cytokine IL-10 in mice and Treg cells in mice and humans, respectively (Sasajima et al. 2009, Mu et al. 2018). Analysis of the gut microbiota revealed that UK4 increased alpha diversity in the cecum, but no other changes in alpha diversity were observed in any other region or comparison. Analysis of beta diversity revealed a trend towards significant separation induced by both Fr1 and UK4, which resulted in a significant separation induced by kefir overall. Changes in the overall composition of the gut microbiota induced by kefir, or kefir-associated bacterial strains have been reported previously (Carasi et al. 2015, Kim et al. 2017, Hsu et al. 2018, Gao et al. 2019, Yilmaz et al. 2019). Notably, none of the bacterial strains present in the kefir microbiota was detected in the gut microbiota of mice receiving kefir, indicating that the kefir microbiota did not colonise to high levels. This has parallels with the fact that probiotics most frequently do not colonise the gut (Derrien and van Hylckama Vlieg 2015). Our analysis additionally revealed that both kefirs increased the prevalence of L. reuteri, E. plexicaudatum, B. pseudolongum, while decreasing of L. bacterium 3_1_46FAA, P. acnes, and B. amyloliquefaciens. Notably, L. Discussion reuteri is a bacterial strain that has been ascribed numerous beneficial effects on host immunity and metabolism (Mobini et al. 2017, Mu et al. 2018). Notably, previous reports have also demonstrated an increased prevalence of Lactobacillus species in response to kefir supplementation (Kim et al. 2017), as well as L. kefiri CIDCA 8348 administration, a species frequently found in kefir (Carasi et al. 2015). Our data also reveals that both kefirs significantly modulated the functional capacity of the gut microbiota and altered the levels of GBMs, which was related to the prevalence of specific bacterial strains. Importantly, kefir-induced increases in L. reuteri levels were linked to an increased capacity to produce GABA. Indeed, Lactobacillus strains have previously been reported to produce GABA (Bienenstock et al. 2010, Barrett et al. 2012, Lin 2013). Our data additionally reveals that L. reuteri encodes enzymes and antiporters associated with GABA production. Furthermore, levels of faecal metabolites associated with GABA synthesis (i.e. 2- oxoglutarate, glutamate and succinate) correlate with L. reuteri abundances. GABA is the primary inhibitory neurotransmitter in the brain and central GABA levels have been linked to anxiety and depression (Bienenstock et al. 2010), indicating that enhancing GABA production in the gut might be associated with affect anxiety- and depressive-like behaviour. Indeed, two GABA-producing Lactobacillus strains have recently been shown to reduce depressive-like behaviour in high fat diet-induced obese mice (Patterson Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 7/28 Page 7/28 et al. 2019). Various Lactobacillus strains, or Lactobacillus-containing supplements, have been shown to reduce depressive-like behaviour in rodents (Bravo et al. 2011, Liang et al. 2015, Abildgaard et al. 2017, Liang et al. 2017, Dhaliwal et al. 2018) and improve measures of depression in humans (Steenbergen et al. 2015, Akkasheh et al. 2016, Bagga et al. 2018, Kazemi et al. 2019). Furthermore, abundances of faecal GABA-producers, such as Bacteroides, correlate negatively with brain signatures associated with depression (Strandwitz et al. 2019). Notably, one of the key features of depression is decreased reward-seeking behaviour (i.e. anhedonia) and that both kefirs increased reward-seeking behaviour. It is, however, important to note that it is still unclear whether gut-derived GABA can cross the blood-brain barrier and whether increasing gut-derived GABA levels can improve mood (Boonstra et al. 2015). et al. 2019). Various Lactobacillus strains, or Lactobacillus-containing supplements, have been shown to reduce depressive-like behaviour in rodents (Bravo et al. 2011, Liang et al. 2015, Abildgaard et al. Methodology Animals This study used male C57BL/6j mice (8 weeks of age on arrival; Envigo, UK; n = 12/group, n = 48 in total). Treatment groups were divided into 1) (cow’s) Milk control, 2) Kefir gavage – Fr1, 3) Kefir gavage UK4, 4) Undisturbed control. The last group was added to control for the fact that chronic oral gavage or milk administration could affect behaviour or physiology (Walker et al. 2012). Food and drinking water were provided ad libitum throughout the study. Animals were housed in groups of 4. The holding room had a temperature of 21 ± 1 °C and humidity of 55 ± 10% with a 12-hour light/dark cycle (lights on at 7:00 am). Bodyweight was monitored on a weekly basis. Experiments were conducted under the project authorization license B100/3774 in accordance with the European Directive 86/609/EEC and the Recommendation 2007/526/65/EC and approved by the Animal Experimentation Ethics Committee of University College Cork. All efforts were made to reduce the number of animals used and to minimise the suffering of these animals. Experimental timeline and behavioural testing Animals were habituated for one week prior to the onset of daily kefir administration by oral gavage. After three weeks of treatment, animals were assessed for various their behavioural phenotype using various tests, which were formed in order of least stressful to most stressful to reduce the likelihood of prior behavioural tests influencing subsequent ones (Figure 9). In addition, there was a minimum of 36-hours between tests. The order of testing was as follows: 1) Marble burying test, 2) 3-Chamber social interaction test, 3) Elevated plus maze, 4) Open field test, 5) Tail-suspension test, 6) Saccharin preference test, 7) Female urine sniffing test, 8) Stress-induced hyperthermia test, 9) Intestinal motility test, 10) Assessment of faecal water content and weight, 11) Appetitive Y-maze, 12) Fear conditioning, 13) Forced swim test. At the end of the study, body composition (i.e., percentage lean, fat and fluid mass) was assessed (Minispec mq 7.5), after which animals were immediately sacrificed by decapitation. Discussion 2017, Liang et al. 2017, Dhaliwal et al. 2018) and improve measures of depression in humans (Steenbergen et al. 2015, Akkasheh et al. 2016, Bagga et al. 2018, Kazemi et al. 2019). Furthermore, abundances of faecal GABA-producers, such as Bacteroides, correlate negatively with brain signatures associated with depression (Strandwitz et al. 2019). Notably, one of the key features of depression is decreased reward-seeking behaviour (i.e. anhedonia) and that both kefirs increased reward-seeking behaviour. It is, however, important to note that it is still unclear whether gut-derived GABA can cross the blood-brain barrier and whether increasing gut-derived GABA levels can improve mood (Boonstra et al. 2015). Conclusion These data demonstrate that kefir can modulate specific aspects of the microbiota-gut-brain axis in mice, supporting the recent broadening of the definition of psychobiotics to include fermented foods, such as the fermented milk drink kefir (Sarkar et al. 2016). In addition, both kefirs differentially affected repetitive behaviour and reward-associated behaviour. In line with these findings, kefirs differentially impacted systemic immunity and colonic serotonergic signalling. Furthermore, kefir influenced specific gut microbial functional capacities, including the biosynthesis of various neuroactives such as GABA. These changes in the gut microbiota function and peripheral immunity might contribute to the kefir-induced behavioural phenotype, even though more research is warranted to validate whether these specific microbiota-gut-brain axis pathways are involved at all. Moreover, studies on the validation of kefir as a dietary intervention to improve mood in humans is now warranted. Marble burying test Mice were tested for repetitive behaviour with the marble burying test (Thomas et al. 2009), which was conducted as previously described (Burokas et al. 2017). Animals were individually placed in a novel Plexiglas cage (35 × 28 × 18.5 cm, L × W × H), which was filled with sawdust (5 cm) and had 20 equally spread marbles placed on top (5 x 4 rows). After mice had spent 30 minutes in the cage, the number of buried marbles was counted by two researchers and averaged. A buried marble was defined as 2/3 of the marble not being visible anymore. Sawdust was renewed, and marbles cleaned with 70% ethanol in-between animals. Kefir culturing and administration Kefir grains were cultured in Irish whole full fat cow’s milk (2% w/v) at 25 °C and milk were renewed every 24 hours using a sterile Buchner funnel and sterile Duran bottle, as previously described (Dobson et al. 2011, Walsh et al. 2016). Grains were rinsed with deionised water prior to the renewal of milk. The fermented milk (i.e., kefir) collected after 24-hour culturing, or unfermented milk Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js deionised water prior to the renewal of milk. The fermente Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 8/28 Page 8/28 control, were administered to the mice within one hour by oral gavage (0.2 mL). The same milk was used for the unfermented milk control and was, similar to the kefir, also incubated for 24 hours at 25 °C. Daily kefir administration was performed after the behavioural test, if one was performed that day, between 4.00 and 7.00 p.m. To analyse the kefir microbiota over time, aliquots from the kefir administered to the mice were taken on a weekly basis and stored at −80 °C for subsequent analysis. control, were administered to the mice within one hour by oral gavage (0.2 mL). The same milk was used for the unfermented milk control and was, similar to the kefir, also incubated for 24 hours at 25 °C. Daily kefir administration was performed after the behavioural test, if one was performed that day, between 4.00 and 7.00 p.m. To analyse the kefir microbiota over time, aliquots from the kefir administered to the mice were taken on a weekly basis and stored at −80 °C for subsequent analysis. 3-Chamber social interaction test The three-chamber sociability test was used to assess social preference and recognition and was conducted as previously described (Desbonnet et al. 2014). The testing apparatus was a three-chambered, rectangular box. The dividing walls between each chamber (20 × 40 × 22 cm, L × W × H) had small circular openings (5 cm diameter), allowing for access to all chambers. The two outer chambers contained wire cup-like cages (10 cm bottom diameter, 13 cm height), allowing for auditory, olfactory and visual, but not physical contact. The test consisted of 10-minute three phases: 1) Habituation, 2) Social preference, 3) Social recognition. In the first phase (Habituation), mice were allowed to explore the entire box with both wire cup-like cages left empty to allow for habituation to the novel environment. In the second phase (Social preference), one wire cup-like cage contained a novel, age-matched, conspecific, male mouse, whereas the other cage contained an object (rubber duckie). In the third phase (Social recognition), the mouse of the previous trial was left in the wire cup-like cage (Familiar mouse), while the object was replaced with a conspecific mouse (Novel mouse). The test mouse was held in the middle chamber while the conspecific mouse and object were placed in the cup wire-like cages. The location of the conspecific mice and object were systemically altered in-between test mice. The three-chamber test apparatus and wire cup-like cages were cleaned with 70% ethanol after each test mouse and left to dry for a few minutes. To reduce potential anxiogenic factors, all mice were habituated to the testing room 40 minutes before the test, the floor of the testing arena was covered with sawdust and testing was performed under dim light (60 lux). All experiments were videotaped using a ceiling camera and were scored blinded for the time interacted with the wire cup-like cages. The discrimination index was calculated as follows: Time spent interacting with object or mouse / Total time spent interacting 100%. Elevated plus maze The elevated plus maze test was used to assess anxiety-like behaviour and was conducted as previously described (Burokas et al. 2017). The elevated plus maze apparatus was elevated 1 meter above the ground and consisted of a grey cross-shaped maze with two open arms and two closed arms (50 × 5 cm with 15 cm walls in the closed arms and 1 cm walls in the open arms). Mice were allowed to explore the maze for 5 min. Mice were habituated to the room 30 minutes prior to the test. Experiments were conducted in red light (5 lux). The elevated plus maze apparatus was cleaned with 70% ethanol in-between animals. Experiments were videotaped using a ceiling camera and videos were scored blinded for time spent in the open arms, which was defined as all paws in the open arm. Open field test Mice were assessed for locomotor activity and response to a novel environment in the open field test, which was conducted as previously described (Burokas et al. 2017). Animals were placed in an open arena (40 × 32 × 24 cm, L × W × H) and were allowed to explore the arena for 10 minutes. Animals were habituated to the room 30 minutes prior to the test. Testing was performed under dim light (60 lux). The open field test box was cleaned with 70% ethanol in-between animals. Experiments were videotaped Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 9/28 Page 9/28 using a ceiling camera and were analysed for time spent in the virtual centre zone (defined as 50% away from the edges) and total distance travelled using Ethovision version 13 software (Noldus). using a ceiling camera and were analysed for time spent in the virtual centre zone (defined as 50% away from the edges) and total distance travelled using Ethovision version 13 software (Noldus). Saccharin preference test Mice were assessed for reward-seeking behaviour using the saccharin preference test as previously conducted (O'Leary et al. 2014). Mice were first habituated to single housing and having two drinking water bottles for 3 days. Drinking water intake and food intake was measured during the habituation phase of the test. Hereafter, one drinking water bottle was replaced by one containing a saccharin solution (0.1% w/v) for 24 hours. Drinking water bottles were weighed every 12 hours during the testing phase to calculate saccharin preference. The side on which the regular drinking water bottle and the one containing saccharine solution was, were randomised and counterbalanced between groups. During the habituation phase, drinking water bottles were alternated every 24 hours, whereas bottles were alternated every 12 hours during the testing phase. Saccharin preference was calculated using the following formula: Total Sucrose Intake / Total fluid intake 100%. Tail-suspension test The tail-suspension test was used to assess depressive-like behaviour and was conducted as previously described (Burokas et al. 2017). Mice were hung by their tail using adhesive tape (2 cm from the tip of the tail) to a 30 cm-elevated grid bar for 6 min. Experiments were videotaped using a numeric tripod-fixed camera and videos were scored blinded for the time mice spent immobile. Female urine sniffing test Mice were assessed for hedonic and reward-seeking behaviour in the female urine sniffing test, which was performed as previously described (Finger et al. 2011). Prior to this experiment, vaginal smears from age-matched female C57BL/6 mice (n=20; Envigo, UK) were taken and assessed for their estrous cycle. Urine from female mice in the esterus stage was collected and pooled. Male mice were habituated 45 min before the start of the test to the test room, with a cotton bulb attached to the lid of their housing cage. The test mice were subsequently introduced to a new cotton bulb containing 60 μl sterile water. After a 45 min intertrial-interval, mice were introduced to a new cotton bulb containing 60 μl urine from a female mouse in esterus for 3 min. The experiment was conducted in red light (5 lux). All tests were videotaped using a ceiling camera and interaction time with the cotton bulbs was scored blinded. Stress-induced hyperthermia test The stress-induced hyperthermia test was used to assess stress-responsiveness, which was conducted as previously described (Burokas et al. 2017). Body temperature was determined at baseline (T1) and 15 minutes later (T2) by gently inserting a Vaseline- covered thermometer 2.0 cm into the rectum. The temperature was noted to the nearest 0.1 °C after it stabilised (~10 s). Mice were restrained by scruffing during this procedure which was the stressor. Animals were habituated to the testing room 1 hour prior to the test. The difference between T1 and T2 reflected the stress-induced hyperthermia. Appetitive Y-maze Finally, mice were individually placed on the Y-maze until they were running and collected the food reward (each arm contained 0.1 mL food reward), which took 4 days. During the first phase (Initial learning), mice were assigned a goal arm according to the position in the room, which was counter- balance between groups. The maze was rotated 120° every trial to prevent potential associations of the correct goal arm with the texture or smell of the arm. The starting position for each trial was determined by a pseudorandomised computer sequence, which was different for each mouse but was the same across treatment groups. This sequence did not contain more than three consecutive starts in the same position to avoid temporary position preferences. Animals were tested in groups of eight, with four animals of two experimental group (i.e. two home cages). Each mouse received ten trials per day with an intertrial interval of approximately 10 minutes. The time of testing was counterbalanced between groups and rotated each day to reduce the effect of testing during a specific time of the day. Mice received eight consecutive days of initial learning, resulting in a total of 80 trials. During the second phase (reversal learning), the goal arm was changed to a different arm, and the placement of the mice was changed accordingly. This phase lasted 5 days, resulting in a total of 50 trials. For each trial, the food well on the goal arm was filled with 0.1 mL food reward (sweetened condensed milk diluted in water 1:1). The mouse was placed at the end of the start arm and was allowed to run freely on the maze. The entries into each arm were counted, as well as when the mouse went into the goal arm immediately, of which the latter was counted as a successful trial. The mouse was placed back into the home cage after it consumed the food reward. In the rare occasion that the mouse did not walk into the goal arm and collect the food reward within 90 seconds, then the mouse was gently guided towards the goal arm and given a chance to collect the food reward, after which it was also returned to the home cage. A trial where the mouse did not walk into any arm was excluded from the analysis, as this indicates that the mouse was anxious. Appetitive Y-maze An entry was counted when the tail of the animal passed the entry of the arm. Between mice, the food wells were not cleaned so that a slight odour of milk reward remained at all times, ensuring mice found the goal arm based on spatial cues, rather the olfactory cues. Fear conditioning Fear conditioning was used to assess amygdala-dependent learning memory and was conducted as previously described (Izquierdo et al. 2006). The test consisted of 3 days/phases; 1) Training, 2) Assessment of cued memory, 3) Assessment of contextual memory, each of which was carried on successive days with a 24-hour interval. In phase 1 (training), animals were recorded for 3 minutes (baseline), followed by 6 tone-conditioned stimuli (70 dB, 20 s), followed by a foot shock (0.6 mA, 2 s), with a 1-minute interval. In phase 2 (Assessment of cued memory), mice were placed in a novel context (i.e. black-checkered walls with a solid Plexiglas opaque floor, under which paper was placed containing a 400 μl vanilla solution (79.5% water/19.5% ethanol/1% vanilla-extract solution), and after an initial acclimation period of 2 minutes, mice received 40 presentations of the tone- conditioned stimuli, each lasting 30 seconds with a 5-second interval. In phase 3 (Assessment of contextual memory), mice were placed in the context of day 1 and recorded for 5 minutes, without the presentation of any tone-conditioned stimuli. The fear conditioning apparatus was cleaned with 70% ethanol in-between animals. Appetitive Y-maze The appetitive Y-maze was used to assess long-term spatial learning and was performed as previously described (Finger et al. 2010). The test consisted of two phases; the initial learning phase, where the first association between the location of the food reward and spatial reference cues were formed, and the reversal learning phase, where the location of the food reward was altered in reference to the spatial reference cues, in which the relearning of a context was measured. The Y-maze apparatus was elevated 80 cm above the ground and consisted of three arms (50 x 9.5 cm, L x W, with a 0.5 cm-high rim) arranged at an angle of 120° of each other (Figure S10A). The apparatus could be rotated during testing. A small plastic food Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 10/28 well (a cap of a 15 mL tube) was placed at the distal end of each arm. Testing was performed under dim light (30 lux). well (a cap of a 15 mL tube) was placed at the distal end of each arm. Testing was performed under dim light (30 lux). Prior to testing, mice were food-restricted (3-4 gram food per day) and kept between 90-95% of their free-feeding body weight (Figure S10B). Two days later, animals were habituated in their home cage to the small plastic well containing 1 mL food reward (sweetened condensed milk diluted in water 1:1) per mouse before the onset of the active phase. Mice were subsequently habituated on the Y-maze apparatus in home cage groups until mice were freely running around and readily collecting the food reward (each arm contained 1 mL food reward), which took 2 days. Finally, mice were individually placed on the Y-maze until they were running and collected the food reward (each arm contained 0.1 mL food reward), which took 4 days. Prior to testing, mice were food-restricted (3-4 gram food per day) and kept between 90-95% of their free-feeding body weight (Figure S10B). Two days later, animals were habituated in their home cage to the small plastic well containing 1 mL food reward (sweetened condensed milk diluted in water 1:1) per mouse before the onset of the active phase. Mice were subsequently habituated on the Y-maze apparatus in home cage groups until mice were freely running around and readily collecting the food reward (each arm contained 1 mL food reward), which took 2 days. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Intestinal motility assay Gastrointestinal motility was assessed as previously described (Golubeva et al. 2017). Briefly, mice were single-housed at 8.00 a.m. with ad libitum access to food and drinking water. Three hours later, 0.2 mL of non-absorbable 6% carmine red in 0.5% methylcellulose dissolved in sterile phosphate-buffered saline was administered by oral gavage, after which drinking water was removed. The latency for the excretion of the first red-coloured faecal pellet was subsequently timed as a measure of gastrointestinal motility. Tissue collection Collection of faecal samples for metabolomics was done one week prior to euthanasia. This was done by single housing mice until 2 pellets were dropped between 10.00 and 12.00 a.m. The order faecal pellet collection was counterbalanced between groups to minimise the effect of circadian rhythm. Pellets were snap-frozen on dry ice within 3 minutes after excretion and subsequently stored at -80 °C. Animals were sacrificed by decapitation in a random fashion regarding test groups between 9.00 a.m. and 2.00 p.m. Trunk blood was collected in 3 mL EDTA-containing tubes (Greiner bio-one, 454086) and 100 μl was put in a separate Eppendorf for flow cytometry. Both tubes were centrifuged for 10 min at 3,500 g at 4°C, after which plasma was collected and stored at −80 °C for cytokine quantification. The remaining cell pellet of the Eppendorf containing 100 μl blood was stored on ice and subsequently used for flow cytometry. Mesenteric lymph nodes (MLNs) were extracted, fat tissue was removed and stored in RPMI-1640 medium with L-glutamine and sodium bicarbonate (R8758, Sigma), supplemented with 10% FBS (F7524l, Sigma) and 1% Pen/strep (P4333, Sigma) on ice for subsequent flow cytometry. The faecal pellets, cecum, and contents of the distal part of the ileum (2 cm) were collected, snap-frozen on dry ice and stored at −80 °C for shotgun sequencing. Repeated plasma sampling for corticosterone quantification Plasma from each animal was sampled by tail-tip five minutes before the forced swim test, and repeatedly after the test in 30-min intervals up to 120 minutes, as previously discussed (Burokas et al. 2017). For the tail-tip, the end of the tail was gently held with two fingers without restraining the mouse. Using a single edge razor blade, a 2-4 mm long diagonal incision was made at the end of the tail. Approximately 40 μl of whole blood was taken per time point using an EDTA-containing capillary (Fisher Scientific, 749311), deposited in an Eppendorf and centrifuged for 10 min at 3,500 g at 4°C. Plasma was collected and stored at −80 °C for later corticosterone quantification. Assessment of faecal water content and weight Mice were single-housed for one hour during which faecal pellets were collected (± 9 per animal). Pellets were subsequently weighed, dried at 50 °C for 24 hours and weighed again. The average weight per pellet and percentage of faecal water content was calculated. Forced swim test The forced swim test was used to assess depressive-like behaviour and was conducted as previously described (Cryan and Mombereau 2004). Mice were individually placed in a transparent glass cylinder (24 × 21 cm diameter) containing 15-cm-depth water (23-25 °C) for 6 minutes. Mice were gently dried after the test, and water was renewed after each animal. Experiments were videotaped using a ceiling camera and videos were scored blinded for immobility time in the last 4 min of the test.fi Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 11/28 Page 11/28 Page 11/28 Flow cytometry Blood and MLNs collected when animals were sacrificed were processed on the same day for flow cytometry, as previously described (Boehme et al. 2019, Gururajan et al. 2019). Blood was resuspended in 10 mL home-made red blood cell lysis buffer (15.5 mM NH4Cl, 1.2 mM NaHCO3, 0.01 mM tetrasodium EDTA diluted in deionised water) for 3 minutes. Blood samples were subsequently centrifuged (1500 g, 5 minutes), split into 2 aliquots and resuspended in 45 μl staining buffer (autoMACS Rinsing Solution (Miltenyi, 130-091-222) supplemented with MACS BSA stock solution (Miltenyi, 130-091-376)) for the staining procedure. MLNs were poured over a 70 µm strainer and disassembled using the plunger of a 1 mL syringe. The strainer was subsequently washed with 10 mL media (RPMI-1640 medium with L-glutamine and sodium bicarbonate, supplemented with 10% FBS and 1% Pen/strep), centrifuged and 2x106 cells were resuspended in 90 μl staining buffer and split into two aliquots for the staining Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 12/28 Page 12/28 procedure. For the staining procedure, 5 μl of FcR blocking reagent (Miltenyi, 130-092-575) was added to each sample. Samples were subsequently incubated with a mix of antibodies (Blood aliquot 1; 5 μl CD11b-VioBright FITC (Miltenyi, 130-109-290), 5 μl LY6C-PE (Miltenyi, 130-102-391), 0.3 μl CX3CR1-PerCP-Cyanine5.5 (Biolegend, 149010) and 5 μl CCR2-APC (Miltenyi, 130-108- 723); Blood aliquot 2 and MLNs 1; 1 μl CD4-FITC (ThermoFisher, 11-0042-82) and 1 μl CD25-PerCP-Cyanine5.5 (ThermoFisher, 45- 0251-80); MLNs 2; 5 μl CD103-FITC (Miltenyi, 130-102-479), 2 μl CD11c-PE (Miltenyi, 130-110-838), 0.3 μl CX3CR1-PerCP- Cyanine5.5 (Biolegend, 149010) and 5 μl MHC-II-APC (Miltenyi, 130-102-139)) and incubated for 30 minutes on ice. Blood aliquot 1 was subsequently fixed in 4% PFA for 30 minutes on ice, whilst Blood aliquot 2 and MLNs underwent intracellular staining using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher, 00-5523-00), according to the manufacturers’ instructions, using antibodies for intracellular staining (2 μl FoxP3-APC (ThermoFisher, 17-5773-82) and 5 μl Helios-PE (ThermoFisher, 12-9883-42)). Fixed samples were resuspended in staining buffer and analysed the subsequent day on the BD FACSCalibur flow cytometry machine. Data were analysed using FlowJo (version 10), see Figure S11 for the gating information. Cell populations were selected as following: Treg cells: CD4+, CD25+, FoxP3+; Neutrophils: CD11b+, LY6Cmid. SSChigh; Monocytes: CD11b+, LY6Chigh; CD103+ Dendritic cells; MHC-II+, CD11c+, CD103+. The investigated cell populations were normalised to PBMC levels. Plasma corticosterone and cytokine assessment Corticosterone quantification of plasma samples (20 μl) obtained in the forced swim test was performed using a corticosterone ELISA (Enzo Life Sciences, ADI-901-097) according to the manufacturer's guidelines. A multi-mode plate reader (Synergy HT, BioTek Instruments) was used to measure light absorbance. Cytokine levels from plasma samples collected during euthanasia were quantified using the V-PLEX Proinflammatory Panel 1 Mouse Kit (MSD, K15048D). Cytokine quantification was done according to the manufacturer's guidelines with one modification, where 20 μl plasma sample was added onto the plate and incubated overnight (15 hours) at 4 °C, after which the rest of the protocol was carried out as suggested by the guideline. Values under the fit curve range and detection range were excluded. High-performance liquid chromatography 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations were determined using HPLC based on methodology previously described (Clarke et al. 2013). Briefly, mobile phase consisted of HPLC-grade 0.1M citric acid, 0.1M sodium dihydrogen phosphate monohydrate, 0.01mM EDTA disodium salt (Alkem/Reagecon), 5.6mM octane-1-sulphonic acid (Sigma Aldrich), and 9% (v/v) methanol (Alkem/Reagecon).The pH of the mobile phase was adjusted to 2.8 using 4N sodium hydroxide (Alkem/Reagecon). Homogenization buffer consisted of mobile phase with the addition of 20 ng/20 µl of the internal standard, N-methyl 5-HT (Sigma Aldrich). Briefly, tissue samples were sonicated (Sonopuls HD 2070) for 4 seconds in 500µl cold homogenization buffer during which they were kept chilled. Tissue homogenates were then centrifuged at 14,000g for 20min at 4oC. The supernatant was collected and the pellet was discarded. The supernatant was then briefly vortexed and 30 µl of supernatant was spiked into 270 µl of mobile phase. 20 µl of the 1:10 dilution was injected into the HPLC system (Shimadzu, Japan) which was comprised of a SCL 10-Avp system controller, LC-10AS pump, SIL-10A autoinjector, CTO-10A oven, LECD 6A electrochemical detector, and Class VP-5 software. The chromatographic conditions were flow rate of 0.9mL/min using a Kinetex 2.6u C18 100A x 4.6mm column (Phenomenex), oven temperature of 30oC, and detector settings of +0.8V. 5-HT and 5-HIAA external standards (Sigma Aldrich, H7752 and H8876, respectively) were injected at regular intervals during sample analyses. Monoamines in unknown samples were determined by their retention times compared to external standards. Peak heights of the analyte: internal standard ratio were measured and compared with external standards, results were expressed as µg of neurotransmitter per gram of tissue. Monoamines in unknown samples were determined by their retention times compared to external standards. Peak heights of the analyte: internal standard ratio were measured and compared with external standards, results were expressed as µg of neurotransmitter per gram of tissue. DNA extractions and sequencing For analysis of the kefir microbiome, DNA was extracted from the fermented milk using the PowerSoil DNA Isolation Kit, as described previously (Walsh et al. 2016). For analysis of the murine gut microbiome, DNA was extracted from the total ileal contents, cecal contents and faecal pellets using the QIAamp PowerFaecal DNA Kit. Whole-metagenome shotgun libraries were Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 13/28 Page 13/28 prepared using the Nextera XT kit in accordance with the Nextera XT DNA Library Preparation Guide from Illumina, with the exception that tagmentation time was increased to 7 minutes. Kefir libraries were sequenced on the Illumina MiSeq sequencing platform with a 2 x 300 cycle v3 kit. Gut libraries were sequenced on the Illumina NextSeq 500 with a NextSeq 500/550 High Output Reagent Kit v2 (300 cycles). All sequencing was performed at the Teagasc sequencing facility in accordance with standard Illumina protocols. Bioinformatics Raw microbiota reads have been deposited to the European Nucleotide Archive under the project accession number PRJEB35751 Raw microbiota reads have been deposited to the European Nucleotide Archive under the pr Faecal metabolomics The faecal metabolome was analysed by chromatography–mass spectrometry (GC-MS) by MS-Omics, Copenhagen. Samples were derivatized using methyl chloroformate. For SCFA quantification, samples were acidified with hydrochloric acid. Bioinformatics were removed from the raw sequencing files using the NCBI Best Match Tagger (BMTagger) q g g gg ( gg ) (ftp://ftp.ncbi.nlm.nih.gov/pub/agarwala/bmtagger/), and fastq files were converted to unaligned bam files using SAMtools (Li et al. 2009). Duplicate reads were subsequently removed using Picard Tools (https://github.com/broadinstitute/picard). Next, low- quality reads were removed using the trimBWAstyle.usingBam.pl script from the Bioinformatics Core at UC Davis Genome Center (https://github.com/genome/genome/blob/master/lib/perl/Genome/Site/TGI/Hmp/HmpSraProcess/trimBWAstyle.usingBam.pl). Specifically, MiSeq reads were filtered to 200 bp, while NextSeq were filtered to 105 bp. All reads with a quality score less than Q30 were discarded. The resulting fastq files were then converted to fasta files using the fq2fa option from IDBA-UD (Peng et al. 2012). Compositional analysis was performed using MetaPhlAn2 (Truong et al. 2015). Strain-level metagenomic analysis was performed using StrainPhlAn (Truong et al. 2017) and PanPhlAn (Scholz et al. 2016) StrainPhlAn outputs were visualised using GraPhlAn (Asnicar et al. 2015). Custom PanPhlAn databases were constructed from complete genome assemblies which were annotated using Prokka (Seemann 2014). See Table S1 for the list of reference genomes used in this study. Functional analysis was performed with HUMAnN2 (Abubucker et al. 2012), using the --bypass-translated-search option, and PanPhlAn. HUMAnN2 gene families were mapped to level-4 enzyme commission (EC) categories using HUMAnN2 utility mapping files. Sequence data have been deposited in the European Nucleotide Archive (ENA). Correlations between gut microbial species and significantly altered behavioural and immunological parameters were investigated using HAllA (https://bitbucket.org/biobakery/halla/wiki/Home). Analysis of gut-brain modules (GBMs) was performed as previously described (Valles-Colomer et al. 2019). Briefly, the UniRef gene families that were detected by HUMAnN2 were mapped to KEGG Orthogroups (KOs) using the humann2_regroup_table function, and the abundances of KOs were normalised using the humann2_renorm_table function. Next, these KOs were further mapped to GBMs using Omixer-RPM. Metagenome co-assembly was performed using MEGAHIT (Li et al. 2015). MetaBAT 2 (Kang et al. 2015) was used to recover genomes from the metagenome. CheckM (Parks et al. 2015) was used to assess the quality of the MAGs. The Lactobacillus reuteri genome was identified using PhyloPhlAn (Segata et al. 2013). Prokka was used to annotate the genome and CarveMe (Machado et al. 2018) was used to construct a metabolic model. COBRApy (Ebrahim et al. 2013) was used to perform flux variability analysis (FVA), with an objective of 95% biomass, of this model. Funding Sources The APC Microbiome Ireland is a research institute funded by Science Foundation Ireland (SFI) through the Irish Government's National Development Plan. J.F.C, T.G.D and P.D.C. are supported by SFI (Grant Nos. SFI/12/RC/2273). MB is supported by an educational grant from Science Foundation Ireland (SFI), Ireland (15/JP‐HDHL/3270; JPI‐HDHL‐NutriCog project ‘AMBROSIAC’). Author Contributions M.vd.W. performed the in vivo study, behavioural analysis, flow cytometry and ELISAs. A.M.W. performed DNA extractions, sequencing and bioinformatics. F.C. assisted with bioinformatics. L.v.L. assisted with the in vivo study and the behavioural analysis. J.M.L performed the HPLC analysis. M.B. assisted with flow cytometry. G.C., T.G.D., P.D.C, and J.F.C contributed to the experimental design and drafting and critical revision of the manuscript. Availability of data and materials Raw microbiota reads have been deposited to the European Nucleotide Archive under the project accession number PRJEB35751. Raw microbiota reads have been deposited to the European Nucleotide Archive under the pr Raw microbiota reads have been deposited to the European Nucleotide Archive under the project accession number PRJEB35751. Consent for publication Not applicable Declarations Not applicable Statistical analysis All behavioural and physiological data were assessed for normality using the Shapiro-Wilk test and Levene's test for equality of variances. The effect of kefir was determined by a one-way ANOVA, followed by Dunnett's post hoc test whenever data were normally distributed. If data were non-parametrically distributed, then a Kruskal-Wallis test followed by a Mann-Whitney U test was used. Undisturbed control and milk control datasets were assessed for statistical significance using an unpaired Student’s t-test or a Mann-Whitney U test to investigate the impact of milk gavage. Bodyweight, fear conditioning and appetitive Y-maze data were Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 14/28 Page 14/28 assessed using repeated-measures ANOVA, followed by a Dunnett's post hoc test. The presence of social preference and recognition in the 3-chamber sociability test was assessed using a paired Student’s t-test. Parametric data are depicted as bar graphs with points as individual data points and expressed as mean ± SEM. Non-parametric data is depicted as a box with whiskers plot. Statistical analysis was performed using SPSS software version 24 (IBM Corp). A p-value < 0.05 was deemed significant assessed using repeated-measures ANOVA, followed by a Dunnett's post hoc test. The presence of social preference and recognition in the 3-chamber sociability test was assessed using a paired Student’s t-test. Parametric data are depicted as bar graphs with points as individual data points and expressed as mean ± SEM. Non-parametric data is depicted as a box with whiskers plot. Statistical analysis was performed using SPSS software version 24 (IBM Corp). A p-value < 0.05 was deemed significant Statistical analysis for bioinformatics data was performed using the R package vegan for alpha diversity analysis and principal component analysis (Oksanen et al. 2007). The Wilcoxon rank-sum test was used to measure statistical differences in alpha diversity between groups. The adonis function from vegan was used for PERMANOVA. The linear discriminant analysis (LDA) effect size (LEfSe) method (Segata et al. 2011) was used to investigate if any taxa or HUMAnN2 pathways were differentially abundant between groups. Data were visualised using hclust2 (https://bitbucket.org/nsegata/hclust2), GraPhlAn, and the R package ggplot2 (Wickham 2016). Competing Interests J.F.C and T.G.D have research support from Mead Johnson, Cremo, 4D Pharma, Dupont, and Nutricia. P.D.C has research support from PepsiCo and Danone. J.F.C, T.G.D and P.D.C. have spoken at meetings sponsored by food and pharmaceutical companies. All other authors report no potential conflicts of interest. Acknowledgements Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 15/28 Page 15/28 Flow cytometry analysis was performed at the APC Microbiome Ireland Flow Cytometry Platform located at University College Cork. The authors are also grateful for the technical assistance of P. Fitzgerald and C. Manly, and the assistance with data analysis and study design by Drs G. Moloney, A. Golubeva and K. O’Riordan. Abbreviations Treg: T regulatory cell MLN: Mesenteric lymph node pTreg: Peripheral-derived T regulatory cells 5-HT: Serotonin 5HIAA: 5-hydroxyindoleacetic acid SCFA: Short-chain fatty acid GC-MS: Chromatography–mass spectrometry EC: Enzyme commission GBM: Gut-brain modules KO: KEGG Orthogroup GABA: Gamma aminobutyric acid GABA: Gamma aminobutyric acid 9. Boonstra, E., R. de Kleijn, L. S. Colzato, A. Alkemade, B. U. Forstmann and S. Nieuwenhuis (2015). "Neurotransmitters as food supplements: the effects of GABA on brain and behavior." Front Psychol6: 1520. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js References Bourrie, B. C. T., P. D. Cotter and B. P. Willing (2018). "Traditional kefir reduces weight gain and improves plasma and liver lipid profiles more successfully than a commercial equivalent in a mouse model of obesity." Journal of Functional Foods46: 29-37 12. Bourrie, B. C. T., B. P. Willing and P. D. Cotter (2016). 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Turk J Gastroenterol30(3): 242-253. Page 20/28 Figures Figure 1 Kefir modulates repetitive behaviour and reward-seeking behaviour. References Repetitive/anxiety-like behaviour was assessed using the marble burying test (A). Anhedonia and reward-seeking behaviours were investigated using the female urine sniffing test (B, C) and saccharin preference test (D, E). The marble burying test was normally distributed and analysed using a one-way ANOVA, followed by a Dunnett's post hoc test. The female urine sniffing test and saccharin preference test were non-normally distributed and analysed using the Kruskal-Wallis test, followed by the Mann-Whitney test. Significant differences are depicted as: p < 0.05, p < 0.01 and *p < 0.001; Milk control compared to Kefir supplementation, $p < 0.05; Undisturbed control compared to Milk control. All data are expressed as mean ± SEM (n = 11-12). Dots on each graph represent individual animals. Figure 1 Kefir modulates repetitive behaviour and reward-seeking behaviour. Repetitive/anxiety-like behaviour was assessed using the marble burying test (A). Anhedonia and reward-seeking behaviours were investigated using the female urine sniffing test (B, C) and saccharin preference test (D, E). The marble burying test was normally distributed and analysed using a one-way ANOVA, followed by a Dunnett's post hoc test. The female urine sniffing test and saccharin preference test were non-normally distributed and analysed using the Kruskal-Wallis test, followed by the Mann-Whitney test. Significant differences are depicted as: p < 0.05, p < 0.01 and p < 0.001; Milk control compared to Kefir supplementation, $p < 0.05; Undisturbed control compared to Milk control. All data are expressed as mean ± SEM (n = 11-12). Dots on each graph represent individual animals. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 21/28 Figure 2 UK4 enhances fear-dependent contextual memory yet decreases long-term spatial learning. Fear-dependent memory and learning were assessed using fear conditioning. At phase 1 – Acquisition, mice were presented with a tone, followed by a foot shock. Cue- associative learning was assessed by measuring freezing behaviour during the presentation of the tone (A), whereas context- associative learning was determined in-between tones (B). At phase 2 – Cued memory, mice received 40 presentations of the same cue (the first 10 are shown), without foot shock, in a different context, in which fear-dependent cued memory was assessed (C). At phase 3 – Contextual memory, mice were exposed to the same context as day one for 5 minutes and contextual memory was assessed (D). Long-term spatial learning was assessed in the appetitive Y-maze, as determined by the percentage of times the mice made the correct choice as the first choice for reaching the goal (food reward) (E), as well as the number of average entries it took the mice to reach the goal (F). All data were normally distributed and analysed using a repeated measures ANOVA or one-way ANOVA, followed by a Dunnett's post hoc test. Significant differences are depicted as: p < 0.05; Milk control compared to Kefir supplementation, $p < 0.05; undisturbed control compared to Milk control. All data are expressed as mean ± SEM (n = 10-12). Dots on each graph represent individual animals. Figure 2 Figure 2 UK4 enhances fear-dependent contextual memory yet decreases long-term spatial learning. Fear-dependent memory and learning were assessed using fear conditioning. At phase 1 – Acquisition, mice were presented with a tone, followed by a foot shock. Cue- associative learning was assessed by measuring freezing behaviour during the presentation of the tone (A), whereas context- associative learning was determined in-between tones (B). At phase 2 – Cued memory, mice received 40 presentations of the same cue (the first 10 are shown), without foot shock, in a different context, in which fear-dependent cued memory was assessed (C). At phase 3 – Contextual memory, mice were exposed to the same context as day one for 5 minutes and contextual memory was assessed (D). Long-term spatial learning was assessed in the appetitive Y-maze, as determined by the percentage of times the mice made the correct choice as the first choice for reaching the goal (food reward) (E), as well as the number of average entries it took the mice to reach the goal (F). All data were normally distributed and analysed using a repeated measures ANOVA or one-way ANOVA, followed by a Dunnett's post hoc test. Significant differences are depicted as: p < 0.05; Milk control compared to Kefir supplementation, $p < 0.05; undisturbed control compared to Milk control. All data are expressed as mean ± SEM (n = 10-12). Dots on each graph represent individual animals. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 22/28 Figure 3 UK4 increases Treg cells levels, while Fr1 decreases neutrophil levels. T regulatory cells mesenteric lymph nodes (MLNs) (A) and were subsequently analysed for Helios expres Helios+ cells exclusively originate in the thymus. Blood was also assessed for Treg cell 10) levels (D). Similarly, neutrophils (CD11b+, LY6Cmid, SSChigh and CXCL1 levels wer F). All data were normally distributed and analysed using a one-way ANOVA, followed b differences are depicted as: p < 0.05, *p < 0.01; Milk control compared to Kefir supple Undisturbed control compared to Milk control. All data are expressed as mean ± SEM (n Figure 3 UK4 increases Treg cells levels, while Fr1 decreases neutrophil levels. T regulatory cells (CD4+, CD25+, FoxP3+) were assessed in mesenteric lymph nodes (MLNs) (A) and were subsequently analysed for Helios expression (B), to investigate their origin as Helios+ cells exclusively originate in the thymus. Figure 2 Blood was also assessed for Treg cell levels (C), and plasma interleukin 10 (IL- 10) levels (D). Similarly, neutrophils (CD11b+, LY6Cmid, SSChigh and CXCL1 levels were also investigated in peripheral blood (E, F). All data were normally distributed and analysed using a one-way ANOVA, followed by a Dunnett's post hoc test. Significant differences are depicted as: p < 0.05, *p < 0.01; Milk control compared to Kefir supplementation, $p < 0.05 and $$p < 0.01; Undisturbed control compared to Milk control. All data are expressed as mean ± SEM (n = 11-12). Dots on each graph represent individual animals. Figure 3 Figure 3 UK4 increases Treg cells levels, while Fr1 decreases neutrophil levels. T regulatory cells (CD4+, CD25+, FoxP3+) were assessed in mesenteric lymph nodes (MLNs) (A) and were subsequently analysed for Helios expression (B), to investigate their origin as Helios+ cells exclusively originate in the thymus. Blood was also assessed for Treg cell levels (C), and plasma interleukin 10 (IL- 10) levels (D). Similarly, neutrophils (CD11b+, LY6Cmid, SSChigh and CXCL1 levels were also investigated in peripheral blood (E, F). All data were normally distributed and analysed using a one-way ANOVA, followed by a Dunnett's post hoc test. Significant differences are depicted as: p < 0.05, p < 0.01; Milk control compared to Kefir supplementation, $p < 0.05 and $$p < 0.01; Undisturbed control compared to Milk control. All data are expressed as mean ± SEM (n = 11-12). Dots on each graph represent individual animals. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 23/28 Page 23/28 Figure 4 Fr1 modulates serotonergic signaling in the colon, but not ileum. Ileal (A-C) and colonic (D-F) tissues were quantified for 5HIAA and serotonin (5-HT) levels using HPLC. The 5HIAA/5-HT ratio was subsequently calculated. All data was normally distributed and analysed using a one-way ANOVA, followed by a Dunnett's post hoc test. Significant differences are depicted as: p < 0.01; Milk control compared to Kefir supplementation, $p < 0.05, p < 0 01and Figure 4 Figure 4 Fr1 modulates serotonergic signaling in the colon, but not ileum. Ileal (A-C) and colonic (D-F) tissues were quantified for 5HIAA and serotonin (5-HT) levels using HPLC. The 5HIAA/5-HT ratio was subsequently calculated. All data was normally distributed and analysed using a one-way ANOVA, followed by a Dunnett's post hoc test. Significant differences are depicted as: p < 0.01; Milk control compared to Kefir supplementation, $p < 0.05, Fr1 modulates serotonergic signaling in the colon, but not ileum. Ileal (A-C) and colonic (D-F) tissues were quantified for 5HIAA and serotonin (5-HT) levels using HPLC. The 5HIAA/5-HT ratio was subsequently calculated. All data was normally distributed and analysed using a one-way ANOVA, followed by a Dunnett's post hoc test. Significant differences are depicted as: p < 0.01; Milk control compared to Kefir supplementation, $p < 0.05, p < 0.01and $p < 0.001; Undisturbed control compared to Milk control. All data are expressed as mean ± SEM (n = 11-12). Dots on each graph represent individual animals. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 24/28 Figure 5 Kefir modulates the composition of the gastrointestinal microbiota. Alpha diversity (Shannon) of th microbiota was compared between mice receiving kefir (Fr1 or UK4) and mice receiving milk contro diversity was assessed by PERMANOVA to investigate the dissimilarity in the gut microbial compos using MDS plots. Dots represent individual animals (n = 12) (B). Differentially abundant taxa were d Figure 5 Kefir modulates the composition of the gastrointestinal microbiota. Alpha diversity (Shannon) of the ileal, cecal and faecal microbiota was compared between mice receiving kefir (Fr1 or UK4) and mice receiving milk control using violin plots (A). Beta diversity was assessed by PERMANOVA to investigate the dissimilarity in the gut microbial composition, which were depicted using MDS plots. Dots represent individual animals (n = 12) (B). Differentially abundant taxa were determined using LEfSe (C). Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 25/28 Figure 6 Bacterial species in the gastrointestinal microbiota correlate with changes in host immunity and measures of gut serotonin. The prevalence of bacterial species in the ileum, cecum and faeces were correlated with significantly altered changes in host behaviour and immunity using HAllA. Figure 9 Experimental design. Animals received three weeks of treatment lead in, where animals only received kefir or milk supplementation, after which they were assessed for their behavioural phenotype. Treatment groups consisted of: 1) No gavage control, 2) Milk gavage control, 3) Kefir gavage – Fr1, and 4) Kefir gavage – UK4 (n = 12/group). The order of behavioural tests was as following; Week 4: Marble burying test (MB), 3-Chamber social interaction test (3CT) and Elevate plus maze (EPM); Week 5: Open field test (OF) and Tail suspension test (TST); Week 6: Saccharin preference test (SPT); Week 7: Female urine sniffing test (FUST); Week 8: Stress-induced hyperthermia test (SIH); Week 9: Intestinal motility test (IM) and Faecal water content assessment (FWC): Week 9-12: Appetitive Y-maze; Week 13: Fear conditioning; Week 14: Forced swim test; Week 15: Euthanasia. Postmortem, ileal, caecal and faecal microbiota composition and function was investigated by shotgun sequencing and faecal metabolomics. Host immunity was assessed using flow cytometry and by quantification of cytokines. Finally, ileum and colonic serotonergic levels were quantified by high-performance liquid chromatography (HPLC). . Animals received three weeks of treatment lead in, where animals only received kefir or milk Experimental design. Animals received three weeks of treatment lead in, where animals only received kefir or milk supplementation, after which they were assessed for their behavioural phenotype. Treatment groups consisted of: 1) No gavage control, 2) Milk gavage control, 3) Kefir gavage – Fr1, and 4) Kefir gavage – UK4 (n = 12/group). The order of behavioural tests was as following; Week 4: Marble burying test (MB), 3-Chamber social interaction test (3CT) and Elevate plus maze (EPM); Week 5: Open field test (OF) and Tail suspension test (TST); Week 6: Saccharin preference test (SPT); Week 7: Female urine sniffing test (FUST); Week 8: Stress-induced hyperthermia test (SIH); Week 9: Intestinal motility test (IM) and Faecal water content assessment (FWC): Week 9-12: Appetitive Y-maze; Week 13: Fear conditioning; Week 14: Forced swim test; Week 15: Euthanasia. Postmortem, ileal, caecal and faecal microbiota composition and function was investigated by shotgun sequencing and faecal metabolomics. Host immunity was assessed using flow cytometry and by quantification of cytokines. Finally, ileum and colonic serotonergic levels were quantified by high-performance liquid chromatography (HPLC). Figure 8 The kefir-induced increased Lactobacillus reuteri strain has the potential to produce GABA. Spearman rank correlations between Lactobacillus reuteri abundances and levels of faecal 2-oxoglutarate, glutamate and succinate were performed (A). Faecal metabolite levels were quantified using chromatography–mass spectrometry (GC-MS). Flux Variability Analysis was used to assess whether L. reuteri can influence 2-oxoglutarate, glutamate and succinate levels (B). The kefir-induced increased Lactobacillus reuteri strain has the potential to produce GABA. Spearman rank correlations between Lactobacillus reuteri abundances and levels of faecal 2-oxoglutarate, glutamate and succinate were performed (A). Faecal metabolite levels were quantified using chromatography–mass spectrometry (GC-MS). Flux Variability Analysis was used to assess whether L. reuteri can influence 2-oxoglutarate, glutamate and succinate levels (B). assess whether L. reuteri can influence 2 oxoglutarate, glutamate and succinate levels (B). Figure 9 SupplementsKefirMvdW.docx Figure 6 Bacterial species in the gastrointestinal microbiota correlate with changes in host immunity and measures of gut serotonin. The prevalence of bacterial species in the ileum, cecum and faeces were correlated with significantly altered changes in host behaviour and immunity using HAllA. Bacterial species in the gastrointestinal microbiota correlate with changes in host immunity and measures of gut serotonin. The prevalence of bacterial species in the ileum, cecum and faeces were correlated with significantly altered changes in host behaviour and immunity using HAllA. Bacterial species in the gastrointestinal microbiota correlate with changes in host immunity and measures of gut serotonin. The prevalence of bacterial species in the ileum, cecum and faeces were correlated with significantly altered changes in host behaviour and immunity using HAllA. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 26/28 Figure 7 Kefir modulates the functional capacity of the gastrointestinal microbiota. Beta diversity was ass investigate the dissimilarity in the functional capacity of the gut microbiota, which were depicted individual animals (n = 12) (A). Differential abundances were assessed of enzyme categories (EC Gut-brain modules (GBMs) were additionally assessed for their differential abundance (C). Figure 7 Kefir modulates the functional capacity of the gastrointestinal microbiota. Beta diversity was assessed by PERMANOVA to investigate the dissimilarity in the functional capacity of the gut microbiota, which were depicted using MDS plots Dots represent individual animals (n = 12) (A). Differential abundances were assessed of enzyme categories (EC) and depicted as violin plots (B). Gut-brain modules (GBMs) were additionally assessed for their differential abundance (C). Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 27/28 Page 27/28 Figure 8 The kefir-induced increased Lactobacillus reuteri strain has the potential to produce GABA. Spearman rank correlations between Lactobacillus reuteri abundances and levels of faecal 2-oxoglutarate, glutamate and succinate were performed (A). Faecal metabolite levels were quantified using chromatography–mass spectrometry (GC-MS). Flux Variability Analysis was used to assess whether L. reuteri can influence 2-oxoglutarate, glutamate and succinate levels (B). Figure 9 Figure 8 The kefir-induced increased Lactobacillus reuteri strain has the potential to produce GABA. Spearman rank correlations between Lactobacillus reuteri abundances and levels of faecal 2-oxoglutarate, glutamate and succinate were performed (A). Faecal metabolite levels were quantified using chromatography–mass spectrometry (GC-MS). Flux Variability Analysis was used to assess whether L. reuteri can influence 2-oxoglutarate, glutamate and succinate levels (B). Figure 9 Experimental design. Figure 6 Animals received three weeks of treatment lead in, where animals only received kefir or milk supplementation, after which they were assessed for their behavioural phenotype. Treatment groups consisted of: 1) No gavage control, 2) Milk gavage control, 3) Kefir gavage – Fr1, and 4) Kefir gavage – UK4 (n = 12/group). The order of behavioural tests was as following; Week 4: Marble burying test (MB), 3-Chamber social interaction test (3CT) and Elevate plus maze (EPM); Week 5: Open field test (OF) and Tail suspension test (TST); Week 6: Saccharin preference test (SPT); Week 7: Female urine sniffing test (FUST); Week 8: Stress-induced hyperthermia test (SIH); Week 9: Intestinal motility test (IM) and Faecal water content assessment (FWC): Week 9-12: Appetitive Y-maze; Week 13: Fear conditioning; Week 14: Forced swim test; Week 15: Euthanasia. Postmortem, ileal, caecal and faecal microbiota composition and function was investigated by shotgun sequencing and faecal metabolomics. Host immunity was assessed using flow cytometry and by quantification of cytokines. Finally, ileum and colonic serotonergic levels were quantified by high-performance liquid chromatography (HPLC). Figure 8 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementsKefirMvdW.docx SupplementsKefirMvdW.docx Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Page 28/28 Page 28/28
https://openalex.org/W2809274910	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Appendix_from_Prognostic_Value_of_Methylator_Phenotype_in_Stage_III_Colon_Cancer_Treated_with_Oxaliplatin-based_Adjuvant_Chemotherapy/22469858/1/files/39921344.pdf	Luxembourgish	null	Prognostic Value of Methylator Phenotype in Stage III Colon Cancer Treated with Oxaliplatin-based Adjuvant Chemotherapy	Clinical cancer research	2,018	cc-by	4,901	Denmark Lone Nørgård Petersen Mette Yilmaz (Aalborg Sygehus-Afsnit Syd, Aalborg); Svend Erik Nielsen (Hillerød Hospital, Hillerød); Lene Vestermark (Odense Universitets Hospital, Odense); Jim Larsen (Roskilde Amtssygehus, Roskilde) France Fédération Francophone de Cancérologie Digestive (FFCD), Jean François Seitz; Fédération Nationale des Centres de Lutte Contre le Cancer (UNICANCER), Marc Ychou; Fédération Nationale des Centres de Lutte Contre le Cancer Association Européenne de Recherche en Oncologie (AERO), Ayman Zawadi Mohamed-Ayman Zawadi (Centre Hospitalier Les Oudairies, La Roche sur Yon); Olivier Bouche (CHU de Reims, Hopital Robert Debre, Reims); Laurent Mineur (Institut Sainte Catherine, Avignon); Jaafar Bennouna-Louridi (CRLCC René Gauducheau, St Herblain); Louis Marie Dourthe, (Clinique Sainte Anne, Strasbourg); Marc Ychou (Centre Regional Val d'Aurelle Paul Lamarque, Montpellier); Eveline Boucher (CRLC Eugène Marquis, Rennes); Julien Taieb Appendix: Supplementary data PETACC-8 study investigators Country Group(s), Coordinator(s) Principle investigator(s) (Centre) Austria Austrian Breast and Colorectal cancer Study Group (ABCSG), Joseph Thaler Josef Thaler (Klinikum Kreuzschwestern Wels, Wels); Richard Greil (LKH Salzburg); Johannes Gaenzer (BKH Hall in Tirol, Hall in Tirol); Wolfgang Eisterer (University Klinik Innsbruck, Innsbruck); Joerg Tschmelitsch (KH Barmherzige Brüder St. Veit / Glan); Felix Keil (LKH Leoben, Leoben); Hellmut Samonigg (Landeskrankenhaus Graz Medizinische Universitätsklinik, Graz); August Zabernigg (BKH Kufstein, Kuftstein); Franz Schmid (LKH Bregenz, Bregenz); Günther Steger (Universitätsklinik für Innere Medizin I, Wein); Robert Steinacher (LKH Wolfsberg, LKH); Johannes Andel (LKH Steyr, Steyr); Björn Jagdt, (a.ö.Krankenhaus d. Barmherz. Schwest. Ried, Ried); Alois Lang (LKH Rankweil, Rankweil), Michael Fridrik (AKH Linz, Linz); Reinhold Függer (A.ö. Krankenhaus d. Elisabethinen Linz); Friedrich Hofbauer (LKH Oberpullendorf, Oberpullendorf); Ewald Woell (KH St. Vinzenz Zams, Zams); Dietmar Geissler (LKH Klagenfurt, Klagenfurt); Alfred Lenauer (KH Wiener Neustadt, Wiener Neustadt); Manfred Prager (A.ö. KH Oberwart, Oberwart) Appendix: Supplementary data PETACC-8 study investigators Country Group(s), Coordinator(s) Principle investigator(s) (Centre) Austria Austrian Breast and Colorectal cancer Study Group (ABCSG), Joseph Thaler Josef Thaler (Klinikum Kreuzschwestern Wels, Wels); Richard Greil (LKH Salzburg); Johannes Gaenzer (BKH Hall in Tirol, Hall in Tirol); Wolfgang Eisterer (University Klinik Innsbruck, Innsbruck); Joerg Tschmelitsch (KH Barmherzige Brüder St. Veit / Glan); Felix Keil (LKH Leoben, Leoben); Hellmut Samonigg (Landeskrankenhaus Graz Medizinische Universitätsklinik, Graz); August Zabernigg (BKH Kufstein, Kuftstein); Franz Schmid (LKH Bregenz, Bregenz); Günther Steger (Universitätsklinik für Innere Medizin I, Wein); Robert Steinacher (LKH Wolfsberg, LKH); Johannes Andel (LKH Steyr, Steyr); Björn Jagdt, (a.ö.Krankenhaus d. Barmherz. Schwest. Ried, Ried); Alois Lang (LKH Rankweil, Rankweil), Michael Fridrik (AKH Linz, Linz); Reinhold Függer (A.ö. Krankenhaus d. Elisabethinen Linz); Friedrich Hofbauer (LKH Oberpullendorf, Oberpullendorf); Ewald Woell (KH St. Vinzenz Zams, Zams); Dietmar Geissler (LKH Klagenfurt, Klagenfurt); Alfred Lenauer (KH Wiener Neustadt, Wiener Neustadt); Manfred Prager (A.ö. KH Oberwart, Oberwart) Appendix: Supplementary data *PETACC-8 study investigators Josef Thaler (Klinikum Kreuzschwestern Wels, Wels); Richard Greil (LKH Salzburg); Johannes Günther Steger (Universitätsklinik für Innere Medizin I, Wein); Robert Steinacher (LKH Wolfsberg, LKH); Johannes Andel (LKH Steyr, Steyr); Björn Jagdt, l l l l a.ö.Krankenhaus d. Barmherz. Schwest. Ried, Ried); Alois Lang (LKH Rankweil, Rankweil), Michael Fridrik (AKH Linz, Linz); Reinhold Függer (A.ö. Krankenhaus d. Elisabethinen Linz); Friedrich Hofbauer (LKH Oberpullendorf, Oberpullendorf); Ewald Woell (KH St. Vinzenz Zams, Zams); Dietmar Geissler (LKH Klagenfurt, Klagenfurt); Alfred Lenauer (KH Wiener Neustadt, Wiener Neustadt); Manfred Prager (A.ö. KH Oberwart, Oberwart) Belgium g Belgian Group of Digestive Oncology (BGDO), Jean-Luc Van Laethem and Eric Van Cutsem Geert D'Haens (Imelda Ziekenhuis, Bonheiden); Gauthier Demolin (Clinique St-Jospeh, Liège); Joseph Kerger (Cliniques Universitaires de Mont-Godinne U.C.L. Yvoir); Guido Deboever (A. Z. St-Jozef, Oostende); Gilbert Ghillebert (Heilig Hart Ziekenhuis, Roeselare); Marc Polus (C. H. U. Sart-Tilman, Liège); Eric Van Cutsem (University Hospitals , Leuven); Hassan Rezaie Kalantari (C. H. Peltzer-La Tourelle, Verviers); Thierry Delaunoit (Centre Hospitalier de Jolimont-Lobbes, La Louvière); Jean Charles Goeminne (Clinique et Maternité Sainte-Elisabeth, Namur); Marc Peeters (Universitair Ziekenhuis Gent, Gent); Philippe Vergauwe (AZ Groeninge-Campus Kennedylaan, Kortrijk); Ghislain Houbiers (Centre médical de L'Avenue, Liege); Yves Humblet (Cliniques Universitaires Saint-Luc, Brussels); Jos Janssens (St-Elisabeth Ziekenhuis, Turnhout); Dirk Schrijvers (ZNA Middelheim, Antwerpen); Erik Vanderstraeten (AZ Maria Middelares, Gent); Jean-Luc Van Laethem (ULB Hôpital Erasme, Brussels); Jan Vermorken (UZ Antwerpen, Edegem); Daniel Van Daele (Clinique Nôtre-Dame de Grâce, Gosselies); Michel Ferrante (AZ Sint-Maarten, Mechelen,); Frederic Forget (Centre Hospitalier de l'Ardenne, Libramont); Alain Hendlisz (Jules Bordetinstituut, Brussels) Denmark Lone Nørgård Petersen Mette Yilmaz (Aalborg Sygehus-Afsnit Syd, Aalborg); Svend Erik Nielsen (Hillerød Hospital, Hillerød); Lene Vestermark (Odense Universitets Hospital, Odense); Jim Larsen (Roskilde Amtssygehus, Roskilde) France Fédération Francophone de Cancérologie Digestive (FFCD), Jean François Seitz; Fédération Nationale des Centres de Lutte Contre le Cancer (UNICANCER), Marc Ychou; Fédération Nationale des Centres de Lutte Contre le Cancer Association Européenne de Recherche en Oncologie (AERO), Ayman Zawadi Mohamed-Ayman Zawadi (Centre Hospitalier Les Oudairies, La Roche sur Yon); Olivier Bouche (CHU de Reims, Hopital Robert Debre, Reims); Laurent Mineur (Institut Sainte Catherine, Avignon); Jaafar Bennouna-Louridi (CRLCC René Gauducheau, St Herblain); Louis Marie Dourthe, (Clinique Sainte Anne, Strasbourg); Marc Ychou (Centre Regional Val d'Aurelle Paul Lamarque, Montpellier); Eveline Boucher (CRLC Eugène Marquis, Rennes); Julien Taieb Belgian Group of Digestive Oncology (BGDO), Jean-Luc Van Laethem and Eric Van Cutsem Geert D'Haens (Imelda Ziekenhuis, Bonheiden); Gauthier Demolin (Clinique St-Jospeh, Liège); Joseph Kerger (Cliniques Universitaires de Mont-Godinne (Jules Bordetinstituut, Brussels) Lone Nørgård Petersen Fédération Francophone de Cancérologie Digestive (FFCD), Jean François Seitz; Fédération Nationale des Centres de Lutte Contre le Cancer (UNICANCER), Marc Ychou; Fédération Nationale des Centres de Lutte Contre le Cancer Association Européenne de Recherche en Oncologie (AERO), Ayman Zawadi Mohamed-Ayman Zawadi (Centre Hospitalier Les Oudairies, La Roche sur Yon); Olivier Bouche (CHU de Reims, Hopital Robert Debre, Reims); Laurent Mineur (Institut Sainte Catherine, Avignon); Jaafar Bennouna-Louridi (CRLCC René Gauducheau, St Herblain); Louis Marie Dourthe, (Clinique Sainte Anne, Strasbourg); Marc Ychou (Centre Regional Val d'Aurelle Paul Lamarque, Montpellier); Eveline Boucher (CRLC Eugène Marquis, Rennes); Julien Taieb Nationale des Centres de Lutte Contre le Cancer (Hôpital Européen Georges Pompidou, Paris); Denis Pezet (CHU Estaing, Clermond Ferrand); Francoise Desseigne (Centre Leon Berard, Lyon); Michel Ducreux (Institut Gustave Roussy Villejuif); Patrick Texereau (Hopital Layne, Mont-de-Marsan); Laurent Miglianico (Centre Hospitalier Privé Saint- Grégoire g p g (Rennes), Saint-Grégoire); Philippe Rougier (Hôpital Européen Georges Pompidou, Paris); Serge Fratte (Centre Hospitalier de Belfort-Montbeliard, Belfort); Fratte (Centre Hospitalier de Belfort-Montbeliard, Belfort); Charles-Briac Levache (Polyclinique Francheville, Perigueux); Yacine Merrouche, (Institut de Cancerologie de la Loire, Saint-Priest-En-Jarez); Stephen Ellis (Clinique Saint Pierre, Perpignan); Christophe Locher (CH Meaux, Meaux); Jean-Francois Ramee (C C h i d Si N ) Cl i G i (Clinique Saint Pierre, Perpignan); Christophe Locher (CH Meaux, Meaux); Jean-Francois Ramee (Centre Catherine de Sienne, Nantes); Claire Garnier (Clinique Saint Pierre, Perpignan); Christophe Locher (CH Meaux, Meaux); Jean-Francois Ramee (Centre Catherine de Sienne, Nantes); Claire Garnier Centre Catherine de Sienne, Nantes); Claire Garnier (UMGEC-Institut Daniel Hollard, Grenoble); Frederic Viret (Institut Paoli Calmettes, Marseille); UMGEC-Institut Daniel Hollard, Grenoble); Frederic Bruno Chauffert (Centre Georges François Leclerc, Dijon); Isabelle Cojean-Zelek (Hopital Croix Saint Simon, Paris); Pierre Michel (Hopital Charles Nicolle, Rouen); Cedric Lecaille (Polyclinique Bordeaux Nord Aquitaine, Bordeaux); Christian Borel (CLCC Paul Strauss, Strasbourg); Jean-Francois Seitz (CHU de la Timone Marseille); Denis Smith (Groupe Hospitalier Aquitaine, Bordeaux); Christian Borel (CLCC Paul Strauss, Strasbourg); Jean-Francois Seitz (CHU de Timone, Marseille); Denis Smith (Groupe Hospitalier Saint-Andre, Bordeaux); Catherine Lombard-Bohas (Hospices Civils de Lyon, Hôpital Edouard Herriot, Lyon); Thierry Andre (Groupe Hospitalier Pitie- Saint-Andre, Bordeaux); Catherine Lombard-Bohas (Hospices Civils de Lyon, Hôpital Edouard p y p Herriot, Lyon); Thierry Andre (Groupe Hospitalier Pitie- Herriot, Lyon); Thierry Andre (Groupe Hospitalier Pitie- Salpetriere, Paris); Jean-Marc Gornet, (Hopital Saint Louis, Paris); Francine Fein (CHU de Besancon, H it l J Mi j B ) M i A d C l Salpetriere, Paris); Jean-Marc Gornet, (Hopital Saint Louis, Paris); Francine Fein (CHU de Besancon, Hopital Jean Minjoz, Besançon); Marie-Aude Coulon- petriere, Paris); Jean-Marc Gornet, (Hopital Saint Lou Salpetriere, Paris); Jean-Marc Gornet, (Hopital Saint Louis, Paris); Francine Fein (CHU de Besancon, Hopital Jean Minjoz, Besançon); Marie-Aude Coulon- Hopital Jean Minjoz, Besançon); Marie-Aude Coulon- Hopital Jean Minjoz, Besançon); Marie-Aude Coulon- Sfairi (Centre Hospitalier du Mans, Le Mans); Marie-Christine Kaminsky (Centre Alexis Vautrin Brabois, Vandoeuvre les Nancy); Jean-Paul Lagasse (CHR d' Sfairi (Centre Hospitalier du Mans, Le Mans); Marie-Christine Kaminsky (Centre Alexis Vautrin Brabois, Vandoeuvre les Nancy); Jean-Paul Lagasse (CHR d' andoeuvre les Nancy); Jean-Paul Lagasse (CHR d' Orléans La Source, Orléans); Dominique Luet (CRLCC Paul Papin, Angiers); Pierre-Luc Etienne (Clinique Armoricaine de Radiologie, Saint-Brieux); Mohamed Gasmi (Hopital Nord de Marseille, Marseille); Andre Vanoli (Clinique Ste-Marie, Chalon Sur Saone); Suzanne Nguyen (Centre Hospitalier de Beauvais, Suzanne Nguyen (Centre Hospitalier de Beauvais, g y ( p , Beauvais); Thomas Aparicio (Hôpital Bichat, Paris); Hervé Perrier (Hopital Saint Joseph, Marseille); g y p Beauvais); Thomas Aparicio (Hôpital Bichat, Paris); Hervé Perrier (Hopital Saint Joseph, Marseille); Noel Stremsdoerfer (Hopital Pierre Oudot, Bourgoin- Beauvais); Thomas Aparicio (Hôpital Bichat, Paris); ) p ( p ) Noel Stremsdoerfer (Hopital Pierre Oudot, Bourgoin- ( p , g Jallieux); Philippe Laplaige (Clinique Saint Côme, La Chaussée St Victor); Dominique Arsene (CHU de Caen Caen); Dominique Auby (Hôpital Robert Boulin p g Jallieux); Philippe Laplaige (Clinique Saint Côme, La Chaussée St Victor); Dominique Arsene (CHU allieux); Philippe Laplaige (Clinique Saint Côme, La Chaussée St Victor); Dominique Arsene (CHU de Caen, Caen); Dominique Auby (Hôpital Robert Boulin, de Caen, Caen); Dominique Auby (Hôpital Robert Boulin, de Caen, Caen); Dominique Auby (Hôpital Robert Boulin, Libourne); Laurent Bedenne (Hopital du Bocage, Dijon); Romain Coriat (Hopital Cochin, Paris); Libourne); Laurent Bedenne (Hopital du Bocage, Dijon); Romain Coriat (Hopital Cochin, Paris); Bernard Denis (Hopital Pasteur, Colmar); Patrick Geoffroy Bernard Denis (Hopital Pasteur, Colmar); Patrick Geoffroy Bernard Denis (Hopital Pasteur, Colmar); Patrick Geoffroy inique Saint-Vincent, Epernay); Gilles Piot (Clinique (CHU de Bordeaux, Bordeaux); Gilbert Bordes (Centre Hospitalier de Digne Les Bains, Digne Les Bains); Gael Deplanque (Hopital Saint Joseph, Paris); ospitalier de Digne Les Bains, Digne Les Bains); Gae Hospitalier de Digne Les Bains, Digne Les Bains); Gael Deplanque (Ho Olivier Dupuis (Clinique Victor Hugo, Le Mans); Frederic Fruge (CHU Poitiers-Hôpital la Milétrie, Poitiers); Rosine Guimbaud (Hopital Purpan,Toulouse); Fruge (CHU Poitiers-Hôpital la Milétrie, Poitiers); Rosine Guimbaud (Hopital Purpan,Toulouse); Thierry Lecomte (Hopital Trousseau - CHU de Tours, Tours); Fruge (CHU Poitiers-Hôpital la Milétrie, Poitiers); Rosine Guimbaud (Hopital Purp y p Gérard Lledo (Hopital Prive Jean Mermoz, Lyon); Iradej Sobhani (Hôpital Henri Mondor (Gastro- y p Gérard Lledo (Hopital Prive Jean Mermoz, Lyon); Iradej Sobhani (Hôpital H Gérard Lledo (Hopital Prive Jean Mermoz, Lyon); Iradej Sobhani (Hôpital Henri Mondor (Gastro- Entérologie), Créteil); Amani Asnacios (Hopital Antoine Entérologie), Créteil); Amani Asnacios (Hopital Antoine Beclere, Clamart); Ahmed Azzedine (Boulat, Michel, Avignon); Christophe Desauw (Hopital Saint Vincent de Paul GHICL Lille Lille); Marie Pierre Galais Beclere, Clamart); Ahmed Azzedine (Boulat, Michel, Avignon); Christophe Desauw (Hopital Saint Beclere, Clamart); Ahmed Azzedine (Boulat, Michel, Avignon); Christophe Beclere, Clamart); Ahmed Azzedine (Boulat, Michel, Avignon); Christophe Desauw (Hopital Saint Vincent de Paul-GHICL Lille, Lille); Marie-Pierre Galais Vincent de Paul-GHICL Lille, Lille); Marie-Pierre Galais Vincent de Paul-GHICL Lille, Lille); Marie-Pierre Galais (Centre François Baclesse, Caen); Dany Gargot (Centre Hospitalier de Blois, Blois); You-Heng Lam (Hopital de Cholet, Cholet); Abakar Abakar-Mahamat (Hopital l'Archet II, Nice); Jean-Francois Berdah (Clinique Sainte Marguerite, Hyères); Sylviane Catteau (Hopital Duchenne, Boulogne sur Mer); Marie- Christine Clavero-Fabri (Clinique Hartman, Levallois Perret); Jean-Francois Codoul (Hopital de D i D i ) J L i L (Hô i l H Draguignan, Draguignan); Jean-Louis Legoux (Hôpital Haut Levêque, Pessac); Denis Goldfain (Centre Hospitalier General de Dreux, Dreux); Pierre Guichard (Hôpital de la Croix Rousse (Gastroentérologie), Lyon); Jocelyne Provencal (Centre Hospitalier d'Annecy, Pringy); Bruno Vedrenne (Hopital E. Lone Nørgård Petersen Marien GmbH, Amberg); Stefan Fuxius (Onkologische Praxis, Heidelberg); Matthias Groschek (Haematologisch-Onkologische Praxis Wuerselen, Wuelselen); Johannes Meiler, Tanja Trarbach (Universitaetsklinikum Essen, Essen); Jacqueline Rauh (Gemeinschaftspraxis Ardeystrasse, Witten); Nicolas Ziegenhagen, Albrecht Kretzschmar (Helios- Kliniken Berlin, Berlin); Ullrich Graeven (Kliniken Maria Hilf GmbH, Moenchengladbach); Arnd Nusch (Onkologische Praxis, Velbert); Goetz von Wichert (Universitaetsklinikum, Ulm); Ralf-Dieter Hofheinz (Klinikum der Stadt Mannheim, Mannheim); Gerhard Kleber (Ostalb-Klinikum Aalen, Aalen); Karl-Heinz Schmidt (Johanniter-Krankenhaus Rheinhausen, Duisberg); Ursula Vehling-Kaiser (Gemeinschaftspraxis, Landshut); Claudia Baum, Jochen Schuette (Marien Hospital Duesseldorf GmbH, Duesseldorf); Georg Martin Haag (Universitaetsklinikum Heidelberg, Heidelberg); Wilhelm Holtkamp (Ammerland-Klinik GmbH, Westerstede); Jochen Potenberg (Evangelisches Waldkrankenhaus, Berlin); Tobias Reiber (Praxis fuer Haematologie/Onkologie, Freiberg); Georg Schliesser (Praxis fuer Haematologie und Onkologie, Giessen); Hans-Joachim Schmoll (Martin-Luther- Universitaet Halle-Wittenberg, Halle); Wolfgang Schneider-Kappus (Arztpraxis, Ulm); Wolfgang Abenhardt (Onkologie Praxis im Elisenhof, Muenchen); Claudio Denzlinger (Marienhospital Stuttgart, Stuggart); Jan Henning, Bartscht Marxsen (Universitaetsklinikum Schleswig-Holstein, Luebeck); Hans Guenter Derigs (Staedtische Kliniken Frankfurt-Hoechst, Frankfurt); Helmut Lambertz (Klinikum Garmisch-Partenkirchen, Garmisch-Partenkirchen); Ingulf Becker-Boost (MVZ Arbeitsgemeinschaft Internistische Onkologie (AIO), Gunnar Folprecht Axel Matzdorff (Caritasklinik St. Theresia, Saarbruecken); Michael Konrad Stahl (Kliniken Essen- Mitte–PS, Essen); Wolfgang Schepp (KKH Muenchen Bogenhausen, Muenchen); Martin Burk (Klinikum der Stadt Hanau, Hanau); Lothar Mueller (Onkologische Schwerpunktpraxis, Leer); Gunnar Folprecht (Universitaetsklinikum Carl Gustav Carus, Dresden); Michael Geissler (Staedtische Kliniken Esslingen, Esslingen); Luisa Mantovani-Loeffler (Staedtisches Klinikum St. Georg Leipzig, Leipzig); Thomas Hoehler (Prosper-Hospital, Recklinghausen); Walter Asperger (Krankenhaus St. Elisabeth und St. Barbara, Halle); Hendrik Kroening (Gemeinschaftspraxis, Magdeburg); Ludwig Fischer von Weikersthal (MVZ Gesundheitszentrum St. Lone Nørgård Petersen Muller, d'Annecy, Pringy); Bruno Vedrenne (Hopital E. Muller, Mulhouse); Catherine Brezault-Bonnet (Hopital de Rambouillet, Rambouillet); Denis Cleau (Centre Mulhouse); Catherine Brezault-Bonnet (Hopital de Rambouillet, Rambouillet); Denis Cleau (Centre Hospitalier Intercommunal de Vesoul, Vesoul); Jean-Paul Desir (Clinique Pole Sante Republique Clermont-Ferrand); David Fallik (Polyclinic Jeanne d’Arc Mulhouse); Catherine Brezault-Bonnet (Hopital de Rambouillet, Rambouillet); Denis Cleau (Centre Hospitalier Intercommunal de Vesoul, Vesoul); Jean-Paul Desir (Clinique Pole Sante Republique, Clermont-Ferrand); David Fallik (Polyclinic Jeanne d’Arc Gien); Bruno Garcia (Clinique de Courlancy, Reims); Marie- Hélène Gaspard (Clinique Claude Bernard, Albi); Dominique Genet (Centre de Cancerologie Chenieux, Limoges); Johannes Hartwig (Infirmerie Protestante de L C l i t C i ) Y K l (C t H it li S l t t S l t t) T M t i k Hélène Gaspard (Clinique Claude Bernard, Albi); Dominique Genet (Centre de Cancerologie Lyon, Caluire et Cuire,); Yves Krummel (Centre Hospitalier Selestat, Selestat); Tamara Matysiak Budnik (CH de Nantes Hotel Dieu, Nantes); Vanessa Palascak- Juif (Hopital de Hautepierre, Strasbourg); Harizo Randrianarivelo (Centre Frederic Joliot, Rouen); Yves Rinaldi (Clinique Clairval, Marseille); Albert Aleba Yves Rinaldi (Clinique Clairval, Marseille); Albert Aleba (Centre Hospitalier de Niort, Hopital Georges Renon, Niort); Ariane Darut-Jouve (Centre d'oncologie et Radiotherapie, Dijon); Aimery de Gramont (Hopital Saint Antoine, Paris); Herve Hamon (Centre Hospitalier de Valence, Valence); Frederic Wendehenne (Clinique Charcot, Sainte-Foy-les-Lyon) G (Centre Hospitalier de Niort, Hopital Georges Renon, Niort); Ariane Darut-Jouve (Centre d'oncologie et Radiotherapie, Dijon); Aimery de Gramont (Hopital Saint Antoine, Paris); Herve Hamon (Centre Hospitalier de Valence, Valence); Frederic Wendehenne (Clinique Charcot, Sainte-Foy-les-Lyon) y Arbeitsgemeinschaft Internistische Onkologie (AIO), Gunnar Folprecht Axel Matzdorff (Caritasklinik St. Theresia, Saarbruecken); Michael Konrad Stahl (Kliniken Essen- Mitte–PS, Essen); Wolfgang Schepp (KKH Muenchen Bogenhausen, Muenchen); Martin Burk (Klinikum der Stadt Hanau, Hanau); Lothar Mueller (Onkologische Schwerpunktpraxis, Leer); Gunnar Folprecht (Universitaetsklinikum Carl Gustav Carus, Dresden); Michael Geissler (Staedtische Kliniken Esslingen, Esslingen); Luisa Mantovani-Loeffler (Staedtisches Klinikum St. Georg Leipzig, Leipzig); Thomas Hoehler (Prosper-Hospital, Recklinghausen); Walter Asperger (Krankenhaus St. Elisabeth und St. Barbara, Halle); Hendrik Kroening (Gemeinschaftspraxis, Magdeburg); Ludwig Fischer von Weikersthal (MVZ Gesundheitszentrum St. Lone Nørgård Petersen Marien GmbH, Amberg); Stefan Fuxius (Onkologische Praxis, Heidelberg); Matthias Groschek (Haematologisch-Onkologische Praxis Wuerselen, Wuelselen); Johannes Meiler, Tanja Trarbach (Universitaetsklinikum Essen, Essen); Jacqueline Rauh (Gemeinschaftspraxis Ardeystrasse, Witten); Nicolas Ziegenhagen, Albrecht Kretzschmar (Helios- Kliniken Berlin, Berlin); Ullrich Graeven (Kliniken Maria Hilf GmbH, Moenchengladbach); Arnd Nusch (Onkologische Praxis, Velbert); Goetz von Wichert (Universitaetsklinikum, Ulm); Ralf-Dieter Hofheinz (Klinikum der Stadt Mannheim, Mannheim); Gerhard Kleber (Ostalb-Klinikum Aalen, Aalen); Karl-Heinz Schmidt (Johanniter-Krankenhaus Rheinhausen, Duisberg); Ursula Vehling-Kaiser (Gemeinschaftspraxis, Landshut); Claudia Baum, Jochen Schuette (Marien Hospital Duesseldorf GmbH Duesseldorf); Georg Martin Haag (Universitaetsklinikum Heidelberg (Universitaetsklinikum Essen, Essen); Jacqueline Rauh (Gemeinschaftspraxis Ardeystrasse, Witten); Nicolas Ziegenhagen, Albrecht Kretzschmar (Helios- (Universitaetsklinikum, Ulm); Ralf-Dieter Hofheinz (Klinikum der Stadt Mannheim, Mannheim); Schmidt (Johanniter-Krankenhaus Rheinhausen, Duisberg); Ursula Vehling-Kais (Gemeinschaftspraxis, Landshut); Claudia Baum, Jochen Schuette (Marien Hospital Duesseldorf GmbH, Duesseldorf); Georg Martin Haag (Universitaetsklinikum Heidelberg, Heidelberg); Wilhelm Holtkamp (Ammerland-Klinik GmbH, Heidelberg); Wilhelm Holtkamp (Ammerland-Klinik GmbH, Westerstede); Jochen Potenberg (Evangelisches Waldkrankenhaus, Berlin); Tobias Reiber (Praxis fuer g g Haematologie/Onkologie, Freiberg); Georg Schliesser Praxis fuer Haematologie und Onkologie, Giessen); H Universitaet Halle-Wittenberg, Halle); Wolfgang Schneider-Kappus (Arztpraxis, Ulm); Wolfgang Abenhardt (Onkologie Praxis im Elisenhof, Muenchen); Claudio Denzlinger (Marienhospital Stuttgart, Stuggart); Jan Henning, Bartscht Marxsen (Universitaetsklinikum Schleswig-Holstein, Luebeck); Hans Guenter Derigs Lambertz (Klinikum Garmisch-Partenkirchen, Garmisch-Partenkirchen); Ingulf Becker-Boost (MVZ Duisburg Sued GmbH, Duisburg); Karel Caca (Klinikum Ludwigsburg, Ludwigsburg); Christian Constantin (Kliniken Lippe-Lemgo GmbH, Lemgo); Thomas Decker (Gemeinschaftspraxis, Ravensburg); Henning Ludwigsburg, Ludwigsburg); Christian Constantin (Kliniken Lippe-Lemgo GmbH, Lemgo); Thom Ludwigsburg, Ludwigsburg); Christian Constantin (Kliniken Lippe-Lemgo GmbH, Lemgo); Thomas Decker (Gemeinschaftspraxis, Ravensburg); Henning dwigsburg, Ludwigsburg); Christian Constantin (Klin ecker (Gemeinschaftspraxis, Ravensburg); Henning Eschenburg (Internistische Gemeinschaftspraxis, Gu Eschenburg (Internistische Gemeinschaftspraxis, Guestrow); Sigrun Gabius (Gemeinschaftspraxis, Rosenheim); Holger Hebart (Klinikum Schwaebisch Rosenheim); Holger Hebart (Klinikum Schwaebisch Italy Senese Policlinico Santa Maria alle Scotte Siena); Massimo Aglietta (Istituto per Stefania Nobili (A.O. Universitaria Careggi, Firenze); Roberto Petrioli (A.O.U. Senese Policlinico Santa Maria alle Scotte, Siena); Massimo Aglietta (Istituto per ( gg , ); Santa Maria alle Scotte, Siena); Massimo Aglietta (Istituto per g p la Ricerca e la Cura del Cancro, Candiolo); Oscar Alabiso (Ospedale Maggiore della Carità, Novara); Federico Capuzzo, Alfredo Falcone (Presidio Ospedaliero erico Capuzzo, Alfredo Falcone (Presidio Ospedalie p , ( p di Livorno, Livorno); Domenico Cristi Corsi (Ospedale "S. Giov. Calibita" Fatebenefratelli, Roma); Roberto Labianca (Ospedali Riuniti di Berga o Berga o); p p di Livorno, Livorno); Domenico Cristi Corsi (Ospedale "S. Giov. Calibita" Fatebenefratelli, Roma); Roberto Labianca (Ospedali Riuniti di Bergamo, Bergamo); p g g Stefania Salvagni (Azienda Ospedaliera di Parma, Parma); Silvana Chiara (Istituto nazionale per la ricerca sul cancro, Genova) Libero Ciuffreda (A.O. San Stefania Salvagni (Azienda Ospedaliera di Parma, Parma); Silvana Chiara (Istituto nazionale per la i l G ) Lib Ci ff d (A O S cerca sul cancro, Genova) Libero Ciuffreda (A.O. San Giovanni Battista, Torino); Francesco Ferraù (Azienda Ospedaliera San Vincenzo, Taormina); Francesco Giuliani (Istituto Oncologico Giovanni Paolo II IRCCS, Francesco Giuliani (Istituto Oncologico Giovanni Paolo II IRCCS, Bari); Sara Lonardi (IOV-Istituto Oncologico Veneto IRCCS, Padova); Nicola Gebbia, Antonio Russo (Azienda Ospedaliero Universitaria Policlinico Paolo Giaccone, Palermo); Giovanni Mantovani, Elena Massa (Università di Cagliari-Presidio Policlinico Monserrato, Monserrato) Italy Mini;Gruppo Oncologico Nord Ovest (GONO), Alfredo Falcone; Gruppo Oncologico Italiano di Ricerca Clinica (GOIRC), Corrado Boni Evaristo Maiello (IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo); Luciano Latini (Ospedale di Macerata, Macerata); Alberto Zaniboni Evaristo Maiello (IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo); Luciano Lati (O d l di M M ) Alb Z ib i Evaristo Maiello (IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo); Luciano Lati (Ospedale di Macerata, Macerata); Alberto Zaniboni (Fondazione Poliambulanza Istituto Ospedaliero, Brescia); Dino Amadori (Istituto Scientifico (Fondazione Poliambulanza Istituto Ospedaliero, Brescia); Dino Amadori (Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola); agnolo per lo Studio e la Cura dei Tumori, Meldola); g p Giuseppe Aprile (Az. Policlinico Universitario di Udine, Udine); Sandro Barni (Azienda Ospedaliera Treviglio Caravaggio Treviglio); Rodolfo Mattioli g p Giuseppe Aprile (Az. Italy Policlinico Universitario di Udine, Udine); Sandro Barni (Azienda Ospedaliera Treviglio-Caravaggio, Treviglio); Rodolfo Mattioli (Ospedale Santa Croce ASUR 3, Fano); Andrea Mar (Ospedale Santa Croce ASUR 3, Fano); Andrea Martoni, Francesca di Fabio (Azienda Ospedaliera (Ospedale Santa Croce ASUR 3, Fano); Andrea Martoni, Francesca di Fabio (A Universitaria Policlinico Sant'Orsola Ma, Bologna); Rodolfo Passalacqua (Azienda Ospedaliera Istituti Ospitalieri di Cremona, Cremona); Mario Nicolini, Enzo P i i (O d l C i C tt li ) C l R bbi E i Passalacqua (Azienda Ospedaliera Istituti Ospitalieri di Cremona, Cr Passalacqua (Azienda Ospedaliera Istituti Ospitalieri di Cremona, Cremona); Mario Nicolini, Enzo Pasquini (Ospedale Cervesi, Cattolica); Carla Rabbi, Enrico Passalacqua (Azienda Ospedaliera Istituti Ospitalieri di Cremona, Cremona); Mario Nicolini, Enzo Pasquini (Ospedale Cervesi, Cattolica); Carla Rabbi, Enrico uini (Ospedale Cervesi, Cattolica); Carla Rabbi, Enric Aitini (Azienda Ospedaliera Carlo Poma, Mantova); Alberto Ravaioli, Emiliano Tamburini (Ospedale Aitini (Azienda Ospedaliera Carlo Poma, Mantova); Alberto Ravaioli, Emiliano Aitini (Azienda Ospedaliera Carlo Poma, Mantova); Alberto Ravaioli, Emiliano Tamburini (Ospedale degli Infermi, Rimini); Carlo Barone (Policlinico Aitini (Azienda Ospedaliera Carlo Poma, Mantova) degli Infermi, Rimini); Carlo Barone (Policlinico Universitario Agostino Gemelli, Roma); Guido Biasco (Ospedale Marcello Malpighi Policlinico Universitario Agostino Gemelli, Roma); Guido Biasco (Ospedale Marcello Malp Universitario Agostino Gemelli, Roma); Guido Bia Sant'Orsola, Bologna); Stefano Tamberi, Angelo Gambi g g (Ospedale Degli Infermi, Faenza); Claudio Verusio (Ospedale di Saronno, Saronno); Marina Marzola, (Ospedale Degli Infermi, Faenza); Claudio Verusio (Ospedale di Saronno, Saronno); Marina Marzola, Gi i L lli (A i d O d li U i it i (Ospedale Degli Infermi, Faenza); Claudio Verusio (Ospedale di Saronno, Saronno); Marina Marzola, Giorgio Lelli (Azienda Ospedaliera Universitaria Giorgio Lelli (Azienda Ospedaliera Universitaria Arcispedale Sant'Anna, Cona (Ferrara)); Corrado Boni (A.O. Arcispedale Santa Maria Nuova, Reggio Emilia); Stefano Cascinu (Azienda Ospedaliero Arcispedale Sant'Anna, Cona (Ferrara)); Corrado Emilia); Stefano Cascinu (Azienda Ospedaliero Universitaria Ospedali Riuniti, Ancona); Paolo Bidoli, Massimo Vaghi (Azienda Ospedaliera San Gerardo, Monza); Giorgio Cruciani (Ospedale Umberto I, Universitaria Ospedali Riuniti, Ancona); Paolo Bidoli, Massimo Vaghi (Azienda Ospedaliera San Gerardo Monza); Giorgio Cruciani (Ospedale Umberto I Universitaria Ospedali Riuniti, Ancona); Paolo Bidoli, Massimo Vaghi (Azienda Ospedaliera San Gerardo, Monza); Giorgio Cruciani (Ospedale Umberto I, Monza); Giorgio Cruciani (Ospedale Umberto I, Luggo); Francesco Di Costanzo (Azienda Ospedaliera Universitaria Careggi, Firenze); Alberto Sobrero (Azienda Ospedaliera San Martino, Genoa); Enrico Mini, nda Ospedaliera San Martino, Genoa); Enrico Mini, Stefania Nobili (A.O. Universitaria Careggi, Firenze); Roberto Petrioli (A.O.U. g Gruppo Cooperativo do Cancro Digestivo da Associação Portuguesa de Investigação Oncológica (GCCD, APIO), Evaristo Sanches Evaristo Sanches (Instituto Português de Oncologia do Porto Francisco Gentil, Porto); Juan Carlos Mellidez (Hospital Distrital de Aveiro, Aveiro); Pedro Santos Evaristo Sanches (Instituto Português de Oncologia do Porto Francisco Gentil, Porto); Juan Carlos Mellidez (Hospital Distrital de Aveiro, Aveiro); Pedro Santos (Hospital São Sebastião, EPE, Santa Maria da Feira); Joao Freire (Instituto Portugues de Oncologia, Lison); Cristina Sarmento (Hospital de Sao Joao, Porto); Luis Costa (Hospital de Santa Maria, Lisbon); Antonio Moreira Pinto (Hospital Geral de Santo Antonio, Porto); Sergio Barroso (Hospital Distrital de Beja, Beja); Jorge Espirito Santo (Hospital do Barreiro, Barreiro); Fátima Guedes (Hospital Distrital Figueira da Foz, EPE, Figueira da Foz); Amélia Monteiro (Hospital São Teotónio, EPE, Viseu); Anabela Sa (Hospitais da Universidade de Coimbra, Coimbra); Irene Furtado (Hospital Distrital de Faro, Faro) S i Evaristo Sanches (Instituto Português de Oncologia do Porto Francisco Gentil, Porto); Juan Carlos Mellidez (Hospital Distrital de Aveiro, Aveiro); Pedro Santos (Hospital São Sebastião, EPE, Santa Maria da Feira); Joao Freire (Instituto Portugues de Oncologia, Lison); Cristina Sarmento (Hospital de Sao Joao, Porto); Luis Costa (Hospital de Santa Maria, Lisbon); Antonio Moreira Pinto (Hospital Geral de Santo Antonio, Porto); Sergio Barroso (Hospital Distrital de Beja, Beja); Jorge Espirito Santo (Hospital do Barreiro, Barreiro); Fátima Guedes (Hospital Distrital Figueira da Foz, EPE, Figueira da Foz); Amélia Monteiro (Hospital São Teotónio, EPE, Viseu); Anabela Sa (Hospitais da Universidade de Coimbra, Coimbra); Irene Furtado (Hospital Distrital de Faro, Faro) S i Evaristo Sanches (Instituto Português de Oncologia do Porto Francisco Gentil, Porto); Juan Carlos Mellidez (Hospital Distrital de Aveiro, Aveiro); Pedro Santos (Hospital São Sebastião, EPE, Santa Maria da Feira); Joao Freire (Instituto Portugues de Oncologia, Lison); Cristina Sarmento (Hospital de Sao Joao, Porto); Luis Costa (Hospital de Santa Maria, Lisbon); Antonio Moreira Pinto (Hospital Geral de Santo Antonio, Porto); Sergio Barroso (Hospital Distrital de Beja, Beja); Jorge Espirito Santo (Hospital do Barreiro, Barreiro); Fátima Guedes (Hospital Distrital Figueira da Foz, EPE, Figueira da Foz); Amélia Monteiro (Hospital São Teotónio, EPE, Viseu); Anabela Sa (Hospitais da Universidade de Coimbra, Coimbra); Irene Furtado (Hospital Distrital de Faro, Faro) S i Lison); Cristina Sarmento (Hospital de Sao Joao, Porto); Luis Costa (Hospital de Santa Maria, Lisbon); Antonio Moreira Pinto (Hospital Geral de Santo Antonio, Porto); Sergio Barroso (Hospital Distrital de Beja, Beja); Jorge Espirito Santo (Hospital do Barreiro, Barreiro); Fátima Guedes (Hospital Distrital Figueira da Foz, EPE, Figueira da Foz); Amélia Monteiro (Hospital São Teotónio, EPE, Viseu); Anabela Sa (Hospitais da Universidade de Coimbra, Coimbra); Irene Furtado (Hospital Distrital de Faro, Faro) S i Portugal Portugal Gruppo Cooperativo do Cancro Digestivo da Associação Portuguesa de Investigação Oncológica (GCCD, APIO), Evaristo Sanches Evaristo Sanches (Instituto Português de Oncologia do Porto Francisco Gentil, Porto); Juan Carlos Mellidez (Hospital Distrital de Aveiro, Aveiro); Pedro Santos (Hospital São Sebastião, EPE, Santa Maria da Feira); Joao Freire (Instituto Portugues de Oncologia, Lison); Cristina Sarmento (Hospital de Sao Joao, Porto); Luis Costa (Hospital de Santa Maria, Lisbon); Antonio Moreira Pinto (Hospital Geral de Santo Antonio, Porto); Sergio Barroso (Hospital Distrital de Beja, Beja); Jorge Espirito Santo (Hospital do Barreiro, Barreiro); Fátima Guedes (Hospital Distrital Figueira da Foz, EPE, Figueira da Foz); Amélia Monteiro (Hospital São Teotónio, EPE, Viseu); Anabela Sa (Hospitais da Universidade de Coimbra, Coimbra); Irene Furtado (Hospital Distrital de Faro, Faro) Spain Grupo Español para el Tratamiento de los Tumores Digestivos (TTD), Josep Tabernero Ramon Salazar (ICO l´Hospitalet Hospital Duran i Reynals, Barcelona); Enrique Aranda Aguilar (Hospital Reina Sofia, Cordoba); Fernando Rivera Herrero (Hospital Universitario Marques de Valdecilla, Santander); Josep Tabernero (Hospital. Vall d´Hebron, Barcelona); Javier Sastre Valera (Hospital Clinico San Carlos, Madrid); Manuel Valladares Ayerbes (Complejo Hospitalario Universitario A Coruña, A Coruña); Jaime Feliu Batlle (Hospital Universitario La Paz, Madrid); Silvia Gil (Hospital Carlos Haya, Malaga ; Albert Abad Esteve (ICO Badalona-Hospital Germans Trias i Pujol, Barcelona); Carlos Garcia-Giron Spain Dr. Peset, Valencia); Pedro Valero Jimenez (Clínica Infanta Luisa, Sevilla); Antonio Galan Brotons (Hospital Sagunto, Sagunto); Santiago Albiol g p Universitario. Dr. Peset, Valencia); Pedro Valero Jimenez (Clínica Infanta Luisa, Sevilla); Antonio Galan Brotons (Hospital Sagunto, Sagunto); Santiago Albiol Universitario. Dr. Peset, Valencia); Pedro Valero Jimenez (Clínica Infanta Luisa, Sevilla); Antonio G l ( i l S S ) S i Albi l Universitario. Dr. Peset, Valencia); Pedro Valero Jimenez (Clínica Infanta Luisa, Sevilla); A Galan Brotons (Hospital Sagunto, Sagunto); Santiago Albiol Galan Brotons (Hospital Sagunto, Sagunto); Santiago Albiol Rodriguez (Hospital Espiritu Santo, Barcelona); Jose Ales Martinez (Hospital Ruber Internacional de Madrid, Madrid); Liliana Canosa Ruiz (Hospital Rodriguez (Hospital Espiritu Santo, Barcelona); Jose Ales Martinez (Hospital Ruber Rodriguez (Hospital Espiritu Santo, Barcelona); J Madrid, Madrid); Liliana Canosa Ruiz (Hospital Torrecardenas, Almeria); Margarita Centelles Ruiz (Hospital Sagrat Cor, Barcelona) Spain Grupo Español para el Tratamiento de los Tumores Digestivos (TTD), Josep Tabernero Ramon Salazar (ICO l´Hospitalet Hospital Duran i Reynals, Barcelona); Enrique Aranda Aguilar (Hospital Reina Sofia, Cordoba); Fernando Rivera Herrero (Hospital Universitario Marques de Valdecilla, Santander); Josep Tabernero (Hospital. Spain Vall d´Hebron, Barcelona); Javier Sastre Valera (Hospital Clinico San Carlos, Madrid); Manuel Valladares Ayerbes (Complejo Hospitalario Universitario A Coruña, A Coruña); Jaime Feliu Batlle (Hospital Universitario La Paz, Madrid); Silvia Gil (Hospital Carlos Haya, Malaga ; Albert Abad Esteve (ICO Badalona-Hospital Germans Trias i Pujol, Barcelona); Carlos Garcia-Giron (Hospital Universitario de Burgos, Burgos); Guillermo Lopez Vivanco (Hospital de Cruces, Vizcaya); Antonia Salud Salvia (Hospital Universitario de Lleida Arnau de Vilanova, Lleida); Vicente Alonso Orduña (Hospital Miguel Servet, Zaragoza); Ruth Vera Garcia (Complejo Hospitalario de Navarra, Pamplona); Javier Gallego (HGU de Elche, Elche-Alicante); Bartomeu Massuti Sureda (Hospital General Universitario de Alicante, Alicante); Jordi Remon (Hospital de Mataro, Barcelona); Maria Jose Safont Aguilera (Hospital General Universitario de Valencia, Valencia); Luis Cirera Nogueras (Hospital Mutua de Terrassa, Barcelona); Bernado Queralt Merino (Hospital Universitari de Girona Dr Josep Trueta, Girona); Cristina Gravalos Castro (Hospital 12 de Octubre, Madrid); Purificacion Martinez de Prado (Hospital de Basurto, Bilbao); Carlos Pijaume Pericay (Consorci Hospitalari Parc Tauli, Barcelona); Manuel Constenla Figueiras (Hospital Provincial de Pontevedra, Pontevedra); Inmaculada Guasch Jordan (Hospital Sant Joan de (Hospital del Mar, Cadiz); Amelia Lopez-Ladron Garcia (Hospital El Tomillar-Ntra Sra de Valme, Son Llatzer, Palma Mallorca); Andres Cervantes (Hospital Clinico Universitario de Valencia, Valencia); Carlos Fernandez Martos (Instituto Valenciano de Oncologia, Valencia); Eugenio Marcuello Gaspar (Hospital de la Santa Creu i Sant Pau, Barcelona); Ines Cabezas Montero (Hospital Universitario Sant Joan de Reus, Tarragona); Pilar Escudero Emperador (Hospital Clinico Universitario Lozano Blesa, Zaragoza); Ana Leon Carbonero (Fundacion Jimenez Diaz, Madrid); Reus, Tarragona); Pilar Escudero Emperador (Hospital Clinico Universitario Lozano Blesa, Zaragoza); Ana Leon Carbonero (Fundacion Jimenez Diaz, Madrid); Manuel Gallen Castillo (Hospital del Mar, Barcelona); Teresa Garcia Garcia (Hospital Morales Manuel Gallen Castillo (Hospital del Mar, Barcelona); Teresa Garcia Garcia ( Meseguer, Murcia); Jose Garcia Lopez (Hospital Universitario Ramón y Cajal, Madrid); Encarnacion Gonzalez Flores (Hospital Virgen de las Nieves Ruiz de Alda, Granada); Monica Guillot Morales (Hospital Son Espases, ada); Monica Guillot Morales (Hospital Son Espases p p Palma de Mallorca); Marta Llanos Muñoz (Hospital Universitario de Canarias, Santa Cruz de Palma de Mallorca); Marta Llanos Muñoz (Hospital Universitario de Canarias, Santa Cruz de Tenerife); Ana López Martín (Hospital Severo Ochoa,); Joan Tenerife); Ana López Martín (Hospital Severo Ochoa,); Joan Maurel (Hospital Clinic i Provincial, Barcelona); Juan Carlos Camara (Fundacion Hospital Alcorcon, Maurel (Hospital Clinic i Provincial, Barcelona); Juan Carlos C Madrid); Rosario Dueñas Garcia (Hospital Ciudad de Jaen, Madrid); Rosario Dueñas Garcia (Hospital Ciudad de Jaen, Jaen); Mercedes Salgado (Complejo Hospitalario Ourense, Ourense); Isabel Hernandez Busquier Jaen); Mercedes Salgado (Complejo Hospitalario Ourense, Ourense); Isabel Her (Hospital Provincial Castellon, Castellon); Teresa Checa Ruiz (Hospital Provincial Castellon, Castellon); Teresa Checa Ruiz Sebastian); Miquel Nogue Aliguer (Hospital General de Vic, Vic); Amalia Velasco Ortiz de Taranco (Hospital Universitario de La Princesa, Madrid); Miguel Vic); Amalia Velasco Ortiz de Taranco (Hospital Universitario de La Princesa, Madri Mendez Ureña (Hospital General de Mostoles, Mostoles); Mendez Ureña (Hospital General de Mostoles, Mostoles); Ferran Losa Gaspa (Consorci Sanitari Creu Roja, Barcelona); Jose Juan Ponce (Hospital Virgen de los Ferran Losa Gaspa (Consorci Sanitari Creu Roja, Barcelona); Jose Juan Ponce (Hospital Virgen de los Lirios, Alicante); Carlos Bosch Roig (Hospital Lirios, Alicante); Carlos Bosch Roig (Hospital ) g ( p Universitario. John Allen Bridgewater g John Bridgewater (North Middlesex University Hospital NHS Trust, London); Rob Glynne-Jones (Centre for Cancer Treatment, Mount Vernon Hospital, Northwood, London); Saad Tahir (Broomfield Hospital, Chelmsford); Tamas Hickish (Poole Hospital NHS Trust, Poole and Bournemouth Hospital, Bournemouth); Jim Cassidy (Beatson West of Scotland Cancer Centre, Glasgow); Leslie Samuel (Aberdeen Royal Infirmary, Aberdeen)
https://openalex.org/W2977036063	https://hal.science/hal-02300858/document	English	null	Inbreeding depression due to recent and ancient inbreeding in Dutch Holstein–Friesian dairy cattle	Genetics selection evolution	2,019	cc-by	14,356	Inbreeding depression due to recent and ancient inbreeding in Dutch Holstein–Friesian dairy cattle Harmen P. Doekes, Roel F. Veerkamp, Piter Bijma, Gerben de Jong, Sipke J. Hiemstra, Jack J. Windig Inbreeding depression due to recent and ancient inbreeding in Dutch Holstein–Friesian dairy cattle Harmen P. Doekes, Roel F. Veerkamp, Piter Bijma, Gerben de Jong, Sipke J. Hiemstra, Jack J. Windig To cite this version: Harmen P. Doekes, Roel F. Veerkamp, Piter Bijma, Gerben de Jong, Sipke J. Hiemstra, et al.. In- breeding depression due to recent and ancient inbreeding in Dutch Holstein–Friesian dairy cattle. Genetics Selection Evolution, 2019, 51 (1), pp.54. 10.1186/s12711-019-0497-z. hal-02300858 Open Access Open Access © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Inbreeding decreases animal performance (inbreeding depression), but not all inbreeding is expected to be equally harmful. Recent inbreeding is expected to be more harmful than ancient inbreeding, because selection decreases the frequency of deleterious alleles over time. Selection efficiency is increased by inbreeding, a process called purging. Our objective was to investigate effects of recent and ancient inbreeding on yield, fertility and udder health traits in Dutch Holstein–Friesian cows. Methods: In total, 38,792 first-parity cows were included. Pedigree inbreeding ( FPED ) was computed and 75 k geno- type data were used to compute genomic inbreeding, among others based on regions of homozygosity (ROH) in the genome ( FROH). Results: Inbreeding depression was observed, e.g. a 1% increase in FROH was associated with a 36.3 kg (SE = 2.4) decrease in 305-day milk yield, a 0.48 day (SE = 0.15) increase in calving interval and a 0.86 unit (SE = 0.28) increase in somatic cell score for day 150 through to 400. These effects equalled − 0.45, 0.12 and 0.05% of the trait means, respec- tively. When FPED was split into generation-based components, inbreeding on recent generations was more harmful than inbreeding on more distant generations for yield traits. When FPED was split into new and ancestral components, based on whether alleles were identical-by-descent for the first time or not, new inbreeding was more harmful than ancestral inbreeding, especially for yield traits. For example, a 1% increase in new inbreeding was associated with a 2.42 kg (SE = 0.41) decrease in 305-day fat yield, compared to a 0.03 kg (SE = 0.71) increase for ancestral inbreeding. There were no clear differences between effects of long ROH (recent inbreeding) and short ROH (ancient inbreeding). Conclusions: Inbreeding depression was observed for yield, fertility and udder health traits. For yield traits and based on pedigree, inbreeding on recent generations was more harmful than inbreeding on distant generations and there was evidence of purging. Across all traits, long and short ROH contributed to inbreeding depression. In future work, inbreeding depression and purging should be assessed in more detail at the genomic level, using higher density information and genomic time series. inbreeding depression is increased homozygosity with inbreeding, which increases the frequency of unfavour- able genotypes [5–7]. Although overdominance and epistasis may contribute to inbreeding depression, partial dominance is expected to account for the major propor- tion of inbreeding depression [6, 8, 9]. Inbreeding depression due to recent and ancient inbreeding in Dutch Holstein– Friesian dairy cattle Harmen P. Doekes1,2* , Roel F. Veerkamp1, Piter Bijma1, Gerben de Jong3, Sipke J. Hiemstra2 and Jack J. Windig1,2 Harmen P. Doekes1,2* , Roel F. Veerkamp1, Piter Bijma1, Gerben de Jong3, Sipke J. Hiemstra2 and Jack J. Windig1,2 Correspondence: harmen.doekes@wur.nl 1 Wageningen University & Research, Animal Breeding and Genomics, P.O. Box 338, 6700 AH Wageningen, The Netherlands Full list of author information is available at the end of the article HAL Id: hal-02300858 https://hal.science/hal-02300858v1 Submitted on 30 Sep 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Doekes et al. Genet Sel Evol (2019) 51:54 https://doi.org/10.1186/s12711-019-0497-z Ge n e t i c s Selection Evolution Ge n e t i c s Selection Evolution Animals and datai In total, 38,792 first-parity cows (fraction Holstein–Frie- sian > 87.5%, either red or black) from 233 herds were included. These cows calved in the period 2012–2016 and were from herds with a data-agreement with the Dutch-Flemish cattle improvement cooperative (CRV; Arnhem, the Netherlands). Initially, 47,254 first-parity cows from 440 herds during the 2012–2016 period were considered. From this initial dataset, herds with less than 10 genotyped cows per year were discarded ( nherds = 207; ncows = 8462) in order to exclude herds in which only a few cows were occasionally genotyped. Pedigree, genotype and phenotype data were provided by CRV. The total pedigree comprised 167,924 individu- als. To assess pedigree completeness, the number of com- plete generations (NCG) and the complete generation equivalent (CGE) were computed. The CGE was com- puted as the sum of 1 2 n of all known ancestors of an individual, with n being the number of generations between the individual and a given ancestor. To limit the effect of missing pedigree information on results, cows with a NCG lower than 3 and/or a CGE lower than 10 were excluded from pedigree-based analyses (n = 1731). The mean NCG and CGE in the remaining cows equalled 6.5 generations and 12.5 generation-equivalents, respectively. With pedigree data, recent inbreeding may be distin- guished from ancient inbreeding by including only a limited number of ancestral generations in the computa- tion of inbreeding coefficients [18, 20]. Alternatively, one may use a purging-based approach to split the classical inbreeding coefficient into a new and an ancestral com- ponent, based on whether alleles are identical-by-descent (IBD) for the first time or have also been IBD in previous generations [21, 22]. The few studies that have applied the latter approach to commercial cattle populations found that the new inbreeding component was more harmful than the ancestral component, suggesting the presence of purging in these populations [4, 23]. With genomic data, age of inbreeding may be derived from the length of ROH [1, 17, 24]. Longer ROH reflect more recent inbreeding, because they have not yet been broken up by recombination. More specifically, the length of ROH derived from a common ancestor G gen- erations ago roughly follows an exponential distribution with a mean of 1/2G Morgan [24, 25]. Background Inbreeding depression is the decrease in mean perfor- mance due to mating between relatives. Many impor- tant traits in dairy cattle, such as yield and fertility traits, show inbreeding depression [1–4]. The genetic basis of A variety of methods can be used to assess inbreeding depression. Traditionally, inbreeding depression has been assessed by regression of phenotypes on pedigree-based Correspondence: harmen.doekes@wur.nl 1 Wageningen University & Research, Animal Breeding and Genomics, P.O. Box 338, 6700 AH Wageningen, The Netherlands Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Doekes et al. Genet Sel Evol (2019) 51:54 Page 2 of 16 inbreeding coefficients [10–12]. Nowadays, with the wide availability of genotype data, pedigree-based inbreed- ing coefficients can be replaced by genomic inbreeding coefficients [1–3]. Genomic inbreeding can be computed from a genomic relationship matrix (GRM) or from the proportion of the genome covered by regions (or runs) of homozygosity (ROH) [13, 14]. Genomic inbreeding coef- ficients are expected to be more accurate than pedigree- based coefficients, because they account for Mendelian sampling variation (e.g. [15]) and do not depend on pedi- gree completeness and quality (e.g. [16]). Moreover, the use of ROH provides additional opportunities to distin- guish recent from ancient inbreeding [1, 17, 18]. to find stronger unfavourable effects for recent inbreed- ing compared to ancient inbreeding, because of selec- tion against deleterious alleles over time (strengthened by purging). For a population of almost 40,000 genotyped cows, we determined the degree of inbreeding depression for yield, fertility and udder health traits. We used vari- ous pedigree-based and genomic inbreeding measures to compare these measures in terms of inbreeding depres- sion. This study was performed in the context of artifi- cial selection, meaning that all traits were under artificial selection and that natural selection will have had a rela- tively small contribution (or no contribution at all). Methods Not all inbreeding is expected to be equally harmful. Recent inbreeding (i.e. inbreeding arising from recent common ancestors) is expected to have a larger unfavour- able effect than ancient inbreeding (i.e. inbreeding arising from more distant common ancestors). This hypothesis is based on the expected decrease in frequency of deleteri- ous alleles over time, which is the result of (natural and/ or artificial) selection. Since most deleterious alleles are (partially) recessive, inbreeding increases the efficiency of selection against these alleles by increasing homozygo- sity, which is called purging [9]. Purging is more likely to occur when there is strong selection pressure and when inbreeding accumulates slowly over many generations [9, 19]. Animals and datai Only a few studies have investigated the effect of ROH of different lengths on phenotypes in livestock, and the results of these stud- ies vary [1, 18, 26].h Cows were genotyped with the Illumina BovineSNP50 BeadChip (versions v1 and v2) or the CRV custom-made 60 k Illumina panel (versions v1 and v2). Genotypes were imputed to 76 k from the different panels, follow- ing Druet et al. [27]. Prior to imputation, single nucleo- tide polymorphisms (SNPs) with a call rate lower than 0.85, a minor allele frequency (MAF) lower than 0.025 or a difference of more than 0.15 between observed and expected heterozygosity were discarded. In addition, SNPs with an unknown position on the Btau4.0 genome assembly were discarded. The final dataset contained 75,538 autosomal SNPs. The objective of this study was to evaluate the degree of inbreeding depression due to recent and ancient inbreed- ing in Dutch Holstein–Friesian dairy cattle. We expected Doekes et al. Genet Sel Evol (2019) 51:54 Doekes et al. Genet Sel Evol (2019) 51:54 Page 3 of 16 Page 3 of 16 Yield, fertility and udder health traits were considered. For yield, the 305-day milk yield (MY; in kg), 305-day fat yield (FY; in kg), and 305-day protein yield (PY; in kg) were included. For fertility, the calving interval (CI; in days), interval calving to first insemination (ICF; in days), interval first to last insemination (IFL; in days), and con- ception rate (CR; in %) were included. For udder health, the mean somatic cell scores for day 5 through to 150 (SCS150; in units) and day 151 through to 400 (SCS400; in units) were included. Somatic cell scores were calcu- lated as 1000 + 100[log2 of cells/mL]. Kalinowski’s inbreeding coefficients and the AHC were obtained by gene dropping, using 106 replications. The in-house script used for gene dropping is available upon request. To illustrate the differences between all pedigree- based inbreeding measures, two example pedigrees are provided (Fig. 1). In example (1), the FPED of individual X equals 7.03%, since it is the sum of the inbreeding on ancestor A (0.57) and on ancestor D (0.54). Since ances- tor A is in the 5th ancestral generation and D is in the first 4 generations, FPED5−4 equals the partial inbreed- ing on A (i.e. 0.57) and FPED4 equals the partial inbreed- ing on D (0.54). Pedigree purging‑based measures Pedigree purging‑based measures Based on the hypothesis of purging, a few additional pedigree-based measures were calculated. Following Kalinowski et al. [21], the FPED was split into two com- ponents: an ancestral component ( FANC ) and a new com- ponent ( FNEW ). The FANC was defined as the probability that alleles were IBD while they had already been IBD in at least one ancestor, and FNEW was the probability that alleles were IBD for the first time in the pedigree of the individual. The ancestral history coefficient ( AHC ) introduced by Baumung et al. [22] was also calculated. AHC was defined as the number of times that a random allele had been IBD during pedigree segregation [22]. Inbreeding measures Various inbreeding measures were used to assess inbreeding depression and distinguish recent from ancient inbreeding. These measures were divided into four groups: (1) pedigree generation-based measures, (2) pedigree purging-based measures, (3) ROH-based meas- ures, and (4) GRM-based inbreeding. Animals and datai FANC is the probability that X is IBD for an allele that was already IBD in an ancestor, which in example (1) has to be ancestor E (since E is the only inbred ancestor). FANC can be manually calculated by multiplying the probability that E is IBD for an allele of A (0.54) with the probability that X inherits this allele from E given that E is IBD (1) and with the probability that X inherits this allele through D-F-G-X given that D is a carrier of the allele (0.53). Thus, it is equal to 0.78% (i.e. 0.57). In example (2), the FPED of individual X is higher (31.25%) than in example (1), while FPED5−4 equals 0% based on the known information. The calculation of FANC in example (2) depends on both D and E, since both ancestors are inbred. FANC can be derived manually by tracing the possible genotype combinations. Individual A has two alleles, alleles 1 and 2. Consider the scenario in which individual B inherits allele 1 from A such that B has genotype 1/3, with 3 referring to a random allele inherited from the unknown parent of B. The possible genotypes of C are 1/4 and 2/4, where 4 is a random allele inherited from the unknown parent of C. If the genotype of C is 1/4, there are four possible genotypes for D and E (namely 1/1, 1/4, 3/1 and 3/4), resulting in 16 possible combinations of D and E and in 64 genotype possibilities for X. Among these 64 possibilities, there are 12 possi- bilities with X being 1/1 while D and/or E are 1/1 (four of which occur when D and E are both 1/1; the others occur when D or E is 1/1 while the other is 1/3 or 1/4). If C has genotype 2/4, while B is 1/3, there are also 64 genotype possibilities for X, but for none of these possibilities X will be IBD. Thus, if B is 1/3, there are 12 out of 128 pos- sibilities for which X is IBD for allele 1 while D and/or E is also IBD for this allele. Similarly, if B is 2/3, there are 12 out of 128 possibilities for which X is IBD for allele 2 while D and/or E are also IBD for this allele. Therefore, the FANC equals 24 out of 256 (i.e. 9.38%). Pedigree generation‑based measureshfi The classical inbreeding coefficient based on all informa- tion in the pedigree ( FPED ) was calculated with PEDIG [28]. The FPED was defined as the pedigree-based prob- ability that two alleles at a random locus in an individual were IBD [29]. In addition to FPED , inbreeding coefficients based on the first n ancestral generations ( FPEDn ), with n ranging from 4 to 8, were computed with the vanrad.f program in PEDIG [28, 30]. Inbreeding for specific age classes was computed as the difference between succes- sive coefficients (e.g. inbreeding on ancestors from 5 gen- erations ago was computed as FPED5 −FPED4 ; abbreviated as FPED5−4 ). The FPED8−7 was chosen as the most ancient category, because of the limited pedigree complete- ness for more ancient generations (e.g. only 78 cows had a NCG > 8) (see Additional file 1: Figure S1). The FPED4 was chosen as the most recent category, because very few individuals were inbred on ancestors in the first ancestral generations (see Additional file 2: Figure S2). ROH‑based measuresh The scanning window approach implemented in the Plink 2.0 software [31] was used to identify ROH. The fol- lowing criteria were set to define a ROH: (i) a minimum Doekes et al. Genet Sel Evol (2019) 51:54 Page 4 of 16 Inbreeding of X: = 7.03% = 6.25% = 0.78% = 6.25% = 0.78% = 0.031 Example 1 Inbreeding of X: = 31.25% = 31.25% = 0% = 21.87% = 9.38% = 0.125 Example 2 Fig. 1 Example pedigrees illustrating differences between pedigree-based inbreeding measures for individual X. FPED : classical pedigree inbreeding based on all available information; FPED4 : inbreeding based on first 4 generations; FPED5−4 : difference between inbreeding based on 5 and on 4 generations; FNEW : Kalinowski’s new inbreeding, i.e. probability that alleles in X are IBD for the first time; FANC : Kalinowski’s ancestral inbreeding, i.e. probability that X is IBD for allele that has already been IBD in an ancestor; AHC : ancestral history coefficient, i.e. the number of times that a random allele from X has been IBD during pedigree segregation Inbreeding of X: = 7.03% = 6.25% = 0.78% = 6.25% = 0.78% = 0.031 Example 1 Fig. 1 Example pedigrees illustrating differences between pedigree-based inbreeding measures for individual X. FPED : classical pedigree inbreeding based on all available information; FPED4 : inbreeding based on first 4 generations; FPED5−4 : difference between inbreeding based on 5 and on 4 generations; FNEW : Kalinowski’s new inbreeding, i.e. probability that alleles in X are IBD for the first time; FANC : Kalinowski’s ancestral inbreeding, i.e. probability that X is IBD for allele that has already been IBD in an ancestor; AHC : ancestral history coefficient, i.e. the number of times that a random allele from X has been IBD during pedigree segregation was determined for each length category (Fig. 2). The expected age of inbreeding was based on the concept that the length of ROH derived from a common ances- tor G generations ago follows an exponential distribution with mean 1/2G Morgan [24, 25]. For simplicity, a mean genetic distance of 1 Morgan per 100 Mb [32] was used and it was assumed that recombination rates were uni- form across the genome and across sexes. Note that non- uniform recombination rates may result in deviations from the exponential distribution. ROH‑based measuresh For example, Speed and Balding [24] performed extensive simulations for the human genome and found that length of ROH was best physical length of 1 Mb, (ii) a minimum of 10 SNPs, (iii) a minimum density of one SNP per 100 kb, (iv) a maxi- mum of one heterozygous call within a ROH, and (v) a maximum gap of 500 kb between consecutive SNPs. A scanning window of 10 SNPs, with a maximum of one heterozygote per window, was used.iii After identification, ROH were classified into five length classes: (i) > 16 Mb, (ii) 8 to 16 Mb, (iii) 4 to 8 Mb, (iv) 2 to 4 Mb, and (v) 1 to 2 Mb. The expected age of inbreeding increased from the first to the last class, since shorter ROH reflect more ancient inbreed- ing. To illustrate this in more detail, the expected age Fig. 2 Expected age of inbreeding (in ancestral generations) for ROH classes, based on underlying exponential distributions. Note that this figure is an approximation, assuming a uniform distribution of inbreeding across ancestral generations, a uniform recombination rate across the genome and sexes, and a genetic distance of 1 Morgan per 100 Mb Fig. 2 Expected age of inbreeding (in ancestral generations) for ROH classes, based on underlying exponential distributions. Note that this figure is an approximation, assuming a uniform distribution of inbreeding across ancestral generations, a uniform recombination rate across the genome and sexes, and a genetic distance of 1 Morgan per 100 Mb Fig. 2 Expected age of inbreeding (in ancestral generations) for ROH classes, based on underlying exponential distributions. Note that this figure is an approximation, assuming a uniform distribution of inbreeding across ancestral generations, a uniform recombination rate across the genome and sexes, and a genetic distance of 1 Morgan per 100 Mb Doekes et al. Genet Sel Evol (2019) 51:54 Page 5 of 16 where βl is the regression coefficient for Fkl , which was the inbreeding coefficient for the kth cow and the lth inbreeding class, and n is the number of inbreeding classes. approximated with a gamma distribution with a shape parameter of 0.76. Since recombination rates may differ across the bovine genome and across sexes [32], Fig. 2 only provides a rough approximation of the expected length per ROH length class. All analyses were performed with ASReml 4.1 [35]. Results Basic statistics for phenotypes and inbreeding measures Descriptive statistics for the evaluated traits are in Table 1. Heritability estimates, obtained by running Model (1) without an inbreeding effect, were high for yield traits (0.36 to 0.47), moderate for somatic cell scores (0.11 and 0.14) and low for fertility traits (0.03 to 0.11). GRM‑based inbreeding Genomic inbreeding coefficients ( FGRM ) were obtained as a measure of marker homozygosity. First, a genomic relationship matrix (GRM) was computed with calc_grm [33], according to the method of VanRaden [14]. Then, inbreeding coefficients were derived as the diagonal of the GRM minus 1 (since the relationship of an individual with itself equals 1 plus its inbreeding coefficient). When computing the GRM, allele frequencies were fixed to 0.5, such that FGRM was equivalent to the proportion of homozygous SNPs, except for a difference in scale [34]. y Inbreeding based on ROH-coverage ( FROH ) was highly correlated with inbreeding based on marker homozygo- sity ( FGRM ), with a Pearson correlation coefficient of 0.92 (Fig. 3). Pedigree-based inbreeding ( FPED ) was moder- ately correlated with FROH and FGRM , with correlation coefficients of 0.66 and 0.61, respectively. The majority of cows (63%) were not inbred on ancestors in the first four ancestral generations, as illustrated by the distribu- tion for FPED4 (see Additional file 2: Figure S2). For cows that were inbred in the first four ancestral generations, clear peaks were visible at expected FPED4-levels, for example at 0.78% (inbreeding on a single ancestor with an inbreeding loop of eight “steps”) and at 1.56% (a single loop of seven steps, or two loops of eight steps). In line with pedigree-based results, only a few cows had very long ROH (which indicate very recent inbreeding). About a fourth of the cows (26%) had no ROH > 16 Mb, 32% had a single ROH > 16 Mb, 21% had two ROH > 16 Mb and the remaining 21% had three or more ROH > 16 Mb. Pearson correlations suggest that the pedigree genera- tion-based and the ROH-based measures partly captured the same age effects (Fig. 3). For example, FPED4 showed a higher correlation with FROH>16 ( r2 = 0.50) than with FROH8−16 (0.34), FROH4−8 (0.22), FROH2−4 (0.10) and FROH1−2 (− 0.03). Similarly, FPED8−7 showed higher cor- relations with short ROH than with long ROH. Correla- tions among pedigree generation-based classes ranged from − 0.23 to 0.27 and correlations among ROH-classes ranged from − 0.10 to 0.26, suggesting rather independ- ent inbreeding age classes. Notably, the FROH1−2 showed a negative or very low correlation (ranging from − 0.10 to 0.06) with all other calculated inbreeding measures, including overall homozygosity ( FGRM). ROH‑based measuresh Regression coefficients and corresponding standard errors (SE) for inbreeding measures were obtained from output. In addition, P-values for the Wald test were obtained from output and were used to check for signifi- cance of effects. For each ROH length class, the inbreeding coeffi- cient was calculated as the proportion of an individual’s autosome that was covered by ROH of that class (e.g. FROH>16 ). Autosome length was corrected for uncovered regions (i.e. ends of chromosomes and gaps of more than 500 kb without SNPs) and the corrected autosome length was 2469 Mb. A total inbreeding coefficient based on all ROH ( FROH ) was also computed. Statistical analysesh The degree of inbreeding depression was estimated by regressing phenotypes on inbreeding coefficients. For the total inbreeding measures ( FPED , FROH and FGRM ), the following linear mixed model was used: (1) yijk = µ + HYi + monthj + α ∗agek + β ∗Fk + cowk + eijk, (1) yijk = µ + HYi + monthj + α ∗agek + β ∗Fk + cowk + eijk, where HYi is the ith herd-year of calving (1165 classes), monthj is the jth month of calving (12 classes), α is the regression coefficient for agek , which was the age at calv- ing for the kth cow, β is the regression coefficient for Fk , which was the inbreeding coefficient for the kth cow, cowk is the random genetic effect for the kth cow, and eijk is the random error term. The cow-effect was assumed to follow N(0, Aσ 2 a ), where A is the numerator relationship matrix and σ 2 a the additive genetic variance. When FPED or FROH was partitioned into classes based on inbreeding age, Model (1) was extended to fit these classes simultaneously (e.g. FROH>16 , FROH8−16 , FROH4−8 , FROH2−4 and FROH1−2): yijk = µ + HYi + monthj + α ∗agek + n l=1 βl ∗Fkl + cowk + eijk, yijk = µ + HYi + monthj + α ∗agek (2) Doekes et al. Statistical analysesh FPED : pedigree inbreeding based on all generations; FROH : inbreeding based on all regions of homozygosity; FGRM : inbreeding based on genomic relationship matrix computed with allele frequencies of 0.5. FPED4 : pedigree inbreeding based on first 4 generations; FPED5−4 : difference between pedigree inbreeding based on 5 and on 4 generations; FNEW : Kalinowski’s new inbreeding; FANC : Kalinowski’s ancestral inbreeding; AHC : ancestral history coefficient; FROH>16 : inbreeding based on regions of homozygosity longer than 16 Mb; FROH8−16 : inbreeding based on regions of homozygosity of 8 to 16 Mb Fig. 3 Heat map showing Pearson’s correlations between different inbreeding measures. FPED : pedigree inbreeding based on all generations; FROH : inbreeding based on all regions of homozygosity; FGRM : inbreeding based on genomic relationship matrix computed with allele frequencies of 0.5. FPED4 : pedigree inbreeding based on first 4 generations; FPED5−4 : difference between pedigree inbreeding based on 5 and on 4 generations; FNEW : Kalinowski’s new inbreeding; FANC : Kalinowski’s ancestral inbreeding; AHC : ancestral history coefficient; FROH>16 : inbreeding based on regions of homozygosity longer than 16 Mb; FROH8−16 : inbreeding based on regions of homozygosity of 8 to 16 Mb Statistical analysesh Genet Sel Evol (2019) 51:54 Page 6 of 16 Table 1 Number of cows (N), mean, standard deviation (SD), corrected phenotypic standard deviation ( σp ), genetic standard deviation ( σa ) and heritability (h2) for all evaluated traits MY: 305-day milk yield; FY: 305-day fat yield; PY: 305-day protein yield; CI: calving interval; ICF: interval calving to first insemination; IFL: interval first to last insemination; CR: conception rate; SCS150: somatic cell score day 5 to 150; SCS400: somatic cell score day 151 to 400 Trait Trait unit N Mean SD σp σa h2 (SE) MY kg 38,778 8091 1375 1199 825 0.47 (0.02) FY kg 38,778 342 51.8 43.9 28.4 0.42 (0.02) PY kg 38,778 283 44.7 36.6 22.0 0.36 (0.02) CI days 34,864 394 67.2 65.3 18.5 0.08 (0.01) ICF days 34,937 77.6 30.0 27.2 7.9 0.08 (0.01) IFL days 34,937 39.9 56.1 55.4 12.3 0.05 (0.01) CR % 34,774 63.8 36.1 35.7 6.1 0.03 (0.01) SCS150 1000 + 100[log2 of cells/mL] 38,301 1568 138 134 45.5 0.11 (0.01) SCS400 1000 + 100[log2 of cells/mL] 37,068 1581 133 129 48.9 0.14 (0.01) Table 1 Number of cows (N), mean, standard deviation (SD), corrected phenotypic standard deviation ( σp ), genetic standard deviation ( σa ) and heritability (h2) for all evaluated traits Table 1 Number of cows (N), mean, standard deviation (SD), correct standard deviation ( σa ) and heritability (h2) for all evaluated traits MY: 305-day milk yield; FY: 305-day fat yield; PY: 305-day protein yield; CI: calving interval; ICF: interval calving to first insemination; IFL: interval first to last insemination; CR: conception rate; SCS150: somatic cell score day 5 to 150; SCS400: somatic cell score day 151 to 400 Fig. 3 Heat map showing Pearson’s correlations between different inbreeding measures. FPED : pedigree inbreeding based on all generations; FROH : inbreeding based on all regions of homozygosity; FGRM : inbreeding based on genomic relationship matrix computed with allele frequencies of 0.5. FPED4 : pedigree inbreeding based on first 4 generations; FPED5−4 : difference between pedigree inbreeding based on 5 and on 4 generations; FNEW : Kalinowski’s new inbreeding; FANC : Kalinowski’s ancestral inbreeding; AHC : ancestral history coefficient; FROH>16 : inbreeding based on regions of homozygosity longer than 16 Mb; FROH8−16 : inbreeding based on regions of homozygosity of 8 to 16 Mb Fig. 3 Heat map showing Pearson’s correlations between different inbreeding measures. Depression for total inbreeding measures 305-day milk yield of 36.25 kg (P < 0.01), an increase in calv- ing interval of 0.48 day (P < 0.01) and an increase in mean somatic cell score in day 151 to 400 of 0.80 units (P < 0.01). All estimated effects, including those that were not signifi- cant at the 0.05-level (e.g. for ICF), were unfavourable. Inbreeding depression was observed for each of the total inbreeding measures ( FPED , FROH , FGRM ) and the estimated effects were significant for most traits (Table 2). For exam- ple, a 1% increase in FROH was associated with a decrease in Doekes et al. Depression for total inbreeding measures Genet Sel Evol (2019) 51:54 Page 7 of 16 Table 2 Estimates of inbreeding depression for all traits and total inbreeding measures, expressed as the change in expected phenotype per 1% increase in inbreeding MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) FPED : pedigree inbreeding based on all generations; FROH : inbreeding based on all regions of homozygosity; FGRM : inbreeding based on genomic relationship matrix computed with allele frequencies of 0.5 Significance for non-nullity is indicated by stars (P < 0.05; P < 0.01) Trait FPED FROH FGRM Estimate SE Estimate SE Estimate SE MY − 37.95 3.66 − 36.25 2.35 − 48.07 2.83 FY − 1.54 0.14 − 1.34 0.09 − 1.60 0.11 PY − 1.27 0.11 − 1.20 0.07 − 1.55 0.09 CI 0.46* 0.23 0.48 0.15 0.62 0.18 ICF 0.16 0.09 0.08 0.06 0.09 0.07 IFL 0.13 0.19 0.27* 0.12 0.42** 0.15 CR − 0.31* 0.12 − 0.27 0.08 − 0.36 0.09 SCS150 0.58 0.44 0.30 0.28 0.44 0.34 SCS400 0.86* 0.43 0.86 0.28 1.15 0.33 Table 2 Estimates of inbreeding depression for all traits and total inbreeding measures, expressed as the change in expected phenotype per 1% increase in inbreeding Table 3 Difference (Diff) between expected phenotypes of cows with low and high inbreeding, for significant traits and total inbreeding measures MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) FPED : pedigree inbreeding based on all generations; FROH : inbreeding based on all regions of homozygosity; FGRM : inbreeding based on the genomic relationship matrix computed with allele frequencies of 0.5 Low and high inbreeding were defined as the 5% and 95% percentile, respectively. Depression for total inbreeding measures Low and high inbreeding equalled 2.8% and 8.0% for FPED , 8.5% and 16.9% for FROH and 25.9% and 32.4% for FGRM Trait FPED FROH FGRM Low High Diff Low High Diff Low High Diff MY 8175 7977 198 8227 7926 301 8232 7917 315 FY 345.4 337.4 8.0 347.0 335.9 11.1 346.7 336.2 10.5 PY 285.8 279.2 6.6 287.5 277.5 10.0 287.5 277.4 10.1 CI 393.0 395.4 − 2.4 392.2 396.2 − 4.0 392.2 396.2 − 4.0 IFL 39.6 40.3 − 0.7 38.9 41.1 − 2.2 38.7 41.4 − 2.7 CR 64.5 62.9 1.6 64.8 62.6 2.2 64.9 62.5 2.4 SCS400 1579 1583 − 4 1578 1585 − 7 1578 1585 − 7 Table 3 Difference (Diff) between expected phenotypes of cows with low and high inbreeding, for significant traits and total inbreeding measures p ( ); y ( g ) FPED : pedigree inbreeding based on all generations; FROH : inbreeding based on all regions of homozygosity; FGRM : inbreeding based on the genomic relationship matrix computed with allele frequencies of 0.5i p y g FPED : pedigree inbreeding based on all generations; FROH : inbreeding based on all regions of homozygosity; FGRM : inbreeding based on the genomic relationship matrix computed with allele frequencies of 0.5 Low and high inbreeding were defined as the 5% and 95% percentile, respectively. Low and high inbreeding equalled 2.8% and 8.0% for FPED , 8.5% and 16.9% for FROH and 25.9% and 32.4% for FGRM p q Low and high inbreeding were defined as the 5% and 95% percentile, respectively. Low and high inbreeding equalled 2.8% and 8.0% for FPED , 8.5% and 16.9% for FROH and 25.9% and 32.4% for FGRM To compare depression effects across traits, the estimated regression coefficients from Table 2 were also expressed as the percentages of the correspond- ing trait means, as well as in phenotypic and genetic standard deviations (see Additional file 3: Table S1). When expressed in percentages of trait means, yield traits showed a relatively large depression effect (of 0.39 to 0.47%) and somatic cell scores a relatively small effect (of 0.02 to 0.05%). The effect for fertility differed across traits and inbreeding measures. It was relatively high for CR and IFL (0.33 to 0.67%) and intermediate for CI and ICF (0.11 to 0.21%). Depression for total inbreeding measures When compared in phe- notypic standard deviations, yield traits showed the To further illustrate differences in performance asso- ciated with changes in inbreeding, the expected pheno- types of cows with low (5% percentile) and high (95% percentile) inbreeding coefficients were compared (Table 3). These differences were computed only for traits that showed a significant depression effect for each of the total inbreeding measures. Differences between cows with low and high inbreeding coefficients were smaller for pedigree-based inbreeding than for genomic inbreeding measures. For example, the differences in 305-day milk yield between lowly and highly inbred cows were 198, 301 and 315 kg for FPED , FROH and FGRM , respectively. Doekes et al. Genet Sel Evol (2019) 51:54 Page 8 of 16 highest degree of inbreeding depression. When com- pared in genetic standard deviations, yield traits also showed the highest degree of inbreeding depression, in spite of the lower heritability of fertility and udder health traits. Only conception rate, which had a very low heritability of 0.03, showed a depression effect similar to that of yield traits when compared in genetic standard deviations. clear pattern was visible for these traits. For example, the interval between calving and first insemination seemed to be unfavourably affected by all classes, but none of the effects was significant. For all traits, standard errors increased with age of inbreeding. This may be explained by a lower degree of variation for more ancient inbreed- ing (see Additional file 2: Figure S2). Depression for pedigree generation‑based inbreeding components When FPED was split into Kalinowski’s new ( FNEW ) and ancestral ( FANC ) components, new inbreeding sig- nificantly reduced milk, fat and protein yield, whereas ancestral inbreeding did not (Fig. 5). For example, a 1% increase in FNEW was associated with a 2.42 kg (SE = 0.41) decrease in 305-day fat yield, while a 1% increase in FANC was associated with a 0.03 kg (SE = 0.71) increase in fat yield. For fertility and udder health traits, both new and ancestral inbreeding effects were not sig- nificantly different from zero. For most traits (MY, FY, PY, IFL, CR, SCS150, SCS400), the estimated effect of When FPED was split into generation-based classes, recent inbreeding significantly reduced milk, fat and pro- tein yield whereas more ancient inbreeding had a non- significant neutral or even favourable effect (Fig. 4). For example, the estimated effects for 305-day protein yield from the most recent to the most ancient class were equal to − 1.3 kg (for FPED4 ), − 1.4 kg ( FPED5−4 ), − 0.6 kg ( FPED6−5 ), 0.3 kg ( FPED7−6 ) and 0.7 kg ( FPED8−7 ). For fertility and udder health traits, estimated effects were generally not significantly different from zero and no Fig. 4 Effect of a 1% increase in pedigree inbreeding ( FPED ) on phenotypes, for different age classes. Error bars represent one standard error and stars indicate significance for non-nullity (P < 0.05; *P < 0.01). MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) Fig. stars calvi som Fig. 4 Effect of a 1% increase in pedigree inbreeding ( FPED ) on phenotypes, for different age classes. Error bars represent one standard error and stars indicate significance for non-nullity (P < 0.05; *P < 0.01). Depression for pedigree generation‑based inbreeding components MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) Fig. 4 Effect of a 1% increase in pedigree inbreeding ( FPED ) on phenotypes, for different age classes. Error bars represent one standard error and stars indicate significance for non-nullity (P < 0.05; *P < 0.01). MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) Doekes et al. Genet Sel Evol (2019) 51:54 Page 9 of 16 was associated with a decrease in yield, while for shorter ROH this decrease was less pronounced. For fertility and udder health traits, most effects were not significantly different from zero. However, some of these traits did show a trend. For calving interval and for the interval between calving and first insemination, inbreeding based on long ROH seemed to increase these intervals, whereas that based on shorter ROH seemed to decrease these intervals. In contrast, for somatic cell score for day 151 through to 400, there seemed to be a larger unfavourable effect of short ROH compared to long ROH. Across all traits, standard errors were larger for inbreeding based on short ROH compared to long ROH. This may be the result of less variation in inbreeding based on short ROH (see Additional file 2: Figure S2). new inbreeding was more unfavourable than the effect of ancestral inbreeding. For some traits (e.g. IFL), the esti- mated effect of ancestral inbreeding was even slightly favourable, whereas the effect of new inbreeding was always unfavourable. The AHC had no significant effect on traits, except for a favourable effect on 305-day protein yield (Table 4). When AHC was fitted simultaneously with FPED , fat yield also tended to increase with an increase in AHC (P <0.1). Interactions between AHC and FPED were not significant. Depression for pedigree generation‑based inbreeding components Depression for ROH length‑based inbreeding components When FROH was split into classes based on ROH length (> 16, 8–16, 4–8, 2–4 and 1–2 Mb), the effect of these classes differed across traits (Fig. 6). For 305-day milk yield, for example, all five classes showed a significant decrease in yield per 1% increase in inbreeding, with a slightly stronger effect for ancient inbreeding ( FROH1−2 ; effect of − 60 kg) than for more recent inbreeding (longer ROH-classes; effects varying from − 29 to − 40 kg). For 305-day fat yield, an increase in FROH>16 and FROH8−16 Inbreeding depression and its costs Estimates of pedigree-based inbreeding depression were comparable to those reported in previous studies. For example, a 1% increase in pedigree inbreeding has Fig. 5 Effect of a 1% increase in Kalinowski’s new and ancestral inbreeding on phenotypes. Error bars represent one standard error and stars indicate significance for non-nullity (P < 0.05; *P < 0.01). MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) Fig. 5 Effect of a 1% increase in Kalinowski’s new and ancestral inbreeding on phenotypes. Error bars represent one standard error and stars indicate significance for non-nullity (P < 0.05; *P < 0.01). MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) Fig. 5 Effect of a 1% increase in Kalinowski’s new and ancestral inbreeding on phenotypes. Error bars represent one standard error and stars indicate significance for non-nullity (P < 0.05; *P < 0.01). MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) Doekes et al. Inbreeding depression and its costs Genet Sel Evol (2019) 51:54 Page 10 of 16 Table 4 Effect of an increase in the ancestral history coefficient ( AHC ) on all traits, when a model with only the AHC or with the AHC and pedigree-based inbreeding ( FPED ) was used MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) Significance for non-nullity is indicated by stars (P < 0.05; P < 0.01) Trait Model with only AHC Model with AHC and FPED AHC AHC FPED Estimate SE Estimate SE Estimate SE MY 157.1 306.5 403.7 307.0 − 38.3 3.7 FY 9.4 11.1 20.2 11.1 − 1.6 0.14 PY 24.5 9.2 34.1 9.2 − 1.31 0.11 CI 11.5 14.7 7.0 14.9 0.44 0.23 ICF 6.8 6.1 5.3 6.2 0.15 0.09 IFL − 11.2 11.8 − 12.9 12.0 0.17 0.19 CR 3.5 7.2 7.0 7.4 − 0.34** 0.12 SCS150 − 25.3 29.9 − 31.4 30.2 0.64 0.44 SCS400 − 3.2 30.0 − 11.4 30.3 0.88* 0.43 Table 4 Effect of an increase in the ancestral history coefficient ( AHC ) or with the AHC and pedigree-based inbreeding ( FPED ) was used Table 4 Effect of an increase in the ancestral history coefficient ( AHC ) on all traits, when a model with only the AHC or with the AHC and pedigree-based inbreeding ( FPED ) was used MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) Significance for non-nullity is indicated by stars (P < 0.05; P < 0.01) Significance for non-nullity is indicated by stars (P < 0.05; *P < 0.01) gi Fig. 6 Effect of a 1% increase in ROH-based inbreeding ( FROH ) on phenotypes, for different ROH lengths. Inbreeding depression and its costs In an attempt to limit this bias, we decided to include only the individuals with a NCG of at least three generations and a CGE of at least 10 equiva- lents in this study. Costs of inbreeding should be considered in the frame- work of a breeding program. For example, for a trait such as 305-day milk yield, we estimated a reduction of around 38 kg per 1% increase in pedigree-based inbreeding. If we consider that the pedigree-based inbreeding level in Dutch Holstein–Friesian cattle has increased from around 0.5% in 1980 to around 4.5% in 2010 [41–43], this would roughly imply a mean loss of 150 kg due to inbreeding depression. Such a loss is small compared to the realised genetic progress in the same period, which was equal to approximately 2200 kg [44]. Although the rate of inbreeding has increased with the introduction of genomic selection [41], contrary to expectation [45], the increased genetic gains [46] are expected to still outweigh the losses caused by inbreeding depression. It should be noted that overall costs will be larger than the cost for single traits, especially since components of economic return may combine multiplicatively rather than additively [47]. In addition, it is important to real- ise that inbreeding will also affect traits that were not included in the present study, such as stillbirths [23]. Pre- vious economic analyses of inbreeding depression sug- gested lifetime losses per cow in the order of tens of US dollars per 1% increase in inbreeding [10, 12, 39]. These analyses confirm that, by affecting various traits, inbreed- ing depression reduces net income. Combined with the importance of conserving genetic diversity for future adaptability, the costs of inbreeding depression provide incentive to monitor and manage inbreeding in dairy cat- tle populations. Estimates of inbreeding depression based on genomic inbreeding measures were similar to those estimated for pedigree-based inbreeding and to those reported in other studies. In US Holstein–Friesian cattle, Bjelland et al. [2] found a decrease in 205-day milk yield of 20 and 47 kg for a 1% increase in FROH and FGRM , respectively. They also observed an increase in days open (a trait similar to calv- ing interval) of 1.72 and 1.06 days for FROH and FGRM , respectively. They did not observe an effect on SCS. In Australian Holstein–Friesian cattle, Pryce et al. Inbreeding depression and its costs [1] esti- mated inbreeding depression based on a FGRM measure that was corrected for allele frequencies of the contem- porary population. They found that a 1% increase in their FGRM-estimates was associated with a decrease in lacta- tion yields for milk, fat and protein of 28 kg, 1.3 kg and 0.9 kg, respectively. In addition, they observed a slight increase in calving interval of 0.12 days, although this increase was not significant. As illustrated by the current and previous studies, genomic measures of inbreeding can be effectively used to estimate the effects of inbreed- ing on performance. In fact, we found that FROH and FGRM captured more phenotypic differences between lowly and highly inbred cows than FPED (Table 3), in spite of the larger estimated change in phenotype per 1% increase in FPED compared to FROH (Table 2). This finding was in line with the results of Bjelland et al. [2] and is the direct result of a wider distribution for FROH compared to FPED (see Additional file 2: Figure S2). The finding that FPED captures less inbreeding depression than FROH and FGRM may be explained by the random nature of recombination and segregation, which is cap- tured with genomic measures but not with pedigree. Since there will be more measurement errors in pedigree inbreeding than in genomic inbreeding, there will be more attenuation or “flattening” of the slope towards zero for FPED (a statistical phenomenon known as regression dilution). For the various inbreeding measures, which Inbreeding depression and its costs Error bars represent one standard error and stars indicate significance for non-nullity (P < 0.05; *P < 0.01). MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) Fig. stars calvi som Fig. 6 Effect of a 1% increase in ROH-based inbreeding ( FROH ) on phenotypes, for different ROH lengths. Error bars represent one standard error and stars indicate significance for non-nullity (P < 0.05; *P < 0.01). MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305-day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: conception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100[log2 of cells/mL]) Doekes et al. Genet Sel Evol (2019) 51:54 Doekes et al. Genet Sel Evol (2019) 51:54 Page 11 of 16 Page 11 of 16 Keller et al. [17] investigated in their simulation study, ROH-based inbreeding showed the highest correlation with the homozygous mutation load. Our results suggest that FGRM and FROH capture similar effects of inbreeding depression at the population level, which is not surpris- ing because of the high correlation between these two measures ( r2 = 0.93 in this study). previously been associated with a reduction in 305-day milk yield of 20 to 30 kg [12, 36, 37] and with an increase in calving interval of 0.2 to 0.7 days [10, 36, 37]. Inbreed- ing depression for somatic cell score has also been observed before [37–39], but estimates were not directly comparable because of different scales and because of the use of separate measures for early (SCS150) and late (SCS400) lactation in the current study. In general, the accuracy of pedigree-based results depends largely on pedigree quality and completeness. Incomplete pedigrees may lead to downward bias of inbreeding coefficients and, therefore, to misleading estimates of inbreed- ing depression [40]. Recent inbreeding is more harmful than ancient inbreeding and evidence of purgingh They found that a 1% increase in FNEW was associated with a decrease in 305-day milk, fat and protein yields of 32.4 kg, 2.4 kg and 1.1 kg, respectively. They also found unfavourable effects for FANC , but these effects were less strong, namely 8.9 kg, 0.5 kg and 0.3 kg, respectively. For calving interval, they estimated an increase of 4.1 and 0.6 days for FNEW and FANC , respectively. Differences across studies may be partly explained by the way that FNEW and FANC have been fitted. In this study and in the study of Hinrichs et al. [23], the FNEW and FANC were fitted simultane- ously in the model, thereby accounting for the correlation between the two measures ( r2 = 0.67 in this study). In the study of Mc Parland et al. [4], however, FNEW and FANC were fitted individually.ff time for selection to act on alleles that affect fertility and udder health traits compared to alleles that affect yield traits. In addition to Kalinowski’s new and ancestral inbreed- ing, we also considered the ancestral history coefficient ( AHC ). AHC is defined as the number of times that a random allele in an individual has been IBD in the indi- vidual’s pedigree [22]. The rationale behind this recently introduced measure is that purging is not fully effi- cient and that the probability of purging increases with the number of times the alleles have been IBD. In other words, an allele that has been IBD many times in an indi- vidual’s pedigree is more likely to have a neutral or posi- tive effect on traits under selection, compared to an allele that has been IBD only once or never before. An increase in AHC , therefore, is expected to be associated with a favourable effect on the phenotype. Indeed, we observed a few favourable effects, i.e. an increase in protein yield and a tendency for an increase in fat yield (Table 4). Most traits showed no significant effect, but the estimate was generally favourable. In Thoroughbred horses, Todd et al. [48] found a strong positive association between AHC and racing performance. Compared to their study, where the mean AHC was 1.97 (SD = 0.09), the mean AHC in the current study was rather low at 0.31 (SD = 0.05). Recent inbreeding is more harmful than ancient inbreeding and evidence of purgingh The main objective of this study was to evaluate the hypothesis that recent inbreeding is more harmful than ancient inbreeding. This hypothesis was based on the expected decrease in frequency of deleterious alleles over time as a result of selection, strengthened by purging. Computer simulations have shown that purging is more effective when selection pressure is strong and when inbreeding accumulates slowly over many generations [9, 19]. We expected that purging would have occurred in the Dutch Holstein–Friesian population, because the population has undergone decades of intense artificial selection and inbreeding has accumulated (at least until 2012) at approximately 0.13% per year [41–43]. Doekes et al. Genet Sel Evol (2019) 51:54 Doekes et al. Genet Sel Evol (2019) 51:54 Page 12 of 16 Pedigree-based results support our hypothesis. For yield traits, inbreeding on recent generations was more harmful than inbreeding on more distant generations (Fig. 4). In addition, there was evidence of purging for these traits (Fig. 5). For most traits, Kalinowski’s FNEW was more harmful than Kalinowski’s FANC (Fig. 5). For some traits, the estimated effect of FANC was even favourable. In other words, to be IBD for alleles that were already IBD in the past had a neutral or favourable effect, whereas to be IBD for alleles for the first time was gen- erally unfavourable. These findings are in line with the hypothesis of purging, which states that loci that have undergone inbreeding in the past have been exposed to an increased selection efficiency (against deleterious recessive alleles), compared to loci that have not under- gone inbreeding before. Our results are largely in line with previous studies that have investigated purging in commercial cattle populations [4, 23]. In German Hol- stein–Friesian cattle, Hinrichs et al. [23] studied the effects of new and ancestral inbreeding on reproductive traits. They found that a 1% increase in FNEW was asso- ciated with a decrease in birthweight of 11.9 kg, while a 1% increase in FANC was associated with an increase in birthweight of 41.6 kg. They also observed a significant increase in the rate of stillbirths for FNEW , while FANC showed a slight reduction in stillbirths that was not sig- nificant. In Irish Holstein–Friesian cattle, Mc Parland et al. [4] investigated the effects of new and ancestral inbreeding on yield and fertility traits. Recent inbreeding is more harmful than ancient inbreeding and evidence of purgingh [53] showed that inbreeding depression estimates based on the IP model are similar to those obtained using Ballou’s approach, with smaller standard errors for the IP model. We con- sidered using the IP model for the current study. Since the model and associated software (PURGd) have been developed outside the context of artificially selected populations, various limitations exist for its application to livestock data. First, random effects cannot be fitted in the model, making it impossible to directly correct for additive genetic relationships. To overcome this limita- tion, one could first run an animal model in a different software environment (e.g. ASReml) and subsequently use the residuals as phenotypes for the IP model. This two-step process is not desirable, because it will affect the inbreeding depression estimates. Second, the model assumes that inbreeding load is due to deleterious alleles that have a low initial frequency in the (base) population. In the context of livestock breeding, where animals are selected based on a breeding goal composed of various traits [42], we do not expect that alleles that are delete- rious for a single trait necessarily segregate at a low fre- quency. Given these limitations, we decided not to use the IP model in the current study. For future research, it would be valuable to explore further the application of the IP model in (livestock) populations undergoing arti- ficial selection. yield when only very long ROH were included than when also shorter ROH were included. To further investigate and compare our results to the findings of Pryce et al. [1], we also ran Model (1) for cumulative ROH-based inbreeding coefficients (i.e. FROH>16 , FROH>8 , FROH>4 , FROH>4 and FROH>1 ). We obtained a similar trend (see Additional file 4: Figure S3) as Pryce et al. [1], with FROH>16 showing the strongest effect and the inclusion of shorter ROHs reducing the effect size. The difference between results for fitting multiple length classes simul- taneously (Fig. 6) and for fitting cumulative measures one by one (see Additional file 4: Figure S3) may be due to the correlations between classes. We believe that fitting length classes simultaneously provides the most accurate estimates, since this approach accounts for the correla- tions between classes. Based on computer simulations, Keller et al. Recent inbreeding is more harmful than ancient inbreeding and evidence of purgingh This can be explained by the very comprehensive pedigree of the Thoroughbred population, which dates back to the late eighteenth century, with individuals from 2000 to 2010 having a mean CGE of 24.6 [48]. A purging-based measure that we did not include in this study is Ballou’s [49] ancestral inbreeding coefficient ( FANC_BAL ). The FANC_BAL is defined as the probability that any allele in an individual has been IBD in an ances- tor at least once [49]. It can be calculated by using an iter- ative formula [49] or with gene dropping [50], where gene dropping provides more robust estimates by account- ing for dependence between FANC_BAL and FPED [50]. To assess the effect of purging, one has to include the prod- uct of FANC_BAL and FPED in the model [4, 49, 51], because FANC_BAL does not consider the IBD-probability for an individual itself. The product of FANC_BAL and FPED is the probability that an individual is IBD for an allele that was already IBD in at least one ancestor, which is in fact the definition of Kalinowski’s FANC [21]. Similarly, the prod- uct of ( 1 −FANC_BAL ) and FPED is equivalent to the FNEW of Kalinowski. Because of this equivalence, we decided to include only Kalinowski’s measures in this study. i Differences between effects of recent and ancient inbreeding (Fig. 4) and between effects of Kalinowski’s FNEW and FANC (Fig. 5) were most apparent for yield traits, which is in accordance with Mc Parland et al. [4]. This finding may be explained by the selection history of Dutch Holstein–Friesian cattle. Targeted selection for fertility and udder health has taken place only since these traits were included in the breeding goal around the year 2000, whereas selection for yield traits has taken place for many more decades [42]. Therefore, there has been less More recently, an inbreeding-purging (IP) model was proposed to assess purging based on genealogical infor- mation [52]. This model, which was developed in a con- servation biology context, predicts how fitness evolves in a population undergoing inbreeding by means of a purged inbreeding coefficient ( g ). g is the traditional Doekes et al. Genet Sel Evol (2019) 51:54 Page 13 of 16 Page 13 of 16 inbreeding coefficient weighted by the reduction in fre- quency of deleterious alleles induced by purging. Using simulations, López-Cortegano et al. Recent inbreeding is more harmful than ancient inbreeding and evidence of purgingh [17] con- cluded that long ROH correlate better with the homozy- gous mutation load than short ROH for a population with an effective population size of 100 (which is the approximate size of the Holstein–Friesian population [41–43]). Functional predictions of deleterious variation have led to inconsistent conclusions as to whether short or long ROH harbour more deleterious genetic variants [54, 55]. For the human genome, Szpiech et al. [54] pre- dicted that long ROH (of several Mb) are enriched with deleterious variants compared to short ROH. In contrast, for four Danish cattle breeds Zhang et al. [55] predicted that short (< 0.1 Mb) and medium (0.1 to 3 Mb) ROH are significantly enriched in deleterious variants compared to long (> 3 Mb) ROH. For domestic dogs, Sams and Boyko [56] recently reported that the relative risk of a ROH car- rying a known deleterious variant is similar across ROH of different lengths, suggesting that ROH of all lengths may contribute to inbreeding depression in dogs. This latter finding is more in line with our results, where both short and long ROH seem to contribute to inbreeding depression. Supplementary information Supplementary information ac org/10.1186/s12711-019-0497-z. Supplementary information accompanies this paper at https://doi. org/10.1186/s12711-019-0497-z. Additional file 1: Figure S1. Distribution of the number of complete generations (NCG) and complete generation equivalent (CGE) for cows included in pedigree-based analyses (n = 37,061). Additional file 2: Figure S2. Distributions of inbreeding measures and the AHC (n = 37,061 for pedigree-based measures and n = 38,792 for genomic measures). The mean ( x ) and standard deviation ( SD ) are also shown. FPED : pedigree inbreeding based on all generations; FROH : inbreeding based on all regions of homozygosity; FGRM : inbreeding based on genomic relationship matrix computed with allele frequencies of 0.5; FPED4 : pedigree inbreeding based on first 4 generations; FPED5−4 : difference between pedigree inbreeding based on 5 and on 4 generations; FNEW : Kalinowski’s new inbreeding; FANC : Kalinowski’s ancestral inbreeding; AHC : ancestral history coefficient; FROH>16 : inbreeding based on regions of homozygosity longer than 16 Mb; FROH8−16 : inbreeding based on regions of homozygosity of 8 to 16 Mb. Additional file 3: Table S1. Estimates of inbreeding depression for all traits1 and total inbreeding measures2, expressed in percentage of trait means (% of x ), in corrected phenotypic standard deviations ( σp ) and in genetic standard deviations ( σa ). The results for σp and σa were multiplied by 100. Estimates correspond to the estimates in Table 2. Additional file 4: Figure S3. Effect of a 1% increase in ROH-based inbreeding ( FROH ) for cumulative measures. Error bars represent one standard error and stars indicate significance for non-nullity (P < 0.05; *P < 0.01). MY: 305-day milk yield (kg); FY: 305-day fat yield (kg); PY: 305- day protein yield (kg); CI: calving interval (days); ICF: interval calving to first insemination (days); IFL: interval first to last insemination (days); CR: con- ception rate (%); SCS150 somatic cell score day 5 to 150 (1000 + 100[log2 of cells/mL]); SCS400: somatic cell score day 151 to 400 (1000 + 100*[log2 of cells/mL]). As sequencing costs continue to decrease, genomic data (including that of cows) will become increasingly available. This offers opportunities to perform large- scale analyses on genomic inbreeding depression based on high-density information, e.g. to identify regions associated with inbreeding depression [1, 18, 61]. Supplementary information In addition, genomic time series (consisting of genomic data of an individual and its ancestors) could be used to study purging in more detail at the genomic level. Long and short ROH contribute to inbreeding depression [26] report such a negative correlation of -0.641 between inbreeding based on short ROH (0.5 to 5 Mb) and based on long ROH (> 5 Mb). In this study, we found some negative correlations between the very short ROH ( FROH1−2 ) and the other classes (Fig. 3). However, these negative correlations could also be an artefact of the unreliable estimation of short ROH. To correct for the masking of ancient inbreeding by recent inbreeding, one could subtract the length of long ROH of the total length of the genome when calculating FROH for short ROH. The effect of this or other correction(s) should be investigated in future studies. Lastly, various approaches can be used to identify ROH. In this study, we applied the sliding window approach implemented in Plink 2.0 [31], with a set of (rather arbitrary) rules to define a ROH. As an alternative to this rule-based approach, one may use a Hidden Markov model (HMM) to identify ROH and infer age of inbreeding [59, 60]. In the future, it would be valu- able to compare the different approaches and investigate the benefit of using linkage maps to infer inbreeding age based on ROH. harmful than inbreeding on distant generations and there was evidence of purging. Based on ROH, there was no clear difference between the effects of long ROH (recent inbreeding) and short ROH (ancient inbreeding). Future work should investigate inbreeding depression and purg- ing in more detail at the genomic level, using higher den- sity information and genomic time series. Funding The research leading to these results has been conducted as part of the IMAGE project, which received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the grant agreement no 677353. The study was co-funded by the Dutch Ministry of Agriculture, Nature and Food Quality (KB-34-013-002). Authors’ contributions HD, JW conceived and designed the experiments; HD performed the analyses and prepared the manuscript; HD, JW, RV, PB, GJ and SH participated in the interpretation of results and revision of the manuscript. All authors read and approved the final manuscript. Acknowledgements The authors gratefully acknowledge the Dutch-Flemish cattle improvement cooperative (CRV) for providing pedigree and genotype data. The authors would also like to thank the anonymous reviewers and editors for their valu- able comments and suggestions. Conclusions b d d Inbreeding depression was observed for yield, fertility and udder health traits in Dutch Holstein–Friesian dairy cattle. Observed inbreeding depression was stronger for yield traits than for fertility and udder health traits, when compared in (phenotypic or genetic) standard deviations. Genomic inbreeding captured more inbreeding depres- sion than pedigree-based inbreeding at the population level. For yield traits and based on pedigree information, inbreeding on recent generations was found to be more Long and short ROH contribute to inbreeding depression However, this is rarely done in practice, since it requires a high-quality linkage map [59]. Third, recent inbreeding may mask more ancient inbreeding [26]. If both chro- mosomes at a position in the genome trace back to a dis- tant common ancestor, you expect to find a short ROH. If the same region also traces back to a recent common ancestor, then you would observe only the long ROH. As a result, one may expect a negative correlation between recent and ancient ROH-based inbreeding. In Iberian pigs, Saura et al. [26] report such a negative correlation of -0.641 between inbreeding based on short ROH (0.5 to 5 Mb) and based on long ROH (> 5 Mb). In this study, we found some negative correlations between the very short ROH ( FROH1−2 ) and the other classes (Fig. 3). However, these negative correlations could also be an artefact of the unreliable estimation of short ROH. To correct for the masking of ancient inbreeding by recent inbreeding, one could subtract the length of long ROH of the total length of the genome when calculating FROH for short ROH. The effect of this or other correction(s) should be investigated in future studies. Lastly, various approaches can be used to identify ROH. In this study, we applied the sliding window approach implemented in Plink 2.0 [31], with a set of (rather arbitrary) rules to define a ROH. As an alternative to this rule-based approach, one may use a Hidden Markov model (HMM) to identify ROH and infer age of inbreeding [59, 60]. In the future, it would be valu- able to compare the different approaches and investigate the benefit of using linkage maps to infer inbreeding age based on ROH. region with low recombination. One may account for this effect by computing ROH based on genetic distances. However, this is rarely done in practice, since it requires a high-quality linkage map [59]. Third, recent inbreeding may mask more ancient inbreeding [26]. If both chro- mosomes at a position in the genome trace back to a dis- tant common ancestor, you expect to find a short ROH. If the same region also traces back to a recent common ancestor, then you would observe only the long ROH. As a result, one may expect a negative correlation between recent and ancient ROH-based inbreeding. In Iberian pigs, Saura et al. Long and short ROH contribute to inbreeding depression Long and short ROH contribute to inbreeding depression We expected that inbreeding based on long ROH (recent inbreeding) would be associated with stronger depres- sion effects than inbreeding based on short ROH (ancient inbreeding). For some traits (e.g. fat yield and calving interval) our results were in line with this hypothesis, but for other traits there was no clear pattern across ROH- length classes or there was even a pattern in the oppo- site direction (Fig. 6). Overall, both long and short ROH seemed to contribute to inbreeding depression. There are various aspects that affect the accuracy of identification of ROH and the inference of inbreeding age based on ROH. First, the density of the SNP panel determines the size of ROH that can be accurately identi- fied. Previous studies have shown that the use of a 50 k panel may result in false positive ROH shorter than 5 Mb and especially in many false positives ROH shorter than 2 Mb [57, 58]. For a more accurate estimation of ancient inbreeding, and to apply this approach to even more gen- erations in the past, high-density SNP data or sequence data is required. Second, in this study we assumed a uni- form recombination rate, while it actually varies across the genome (e.g. [32]). A ROH of a given physical length in a region with high recombination will reflect more ancient inbreeding than a ROH of the same length in a Only a few studies have investigated the effect of ROH of different lengths on phenotypes in livestock popula- tions, with various results [1, 18, 26]. In Austrian Fleck- vieh, Ferenčaković et al. [18] found stronger inbreeding depression for number of spermatozoa when considering both long and short ROH (e.g. > 2 Mb) than when con- sidering only long ROH (e.g. > 16 Mb). 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Received: 17 April 2019 Accepted: 19 September 2019 Received: 17 April 2019 Accepted: 19 September 2019 Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate Not applicable. 20. Silió L, Rodríguez MC, Fernández A, Barragán C, Benítez R, Óvilo C, et al. Measuring inbreeding and inbreeding depression on pig growth from pedigree or SNP-derived metrics. J Anim Breed Genet. 2013;130:349–60. Availability of data and materials All i f i i h l All information supporting the results is included in the text, figures and tables of this article. The dataset is not publicly available due to commercial restrictions. Page 15 of 16 Page 15 of 16 Doekes et al. Genet Sel Evol (2019) 51:54 Doekes et al. Genet Sel Evol Ethics approval and consent to participate Not applicable. References 45. Daetwyler HD, Villanueva B, Bijma P, Woolliams JA. Inbreeding in genome- wide selection. J Anim Breed Genet. 2007;124:369–76. y 57. Purfield DC, Berry DP, McParland S, Bradley DG. Runs of homozygosity and population history in cattle. BMC Genet. 2012;13:70. 46. García-Ruiz A, Cole JB, VanRaden PM, Wiggans GR, Ruiz-López FJ, Van Tas- sell CP. Changes in genetic selection differentials and generation intervals in US Holstein dairy cattle as a result of genomic selection. Proc Natl Acad Sci USA. 2016;113:E3995–4004. 58. Ferenčaković M, Sölkner J, Curik I. Estimating autozygosity from high- throughput information: effects of SNP density and genotyping errors. Genet Sel Evol. 2013;45:42. 47. Kristensen TN, Sørensen AC. Inbreeding–lessons from animal breed- ing, evolutionary biology and conservation genetics. Anim Sci. 2005;80:121–33. 59. Druet T, Gautier M. A model-based approach to characterize indi- vidual inbreeding at both global and local genomic scales. Mol Ecol. 2017;26:5820–41. 59. Druet T, Gautier M. A model-based approach to characterize indi- vidual inbreeding at both global and local genomic scales. Mol Ecol. 2017;26:5820–41. 47. Kristensen TN, Sørensen AC. Inbreeding–lessons from animal breed- ing, evolutionary biology and conservation genetics. Anim Sci. 2005;80:121–33. 48. Todd ET, Ho SY, Thomson PC, Ang RA, Velie BD, Hamilton NA. Founder- specific inbreeding depression affects racing performance in Thorough- bred horses. Sci Rep. 2018;8:6167. 60. Sole M, Gori AS, Faux P, Bertrand A, Farnir F, Gautier M, et al. Age-based partitioning of individual genomic inbreeding levels in Belgian Blue cat- tle. Genet Sel Evol. 2017;49:92. 48. Todd ET, Ho SY, Thomson PC, Ang RA, Velie BD, Hamilton NA. Founder- specific inbreeding depression affects racing performance in Thorough- bred horses. Sci Rep. 2018;8:6167. 49. Ballou JD. Ancestral inbreeding only minimally affects inbreeding depres- sion in mammalian populations. J Hered. 1997;88:169–78. 49. Ballou JD. Ancestral inbreeding only minimally affects inbreeding depres- sion in mammalian populations. J Hered. 1997;88:169–78. 61. Howard JT, Haile-Mariam M, Pryce JE, Maltecca C. Investigation of regions impacting inbreeding depression and their association with the additive genetic effect for United States and Australia Jersey dairy cattle. BMC Genomics. 2015;16:813. 50. Suwanlee S, Baumung R, Sölkner J, Curik I. Evaluation of ancestral inbreeding coefficients: Ballou’s formula versus gene dropping. Conserv Genet. 2007;8:489–95. 51. Boakes EH, Wang J, Amos W. An investigation of inbreeding depression and purging in captive pedigreed populations. Heredity. 2007;98:172–82. References J Dairy Sci. 2006;89:4077–82. 16. Oliehoek PA, Bijma P. Effects of pedigree errors on t 16. Oliehoek PA, Bijma P. Effects of pedigree errors on the efficiency of conservation decisions. Genet Sel Evol. 2009;41:9. 40. Cassell B, Adamec V, Pearson R. Effect of incomplete pedigrees on estimates of inbreeding and inbreeding depression for days to first service and summit milk yield in Holsteins and Jerseys. J Dairy Sci. 2003;86:2967–76. conservation decisions. Genet Sel Evol. 2009;41:9. 17. Keller MC, Visscher PM, Goddard ME. Quantification of inbreeding due to distant ancestors and its detection using dense single nucleotide polymorphism data. Genetics. 2011;189:237–49. 41. Doekes HP, Veerkamp RF, Hiemstra SJ, Bijma P, van der Beek S, Windig JJ. Genomic selection and inbreeding and kinship in Dutch-Flemish Holstein–Friesian cattle. In Proceedings of the 11th world congress on 18. Ferenčaković M, Sölkner J, Kapš M, Curik I. Genome-wide mapping and estimation of inbreeding depression of semen quality traits in a cattle population. J Dairy Sci. 2017;100:4721–30. 19. Boakes E, Wang J. A simulation study on detecting purging of inbreed- ing depression in captive populations. Genet Res. 2005;86:139–48. Doekes et al. Genet Sel Evol (2019) 51:54 Page 16 of 16 53. López-Cortegano E, Bersabé D, Wang J, García-Dorado A. Detection of genetic purging and predictive value of purging parameters estimated in pedigreed populations. Heredity. 2018;121:38–51. genetics applied to livestock production: 11–16 February 2018; Auckland; 2018;90. genetics applied to livestock production: 11–16 February 2018; Auckland; 2018;90. 42. Doekes HP, Veerkamp RF, Bijma P, Hiemstra SJ, Windig JJ. Trends in genome-wide and region-specific genetic diversity in the Dutch-Flemish Holstein–Friesian breeding program from 1986 to 2015. Genet Sel Evol. 2018;50:15. 54. Szpiech ZA, Xu J, Pemberton TJ, Peng W, Zöllner S, Rosenberg NA, et al. Long runs of homozygosity are enriched for deleterious variation. Am J Hum Genet. 2013;93:90–102. 55. Zhang Q, Guldbrandtsen B, Bosse M, Lund MS, Sahana G. Runs of homozygosity and distribution of functional variants in the cattle genome. BMC Genomics. 2015;16:542. 43. Koopman W. Inteelt blijft aandachtspunt, maar nadelige effecten tot nu toe minimaal. Veeteelt: magazine van het Koninklijk Nederlands Rundvee Syndicaat NRS. 2017;6–9. 44. Veerkamp RF, Kaal L, de Haas Y, Oldham JD. Breeding for robust cows that produce healthier milk: ROBUSTMILK. Adv Anim Biosci. 2013;4:594–9. 56. Sams AJ, Boyko AR. Fine-scale resolution of runs of homozygosity reveal patterns of inbreeding and substantial overlap with recessive disease genotypes in domestic dogs. G3 (Bethesda). 2019;9:117–23. 53. López-Cortegano E, Bersabé D, Wang J, García-Dorado A. Detection of genetic purging and predictive value of purging parameters estimated in pedigreed populations. Heredity. 2018;121:38–51. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 52. García-Dorado A, Wang J, López-Cortegano E. Predictive model and software for inbreeding-purging analysis of pedigreed populations. G3 (Bethesda). 2016;6:3593–601. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? 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https://openalex.org/W4321475703	https://zenodo.org/record/7660384/files/D3.8%20Report%20on%20the%20Developed%20and%20Validated%20Atmospheric%20Thematic%20Services%20?download=1	English	null	Report on the Developed and Validated Atmospheric Thematic Services (Release #2)	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	20,440	Ref. Ares(2022)5503451 - 01/08/2022 Ref. Ares(2022)5503451 - 01/08/2022 NEANIAS Novel EOSC services for Emerging Atmosphere, Underwater and Space Challenges Deliverable Deliverable: 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 31/5/2022 NEANIAS is funded by European Union under Horizon 2020 research and innovation programme via grant agreement No. 863448. www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services WP3 - Atmospheric Research Services Disclaimer NEANIAS is a Research and Innovation Action funded by European Union under Horizon 2020 research and innovation programme, via grant agreement No. 863448. research and innovation programme, via grant agreement No. 863448. NEANIAS is project that comprehensively addresses the ‘Prototyping New Innovative Services’ challenge set out in the ‘Roadmap for EOSC’ foreseen actions. It drives the co-design, delivery, and integration into EOSC of innovative thematic services, derived from state-of-the-art research assets and practices in three major sectors: underwater research, atmospheric research and space research. In each sector it engages a diverse set of research and business groups, practices, and technologies and will not only address its community-specific needs but will also enable the transition of the respective community to the EOSC concept and Open Science principles. NEANIAS provides its communities with plentiful resource access, collaboration instruments, and interdisciplinary research mechanisms, which will amplify and broaden each community’s research and knowledge generation activities. NEANIAS delivers a rich set of services, designed to be flexible and extensible, able to accommodate the needs of communities beyond their original definition and to adapt to neighboring cases, fostering reproducibility and re-usability. NEANIAS identifies promising, cutting-edge business cases across several user communities and lays out several concrete exploitation opportunities. This document has been produced receiving funding from the European Commission. The content of this document is a product of the NEANIAS project Consortium and it does not necessarily reflect the opinion of the European Commission. The editor, author, contributors and reviewers of this document have taken any available measure in order for its content to be accurate and lawful. However, neither the project consortium as a whole nor the individual partners that implicitly or explicitly participated in the creation and publication of this document may be held responsible for any damage, financial or other loss or any other issue that may arise as a result of using the content of this document or any of the project outputs that this document may refer to. NEANIAS is project that comprehensively addresses the ‘Prototyping New Innovative Services’ challenge set out in the ‘Roadmap for EOSC’ foreseen actions. It drives the co-design, delivery, and integration into EOSC of innovative thematic services, derived from state-of-the-art research assets and practices in three major sectors: underwater research, atmospheric research and space research. Document Info Project Information Acronym NEANIAS Name Novel EOSC Services for Emerging Atmosphere, Underwater & Space Challenges Start Date 1 Nov 2019 End Date 31 Oct 2022 Program H2020-EU.1.4.1.3. - Development, deployment and operation of ICT-based e- infrastructures Call ID H2020-INFRAEOSC-2018- 2020 Topic H2020-INFRAEOSC-2019-1 Grant No 863448 Instrument RIA Document Information Deliverable No 3.8 Deliverable Title Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Due Date 31-May-2022 Delivery Date 01 – August – 2022 Lead Beneficiary UNIMIB (11) Beneficiaries (part.) NOA (20); MEEO (17); UBIWHERE (6); ATHENA (2); NKUA (1) Editor(s) Alessandro Tibaldi (UNIMIB) Authors (s) Alessandro Tibaldi (UNIMIB), Spyridon Rapsomanikis (ENTA-ATHENA), Ricardo Vitorino (UBIWHERE) Contributor (s) Nikos Chondros (NKUA), Michalis Konstantopoulos (NKUA), Noemi Corti (UNIMIB), Sofia Bressan (UNIMIB), Olyna Gounari (NKUA), Alekos Falagas (NKUA), Makis Ntouskos (NKUA), Vasilios Tsironis (NKUA), Antonia Kournopoulou (NKUA), Konstantinos Karantzalos (ATHENA), Noela Pina (UBIWHERE) Reviewer(s) Eva Sciacca (INAF) Workpackage No WP3 - Atmospheric Research Services Version V1.0 Stage Final Version details Revision: 1.0. Last save: 2022-08-01 , 09:10 Pages: 59 . Characters: 70084 Distribution Public Type Report Keywords EOSC, Services, Atmospheric Research, 3rd release www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Change Record version Date Change Description Editor Change Location (page/section) 1.0 2022-08-01 Document version submitted to EC Alessandro Tibaldi (UNIMIB) Change Record version Date Change Description Editor Change Location (page/section) 1.0 2022-08-01 Document version submitted to EC Alessandro Tibaldi (UNIMIB) Page 3 of 59 www.neania 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Disclaimer In each sector it engages a diverse set of research and business groups, practices, and technologies and will not only address its community-specific needs but will also enable the transition of the respective community to the EOSC concept and Open Science principles. NEANIAS provides its communities with plentiful resource access, collaboration instruments, and interdisciplinary research mechanisms, which will amplify and broaden each community’s research and knowledge generation activities. NEANIAS delivers a rich set of services, designed to be flexible and extensible, able to accommodate the needs of communities beyond their original definition and to adapt to neighboring cases, fostering reproducibility and re-usability. NEANIAS identifies promising, cutting-edge business cases across several user communities and lays out several concrete exploitation opportunities. This document has been produced receiving funding from the European Commission. The content of this document is a product of the NEANIAS project Consortium and it does not necessarily reflect the opinion of the European Commission. The European Union (EU) was established in accordance with the Treaty on the European Union (Maastricht). There are currently 28 member states of the European Union. It is based on the European Communities and the member states’ cooperation in the fields of Common Foreign and Security Policy and Justice and Home Affairs. The five main institutions of the European Union are the European Parliament, the Council of Ministers, the European Commission, the Court of Justice, and the Court of Auditors (http://europa.eu.int/). Page 4 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 5 of 59 Table of Contents Document Info ............................................................................................................................. 2 Disclaimer .................................................................................................................................... 4 Table of Contents ......................................................................................................................... 5 Tables of Figures & Tables ............................................................................................................. 7 Abstract ....................................................................................................................................... 8 1. Introduction ......................................................................................................................... 9 1.1 Context ........................................................................................................................................... 9 1.2 Content and rationale..................................................................................................................... 9 1.3 Changes from previous release report ......................................................................................... 10 1.4 Structure of the document ........................................................................................................... 10 2. Release overview ............................................................................................................... 11 2.1 General overview.......................................................................................................................... 11 2.2 Releases timeline .......................................................................................................................... 11 3. Services.............................................................................................................................. 13 3.1 A1 – Greenhouse gases flux density monitoring service.............................................................. 13 3.2 A2 – Monitoring atmospheric perturbations and components in active tectonic regions service ............................................................................................................................................................ 13 3.3 A3 – Air quality estimation, monitoring and forecasting service ................................................. 15 4. Atmospheric Services Validation and User Requirements fulfilments.................................... WP3 - Atmospheric Research Services Disclaimer 16 4.1 A1 ATMO-FLUD Validation and Addressed User Requirements .................................................. 16 4.2 A2 ATMO-STRESS and ATMO-SEISM Validation and Addressed User Requirements.................. 17 4.2.1. ATMO-STRESS ....................................................................................................................... 17 4.2.2. ATMO-SEISM Service ............................................................................................................ 21 4.3 A3 ATMO-4CAST Validation and Addressed User Requirements ................................................ 22 5. Service Validation Summary ............................................................................................... 27 5.1. Atmo-FLUD end-user evaluation summary ................................................................................. 31 5.2. Atmo-STRESS end-user evaluation summary .............................................................................. 31 5.3. Atmo-SEISM end-user evaluation summary ............................................................................... 31 5.4. Atmo-4CAST end-user evaluation summary ............................................................................... 31 6. Summary and future steps .................................................................................................. 33 7. References ......................................................................................................................... 34 List of acronyms ......................................................................................................................... 36 Appendix 1 ................................................................................................................................. 37 Appendix 2 ................................................................................................................................. 39 What is your level of expertise in the micro-metorology domain? .............................................. 39 How was your experience using our web interface? .................................................................... 39 Table of Contents www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Please provide your comments on the web interface (if any) ..................................................... 40 How satisfied are you with the results? ........................................................................................ 40 Please provide your comments on the results (if any) ................................................................. 40 Was the time to produce the results reasonable? ........................................................................ 41 How satisfied are you with the presentation of the results?........................................................ 41 Please provide your comments on the presentation of the results (if any) ................................. 41 In what format would you like your results to be delivered? (e.g. .dat, .csv, .txt) .................. 42 Why did you choose to use our service? ........................................................................................ 43 Please provide your general comments you have on our service (if any) ................................... 45 What is your level of expertise in the micro-metorology domain? .............................................. 45 How was your experience using our web interface? .................................................................... 45 Please provide your comments on the web interface (if any) ..................................................... 45 How satisfied are you with the results? ........................................................................................ 46 Please provide your comments on the results (if any) ................................................................. 46 Was the time to produce the results reasonable? ........................................................................ 46 How satisfied are you with the presentation of the results?........................................................ 46 Please provide your comments on the presentation of the results (if any) ................................. 46 In what format would you like your results to be delivered? (e.g. .dat, .csv, .txt) .................. 47 Why did you choose to use our service? ........................................................................................ 47 Please provide your general comments you have on our service (if any) ................................... 49 Page 6 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Tables of Figures & Tables Document Figures Figure 1. Screenshot of the Atmo-Stress service, showing the possibility of selecting data fields from the columns of the input file ............................................................................................... 19 Figure 2. Screenshot of the Atmo-Stress service, showing the estimated parameters based on the selected interpolation method and order. The bar showing the progress of the parameter’s estimation is visible. ........................................................................................................... 20 Figure 3. Screenshot of the Atmo-Stress service showing the output type’s choice and the result of the interpolation. Input data (in blue) and outliers (in red) are visible in the output result. ... 20 Figure 4. Map of the hypocenters, where a scale bar and a basemap could be added. ................... 22 Figure 5. Graph relating longitude and depth of the hypocenters. ................................................ 22 Figure 6. Screenshot of the ATMO-4CAST service, showing the input methods available for the service. .............................................................................................................................. 24 Figure 7. User’s feedback forms. ................................................................................................. 25 Figure 8. Screenshot of the ATMO-4CAST service, showing the results of a weather forecast in the webpage service. ................................................................................................................ 26 Figure 9. Screenshot of the ATMO-4CAST service, showing the updated description of the service. 26 Document Tables Table 1 NEANIAS Atmospheric services released .......................................................................... 11 Table 2 Software development plan of the Atmospheric thematic service releases including estimated dates for pre-releases, validation and production. ............................................... 12 Table 3. List of datasets used for ATMO-FLUD validation. ............................................................. 16 Table 4. List of datasets used for ATMO-STRESS validation. .......................................................... 18 Table 5. List of datasets used for ATMO-SEISM validation. ........................................................... 21 Table 6. List of datasets used for ATMO-4CAST validation. ........................................................... 24 Table 7. User requirements addressed by WP3 service ................................................................. 30 WP3 - Atmospheric Research Services Page 7 of 59 WP3 - Atmospheric Research Services www.neania 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services 1.1 Context The NEANIAS WP3 “Atmospheric Thematic services” is focused on the co-design of four services in the atmospheric environment, addressing land-atmosphere interfaces, appropriate for the Research, Development and Business user-communities. The atmospheric services will deliver a springboard of tools to enrich the workflows of a wide range of targeted users from academic/research institutions and industrial stakeholders, e.g. engineering of air pollution control systems and related technology companies, and also end-users at national institutional level such as Civil Protection Agencies. Interest can arise also from more regional governmental institutions such as Regions and Municipalities, especially in relation to detection and evaluation of atmospheric anthropogenic or natural pollutants releases in the atmosphere. Conversely, the quantitation of their man made or natural sinks. The first service A1, named FLUD, provides the users the ability to calculate atmospheric fluxes of Energy and Green House Gases across the terrestrial and oceanic interfaces, using the “Eddy Covariance” method and related algorithms (A1 ATMO-FLUD service). Explained in detail in the documentation that accompanies this service, are the fundamental scientific principles that support this method, the limitations that apply for these calculations and the format of the data input. The A2 Service comprises two services: one named “ATMO-STRESS”, calculates the trajectories of tectonic stress based on a series of local data of stress, and presents them as a map (stress field). It permits the input of any data related to stress, spanning from earthquake focal mechanism solutions to stress calculated from fault slip data sets to geotechnical measurement of in-situ stresses. The second service, named “ATMO-SEISM”, compares a series of parameters regarding the seismicity of a region and the variation of gas emission in the atmosphere, in order to find possible correlations. It automatically produces a series of graphs that correlate all the possible parameters, spanning from latitude and longitude of earthquakes, their foci, their depth, magnitude, distance from the point of gas measurement, gas concentrations, etc. Finally, the A3 service, named “ATMO-4CAST” provides users the ability to obtain and visualize meteorological, road emissions and air quality simulation and forecasts based on their own datasets. The service is hosted on GARR, which can receive different types of data and the necessary model parameters and, passing them through popular models such as WRF-ARW, QTraffic and AUSTAL2000, visualize the obtained outputs (e.g., a map with a visualization of the forecasted air temperature). 1.1 Context Such a service is useful for users who have their own datasets and play key roles on meteorological, road transport or air quality domains. Abstract This document reports on the 3rd and latest release of the Atmospheric Thematic services and the software underlying each service. This report, NEANIAS deliverable 3.8, comes out right after the third thematic space services release, deliverable D3.7. The A1 Service, named “ATMO-FLUD”, (for flux densities) provides a set of algorithms for the calculation of flux densities of Sensible Heat, Latent Heat, Turbulent Kinetic Energy and other scalars like GHGs. The input data to the service is user provided although validation data are available for trial runs. Real data should be experimentally obtained by the user bearing in mind that the fundamental method employed in this service is the “Eddy Covariance”. The A2 Service comprises two services: one named “ATMO-STRESS” calculates the trajectories of tectonic stress based on a series of local data of stress and presents them as a map (stress field). The second service, named “ATMO-SEISM”, compares a series of parameters regarding the seismicity of a region and the variation of gas emission in the atmosphere, in order to find possible correlations. The A3 Service, named “ATMO-4CAST”, is responsible for Air quality estimation, monitoring and forecasting, providing users with the ability to obtain and visualize meteorological and air quality forecasts based on their own datasets through a central server. This is done by processing the incoming data, passing it through popular models such as WRF-ARW, QTraffic and AUSTAL2000, and processing and creating visualizations based on their outputs. For each released service a point-of-access is provided, as well as an interface through which it may interact with other services and a documentation explaining how to use it. Documentation of all NEANIAS space services is organized under NEANIAS docs service. Services reported here have gone through the third release round. Almost all services have been integrated to NEANIAS authentication system service, NEANIAS logging, accounting and monitoring systems, documentation workflow, and are provided through the GARR infrastructure and MEEO cloud services. As expected, this third services milestone has the services integrated to NEANIAS core services, have achieved stability in cloud environments and are currently scaling for growing user demands, datasets increment, and output products publication. Page 8 of 59 WP3 - Atmospheric Research Services WP3 - Atmospheric Research Services www.neania 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services 1.3 Changes from previous release report The initial content and main structure of this document has been given by Deliverable D3.6 [3] reporting the second release of the Atmospheric services. All the chapters of this document have been updated according to the validation and testing made after the third release of the services. 1.2 Content and rationale This deliverable, D3.8, is a report containing the details of the developed and validated Atmospheric Research Services delivered in the 3rd release cycle, listed in deliverable D3.7 “Atmospheric Thematic Services Release #3” [1]. In the first release, D3.4 [2], the services went through the major steps on adapting the TRL6 software into a cloud environment, linking them to core services, documenting and validating Page 9 of 59 WP3 - Atmospheric Research Services www.neanias. 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) port on the Developed and Validated Atmospheric Thematic Services (Release #3) them in the scope of NEANIAS services. In the second release, D3.6 [3], the services went through the steps on increasing to the TRL7 into to the cloud environment, integrating them to additional core services, further documenting and validating them. This document reports on the evolution of the Atmospheric Thematic Services since the second release, each of them individually as planned in the previous releases and according to the development of the Core services [4] and user validation feedback [5]. 1.3 Changes from previous release report WP3 - Atmospheric Research Services 1.4 Structure of the document This document is organized as follows. In chapter 2, we present a general overview of the NEANIAS Atmospheric services, the release processes and tools, and a summary of the released Atmospheric services. Chapter 3 provides short descriptions of each Atmospheric service released up to the date of this document. Chapter 4 reports the service validation and the user requirements fulfilment. Finally, in Chapter 5 we conclude this report with a summary of achievements, major issues and future steps to accomplish service final validation. This document is part of a series of documents to be released during NEANIAS development as the services develop and go through major releases. Each release milestone got a separate report like this one. Page 10 of 59 WP3 - Atmospheric Research Services www.neania 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 2.1 General overview The NEANIAS Atmospheric research services are aimed at supporting management and analysis of large data volumes in Atmospheric and Earth sciences via assisting the scientific community to establish fluxes of Energy and Green House gases across the terrestrial and oceanic interfaces, using the “Eddy Covariance” method and related algorithms (A1 ATMO- FLUD service), secondly, efficiently producing maps of stress affecting the Earth crust (A2 ATMO-STRESS service) and correlating huge amount of data of different parameters to understand the possible correlation between earthquakes and crustal gas emissions (A2 ATMO-SEISM), and finally, optimizing, automating and validating software modules for air quality estimation along with the novel integrations of satellite, geospatial and in-situ data (A3 ATMO-4CAST). 2.2 Releases timeline The Atmospheric Thematic services are released in three main cycles. Release #1 has been delivered in M12 (Deliverable D3.3 [6] and the relative report in the Deliverable D3.4 [2]). Release #2 has been delivered in M23 (Deliverable D3.5 [7] and relative report in the Deliverable D3.6 [3]). The following table summarizes the Atmospheric services released and the main information related to the release. The Atmospheric Thematic services are released in three main cycles. Release #1 has been delivered in M12 (Deliverable D3.3 [6] and the relative report in the Deliverable D3.4 [2]). Release #2 has been delivered in M23 (Deliverable D3.5 [7] and relative report in the Deliverable D3.6 [3]). The following table summarizes the Atmospheric services released and the main information related to the release. Table 1 NEANIAS Atmospheric services released Table 1 NEANIAS Atmospheric services released Short name Sector1 Lead2 TRL3 Version4 Core Integration5 NEANIAS Catalogue6 EOSC Onboarding7 Atmo- FLUD Atmospheric (A1) ATHENA TRL 8 3.0.0 AAI, Logging, Accounting, Data sharing, Monitoring ATMO-FLUD Calculation of Flux Densities Yes Atmo- Stress Atmospheric (A2) ATHENA TRL 8 3.0.1 AAI, Logging, Accounting, Data sharing, Monitoring ATMO- STRESS Yes Atmo- Seism Atmospheric (A2) NKUA TRL 8 3.0.0 AAI, Logging, Accounting, Data sharing, Monitoring ATMO-SEISM No Atmo- 4CAST Atmospheric (A3) UBIWHER E TRL 8 3.0.0 AAI, Logging, Accounting, Data sharing, Monitoring ATMO-4CAST Atmospheric Forecast Yes Final Release #3 has been delivered in M30 (Deliverable D3.7 [1] and relative report in this document). The following table summarizes the pre-release, validation and production project months and estimated exact dates. Table 1 NEANIAS Atmospheric services released Short name Sector1 Lead2 TRL3 Version4 Core Integration5 NEANIAS Catalogue6 EOSC Onboarding7 Atmo- FLUD Atmospheric (A1) ATHENA TRL 8 3.0.0 AAI, Logging, Accounting, Data sharing, Monitoring ATMO-FLUD Calculation of Flux Densities Yes Atmo- Stress Atmospheric (A2) ATHENA TRL 8 3.0.1 AAI, Logging, Accounting, Data sharing, Monitoring ATMO- STRESS Yes Atmo- Seism Atmospheric (A2) NKUA TRL 8 3.0.0 AAI, Logging, Accounting, Data sharing, Monitoring ATMO-SEISM No Atmo- 4CAST Atmospheric (A3) UBIWHER E TRL 8 3.0.0 AAI, Logging, Accounting, Data sharing, Monitoring ATMO-4CAST Atmospheric Forecast Yes Final Release #3 has been delivered in M30 (Deliverable D3.7 [1] and relative report in this document). The following table summarizes the pre-release, validation and production project months and estimated exact dates. Table 1 NEANIAS Atmospheric services released Final Release #3 has been delivered in M30 (Deliverable D3.7 [1] and relative report in this document). WP3 - Atmospheric Research Services Table 2 Software development plan of the Atmospheric thematic service releases including estimated dates fo pre-releases, validation and production. 2.2 Releases timeline The following table summarizes the pre-release, validation and production project months and estimated exact dates. Final Release #3 has been delivered in M30 (Deliverable D3.7 [1] and relative report in this document). The following table summarizes the pre-release, validation and production project months and estimated exact dates. WP3 - Atmospheric Research Services Page 11 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) From project start Exact dates Deliv erabl e Description Product ion Validati on Pre- release Producti on Validatio n Pre- release D3.3 Atmospheric Thematic Services Release #1 12 11 10 31/10/20 30/09/20 31/08/20 D3.5 Atmospheric Thematic Services Release #2 23 22 21 30/09/21 31/08/21 31/07/21 D3.7 Atmospheric Thematic Services Release #3 30 29 28 30/04/22 31/03/22 28/02/22 Table 2 Software development plan of the Atmospheric thematic service releases including estimated dates for pre-releases, validation and production. WP3 - Atmospheric Research Services www.neanias.e 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 3. Services This chapter provides a tabular overview of each service: status, its endpoints, and links to further information. This chapter provides a tabular overview of each service: status, its endpoints, and links to further information. 3.1 A1 – Greenhouse gases flux density monitoring service Short name Lead partner Contributors ATMO-FLUD ATHENA NKUA Service Status The ATMO-FLUD service, which uses a set of algorithms (Eddy Covariance and Gradient Method) for the calculation of flux densities of momentum, energey, and scalars (such as CO2) is up and running, tested and ready for production use. The service is functionaly complete, with both the Eddy Covariance and the Gradient Method in place, and is integrated with all available Core Services (AAI, Logging, Accounting, Data Sharing, Monitoring). Documentation is in place and users are able to provide feedback using a feedback form, one for each available algorithm. Service Endpoints https://atmo-flud.neanias.eu Software information https://gitlab.neanias.eu/a1-service/webapp https://gitlab.neanias.eu/a1-service/integration https://gitlab.neanias.eu/a1-service/scientific Documentation at: https://docs.neanias.eu/projects/a1-service Current Release: 3.0.0 3.2 A2 – Monitoring atmospheric perturbations and components in active tectonic regions service 3.1 A1 – Greenhouse gases flux density monitoring service Service Status The ATMO-FLUD service, which uses a set of algorithms (Eddy Covariance and Gradient Method) for the calculation of flux densities of momentum, energey, and scalars (such as CO2) is up and running, tested and ready for production use. The service is functionaly complete, with both the Eddy Covariance and the Gradient Method in place, and is integrated with all available Core Services (AAI, Logging, Accounting, Data Sharing, Monitoring). Documentation is in place and users are able to provide feedback using a feedback form, one for each available algorithm. Service Endpoints https://atmo-flud.neanias.eu The ATMO-FLUD service, which uses a set of algorithms (Eddy Covariance and Gradient Method) for the calculation of flux densities of momentum, energey, and scalars (such as CO2) is up and running, tested and ready for production use. The service is functionaly complete, with both the Eddy Covariance and the Gradient Method in place, and is integrated with all available Core Services (AAI, Logging, Accounting, Data Sharing, Monitoring). Documentation is in place and users are able to provide feedback using a feedback form, one for each available algorithm. 3.2 A2 – Monitoring atmospheric perturbations and components in active tectonic regions service Note that the A2 service has been implemented in two separate instances/services. After the initial co-design sessions with the internal and external end-users from the Atmospheric community and based on the D3.1 outcome [8] there was a requirement for two separate tools that able to handle the processing pipeline of the proposed A2 service. More specifically: ✓ the first one that has been implemented in the framework of NEANIAS is “Atmo-STRESS” that produces maps of tectonic stress trajectories based on a series of local stress data. Page 13 of 59 WP3 - Atmospheric Research Services www.neanias 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 14 of 59 ✓ the second one that has been implemented in the framework of NEANIAS is “ATMO- SEISM” service that targets end-users which are more experienced in scripting/programming, to help them explore and discover possible correlations between the seismicity of a region and the variation of gas emission in the atmosphere. Short name Lead partner Contributors ATMO-STRESS UNIMIB ATHENA, NKUA Service Status The core functionalities of ATMO-STRESS were based on the LISSAGE software (Lee & Angelier, 1994 [9]). 3.1 A1 – Greenhouse gases flux density monitoring service User authentication and authorization is handled by the NEANIAS AAI service (C2). Logging and Accounting are handled by the corresponding NEANIAS services (C2). The service is also integrated with the NEANIAS file service for handling user files and file sharing. An API is also offered for use from other NEANIAS services Service Endpoints ATMO-STRESS Documentation: https://docs.neanias.eu/projects/a2-1-service ATMO- STRESS: https://atmo-stress.neanias.eu/ Software information ATMO-STRESS Source Code: https://gitlab.neanias.eu/a2-service/a2-1-service/a2-1-worker https://gitlab.neanias.eu/a2-service/a2-1-service/a2-1-web-app Current Release: 3.0.1 Short name Lead partner Contributors ATMO-SEISM UNIMIB ATHENA, NKUA Service Status ATMO-SEISM has been developed as a Jupyter Notebook service hosted in a dedicated JupyterHub deployment. The ATMO-SEISM service, with the use of a developed Python Package (pyradon), enables users to comparingly process and analyze gas releases and earthquake datasets and plot useful visualizations. User authentication and authorization is handled by the NEANIAS AAI service (C2). Logging and Accounting are handled by the ✓ the second one that has been implemented in the framework of NEANIAS is “ATMO- SEISM” service that targets end-users which are more experienced in scripting/programming, to help them explore and discover possible correlations between the seismicity of a region and the variation of gas emission in the atmosphere. Short name Lead partner Contributors ATMO-STRESS UNIMIB ATHENA, NKUA Service Status The core functionalities of ATMO-STRESS were based on the LISSAGE software (Lee & Angelier, 1994 [9]). User authentication and authorization is handled by the NEANIAS AAI service (C2). Logging and Accounting are handled by the corresponding NEANIAS services (C2). The service is also integrated with the NEANIAS file service for handling user files and file sharing. An API is also offered for use from other NEANIAS services Service Endpoints ATMO-STRESS Documentation: https://docs.neanias.eu/projects/a2-1-service ATMO- STRESS: https://atmo-stress.neanias.eu/ Software information ATMO-STRESS Source Code: https://gitlab.neanias.eu/a2-service/a2-1-service/a2-1-worker https://gitlab.neanias.eu/a2-service/a2-1-service/a2-1-web-app Current Release: 3.0.1 WP3 - Atmospheric Research Services Page 14 of 59 Short name Lead partner Contributors ATMO-SEISM UNIMIB ATHENA, NKUA Service Status ATMO-SEISM has been developed as a Jupyter Notebook service hosted in a dedicated JupyterHub deployment. The ATMO-SEISM service, with the use of a developed Python Package (pyradon), enables users to comparingly process and analyze gas releases and earthquake datasets and plot useful visualizations. User authentication and authorization is handled by the NEANIAS AAI service (C2). Logging and Accounting are handled by the Short name Lead partner Contributors ATMO-SEISM UNIMIB ATHENA, NKUA Service Status ATMO-SEISM has been developed as a Jupyter Notebook service hosted in a dedicated JupyterHub deployment. WP3 - Atmospheric Research Services 3.1 A1 – Greenhouse gases flux density monitoring service The ATMO-SEISM service, with the use of a developed Python Package (pyradon), enables users to comparingly process and analyze gas releases and earthquake datasets and plot useful visualizations. User authentication and authorization is handled by the NEANIAS AAI service (C2). Logging and Accounting are handled by the Page 14 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) corresponding NEANIAS services (C2). The service is also integrated with the NEANIAS file service for handling user files and file sharing. Service Endpoints ATMO-SEISM Documentation: https://docs.neanias.eu/projects/a2-2-service ATMO-SEISM: https://atmo-seism.neanias.eu Software information ATMO-SEISM Source Code: https://gitlab.neanias.eu/a2-service/a2-2-service/pyradon Current Release: 3.0.0 corresponding NEANIAS services (C2). The service is also integrated with the NEANIAS file service for handling user files and file sharing. Service Endpoints ATMO-SEISM Documentation: https://docs.neanias.eu/projects/a2-2-service ATMO-SEISM: https://atmo-seism.neanias.eu Software information ATMO-SEISM Source Code: https://gitlab.neanias.eu/a2-service/a2-2-service/pyradon Current Release: 3.0.0 3.3 A3 – Air quality estimation, monitoring and forecasting service 3.3 A3 – Air quality estimation, monitoring and forecasting service 3.3 A3 – Air quality estimation, monitoring and forecasting service Short name Lead partner Contributors ATMO-4CAST UBIWHERE GARR Service Status ATMO-4CAST is divided in three different modules, which enable: (I) weather simulations; (ii) emission simulations; (iii) air quality simulations. The last module provides air quality forecast by connecting with the emission module and gathering background concentrations from another NEANIAS Core service, named ADAM Platform. Through the API of this service, ATMO- 4CAST can obtain background contributions from CAMS (Copernicus Atmosphere Monitoring Service). These functionalities have been integrated and can be used through the NEANIAS portal. Moreover, the service is available in the NEANIAS Catalogue and EOSC platform. WP3 - Atmospheric Research Services WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 4. Atmospheric Services Validation and User Requirements fulfilments Below, we provide information on service validation including data used to validate and assess service functionality and information about the user requirements addressed behind the service evolution and the software developed. Details on the Validation procedures and results of the validation for all the Atmospheric Services Release #2 can be found in Appendix 1 and 2. 4.1 A1 ATMO-FLUD Validation and Addressed User Requirements The ATMO-FLUD Service has been validated using the following datasets: The ATMO-FLUD Service has been validated using the following datasets: DATASETS – ATMO-FLUD id Name of Dataset Type Source FAIR Size (MBs) 01 20131201.dat .dat Private data N 6.4 02 20131213.dat .dat Private data N 7.2 03 20131220.dat .dat Private data N 7.2 04 20140205.dat .dat Private data N 6.5 05 20140222.dat .dat Private data N 6.4 06 20140402.dat .dat Private data N 6.5 07 20140706.dat .dat Private data N 6.9 08 20140719.dat .dat Private data N 6.5 09 20140727.dat .dat Private data N 6.5 10 20140905.dat .dat Private data N 6.4 11 data_slow.dat .dat Private data N 79 Table 3. List of datasets used for ATMO-FLUD validation. Table 3. List of datasets used for ATMO-FLUD validation. The ATMO-FLUD service has been stable since the second release, and is improved with regard to technology stack updates, quality (more testing), as well as continuous security audit and incorporation of needed updates to address security concerns (CVEs). Furthermore, we have developed a new approach for scientific job execution, that allows jobs to be posted to Kubernetes clusters outside the production one at GARR, aiming for better integration with and usage of EGI resources. We are not releasing this functionality yet, because said resources were only recently acquired by the project, and further work is needed to adapt to the constraints EGI is imposing (such as inability to use the root user in containers, inability to directly mount the Data Sharing service). Moreover, discussions are in place on the security of this approach, as the source code for the scientific part of A1 is not open. Page 16 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 4.2 A2 ATMO-STRESS and ATMO-SEISM Validation and Addressed User Requirements The A2 service has been implemented in two separate instances. The first “Atmo-STRESS” produces maps of tectonic stress trajectories based on a series of local stress data while in the second instance “ATMO-SEISM” service is directed to users which are more experienced in programming, to help them explore and discover possible correlations between the seismicity of a region and the variation of gas emission in the atmosphere. This section discusses the validation of the two deployed services. WP3 - Atmospheric Research Services 4.2.1. ATMO-STRESS 4.2.1. ATMO-STRESS WP3 - Atmospheric Research Services Page 17 of 59 The ATMO-STRESS Service has been validated using the following datasets: DATASETS – ATMO-STRESS id Name of Dataset Type Source FAIR Size 01 Opening_lat_lon3 _theta_NE Rift Etna .txt User private data N 6 KB 02 Op_dir_ThFS_Icel and .txt User private data N 74 KB 03 Op_dir_KFS_Icela nd .txt User private data N 106 KB 04 Opening_lat_lon3 _theta_NE Rift Etna .xls User private data N 40 KB 05 Op_dir_ThFS_Icel and .xls User private data N 185 KB 06 Op_dir_KFS_Icela nd .xls User private data N 165 KB 07 paper3_NS_shma x .xls Anton et al. - 2010 [8] N 30 KB 08 paper3_NESW_sh max .xls Anton et al. - 2010 [10] N 26 KB 09 paper3_EW_shm ax .xls Anton et al. - 2010 [10] N 26 KB 10 Caucasus .xls Tibaldi et al. - 2021 [11] N 47 KB 11 sites_paper1 .xls Faure et al. - 2005 [12] N 28 KB 12 area_1_checked_ paper2 .xls De Vincente et al – 1996 [13] N 30 KB 13 area_2_checked_ paper2 .xls De Vincente et al – 1996 [13] N 25 KB The ATMO-STRESS Service has been validated using the following datasets: DATASETS ATMO STRESS The ATMO-STRESS Service has been validated using the following datasets: www.neanias.eu www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 14 Weil et al. 2014_ Wyoming_def .xls Weil et al.2014 [14] N 30 KB 15 Fuerteventura_ar eaMD1 .xls Fernandez et al 2006 [15] N 28 KB 16 Fuerteventura_ar eaMD2 .xls Fernandez et al 2006 [15] N 26 KB 17 Fuerteventura_ar eaMD3 .xls Fernandez et al 2006 [15] N 26 KB 18 Synthetic_data .xls Synthetic data N 382 KB Table 4. List of datasets used for ATMO-STRESS validation. 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Table 4. List of datasets used for ATMO-STRESS validation. Table 4. List of datasets used for ATMO-STRESS validation. Respect to the last release, the following improvements have been applied to the ATMO- STRESS service (Fig. 1), following the suggestions of the end users: • A progress bar that shows the time required for the parameter estimation has been added; thanks to this improvement the user can verify how much time the service needs to estimate the parameters (Fig. 2). WP3 - Atmospheric Research Services 4.2.1. ATMO-STRESS • Now the preview shows not only the interpolation result, but also the input data used to interpolate the output (in blue) and the data that are not well explained by the fitted model, called outliers (in red) (Fig. 3). • Broken lines or trajectories that did not follow the input data have been adjusted, and now they are correct. • The new trajectories map has a wider spatial coverage than the one presented in the last release. Therefore, areas without input data do not result in vertical lines or empty spaces, as requested by end users after Deliverable D3.6 [3]. Page 18 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Figure 1. Screenshot of the Atmo-Stress service, showing the possibility of selecting data fields from the columns of the input file WP3 - Atmospheric Research Services WP3 - Atmospheric Research Services Figure 1. Screenshot of the Atmo-Stress service, showing the possibility of selecting data fields from the columns of the input file Page 19 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3 8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Figure 2. Screenshot of the Atmo-Stress service, showing the estimated parameters based on the selected interpolation method and order. The bar showing the progress of the parameter’s estimation is visible. Figure 3. Screenshot of the Atmo-Stress service showing the output type’s choice and the result of the interpolation. Input data (in blue) and outliers (in red) are visible in the output result. Figure 2. Screenshot of the Atmo-Stress service, showing the estimated parameters based on the selected interpolation method and order. The bar showing the progress of the parameter’s estimation is visible. Figure 2. Screenshot of the Atmo-Stress service, showing the estimated parameters based on the selected interpolation method and order. The bar showing the progress of the parameter’s estimation is visible. Figure 2. Screenshot of the Atmo-Stress service, showing the estimated parameters based on the selected interpolation method and order. The bar showing the progress of the parameter’s estimation is visible. Figure 3. Screenshot of the Atmo-Stress service showing the output type’s choice and the result of the interpolation. Input data (in blue) and outliers (in red) are visible in the output result. Figure 3. Screenshot of the Atmo-Stress service showing the output type’s choice and the result of the interpolation. Input data (in blue) and outliers (in red) are visible in the output result. Page 20 of 59 WP3 - Atmospheric Research Services WP3 - Atmospheric Research Services www.neanias.e 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Even though the ATMO-stress service has been improved since release #2, based on new feedback from our users it is possible to still observe some problems: • ATMO-stress does not support .xlsx, .shp and .csv format input files yet; • The resulting maps have a different coordinate system than the dataset uploaded as input files; this difference generates a distortion between the input data and the resulting trajectories. 4.2.2. ATMO-SEISM Service WP3 - Atmospheric Research Services 4.2.2. ATMO-SEISM Service The ATMO-SEISM Service has been validated using the following datasets: DATASETS – ATMO-SEISM id Name of Dataset Type Source FAIR Size 01a 01b Test radon_2017 Test earth_2017 .csv .csv Private data http://sismoweb. ct.ingv.it/ N Y 462 KB 1 KB 02a 02b Test radon_2017 Test earth_2017 .xlsx .xlsx Private data http://sismoweb. ct.ingv.it/ N Y 351 KB 10 KB 03a 03b Test radon_2017-2018 Test earth_2017-2018 .csv .csv Private data http://sismoweb. ct.ingv.it/ N Y 3.506 KB 7 KB 04a 04b Test radon_2017-2018 Test earth_2017-2018 .xlsx .xlsx Private data http://sismoweb. ct.ingv.it/ N Y 2.544 KB 18 KB 05a 05b Test radon_2016-2018 Test earth_2016-2018 .csv .csv Private data http://sismoweb. ct.ingv.it/ N Y 5.326 KB 83 KB 06a 06b Test radon_2016-2018 Test earth_2016-2018 .xlsx .xlsx Private data http://sismoweb. ct.ingv.it/ N Y 3.864 KB 97 KB Table 5. List of datasets used for ATMO-SEISM validation. The ATMO-SEISM Service has been validated using the following datasets: Table 5. List of datasets used for ATMO-SEISM validation. Table 5. List of datasets used for ATMO-SEISM validation. In order to validate ATMO-SEISM, two datasets are required: one containing the values of gas emissions, temperature and pression, latitude, longitude and date and hour of the measurements, and the second containing earthquake parameters (date and hour, coordinates, depth of the hypocenter and magnitude), related to the same time interval. In order to validate ATMO-SEISM, two datasets are required: one containing the values of gas emissions, temperature and pression, latitude, longitude and date and hour of the measurements, and the second containing earthquake parameters (date and hour, coordinates, depth of the hypocenter and magnitude), related to the same time interval. The service works well with .csv files, whereas the validation from the users has highlighted some problems using .xlsx files as input data. When this file format is used, in fact, the service gives back an error and does not allow to obtain the desired graphs as outputs. In general, the users are satisfied of the results, and they are satisfied particularly about the time needed to obtain them. 4.2.2. ATMO-SEISM Service Anyway, some improvements can still be made, especially regarding the presentation of the results: regarding the map of the hypocenters, a scalebar Page 21 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) should be added, and it would be better to add a basemap, such as the Open Street Map background, or the basemap from Google Earth (Fig. 4). Furthermore, regarding the graph relating longitude and latitude vs depth (Fig. 5), the dots should be scaled based on magnitude, with the same size they have in the hypocenters map. The users also believe that a graph relating the released energy and the filtered gas values (not only measured gas values) should be presented as a result. Finally, another suggestion given by the users regards the possibility to represent in the resulting graphs not all the measured values, but just the daily average, as in Neri et al. (2016) [16]. Figure 4. Map of the hypocenters, where a scale bar and a basemap could be added. Figure 4. Map of the hypocenters, where a scale bar and a basemap could be added. Figure 5. Graph relating longitude and depth of the hypocenters. Figure 4. Map of the hypocenters, where a scale bar and a basemap could be added. Figure 5. Graph relating longitude and depth of the hypocenters. Figure 5. Graph relating longitude and depth of the hypocenters. WP3 - Atmospheric Research Services Page 22 of 59 4.3 A3 ATMO-4CAST Validation and Addressed User Requirements The ATMO-4CAST Service has been validated using the following datasets: WP3 - Atmospheric Research Services WP3 - Atmospheric Research Services 4.3 A3 ATMO-4CAST Validation and Addressed User Requirements The ATMO-4CAST Service has been validated using the following datasets: WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) DATASETS – ATMO-4CAST id Name of Dataset Type Source FAIR Size 01 EXAMPLE 1_GRIB method, Weather module GRIB files and Vtable from GFS; namelist input files https://files.nean ias.eu/s/M235gyf dFrKrtbN?path=% 2FGRIB%20Data% 2FExample%201 Y 89,4 MB 02 EXAMPLE 2_ GRIB method, Weather module GRIB files and Vtable from GFS; namelist input files https://files.nean ias.eu/s/M235gyf dFrKrtbN?path=% 2FGRIB%20Data% 2FExample%202 Y 279 MB 03 EXAMPLE 1_ JSON method, Weather module Configuration.jso n; matrix. json https://files.nean ias.eu/s/M235gyf dFrKrtbN?path=% 2FJSON%20Data %2FExample%20 1 Y 458 KB 04 EXAMPLE 2_ JSON method, Weather module Configuration.jso n; matrix. json https://files.nean ias.eu/s/M235gyf dFrKrtbN?path=% 2FJSON%20Data %2FExample%20 2 Y 2,32 GB 05 EXAMPLE 1, Emission module v_fleet_distributi on.txt; v_traffic.txt https://files.nean ias.eu/s/PpJWrcd WBPGKNtQ?path =%2FExample%2 01 Y 14 KB 06 EXAMPLE 1, Air Quality module austal2000.txt; emissions_profile .txt; local_weather.txt ; v_fleet_distributi on.txt; v_traffic.txt https://files.nean ias.eu/s/SAmo8Z SrdQcktkz?path= %2FExample%20 1 Y 19 KB 07 EXAMPLE 2, Air Quality module austal2000.txt; emissions_profile .txt; meteo_out.akter m; v_fleet_distributi on.txt; v_traffic.txt https://files.nean ias.eu/s/SAmo8Z SrdQcktkz?path= %2FExample%20 2 Y 20 KB 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 01 www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 08 EXAMPLE 3, Air Quality module austal2000.txt; background_conc entrations; emissions_profile .txt; local_weather.txt ; v_fleet_distributi on.txt; v_traffic.txt https://files.nean ias.eu/s/SAmo8Z SrdQcktkz?path= %2FExample%20 3 Y 19 KB 09 EXAMPLE 4, Air Quality module austal2000.txt; series.dmna https://files.nean ias.eu/s/SAmo8Z SrdQcktkz?path= %2FExample%20 4 Y 42 KB Table 6. List of datasets used for ATMO-4CAST validation. 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 08 EXAMPLE 3, Air Quality module austal2000.txt; background_conc entrations; emissions_profile .txt; local_weather.txt ; v_fleet_distributi on.txt; v_traffic.txt https://files.nean ias.eu/s/SAmo8Z SrdQcktkz?path= %2FExample%20 3 Y 19 KB 09 EXAMPLE 4, Air Quality module austal2000.txt; series.dmna https://files.nean ias.eu/s/SAmo8Z SrdQcktkz?path= %2FExample%20 4 Y 42 KB Table 6. List of datasets used for ATMO-4CAST validation. 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 3 Table 6. List of datasets used for ATMO-4CAST validation. In respect to the second release, the following features have been implemented in the ATMO- 4CAST service: In respect to the second release, the following features have been implemented in the ATMO 4CAST service: In respect to the second release, the following features have been implemented in the ATMO- 4CAST service: WP3 - Atmospheric Research Services ✓ The service now integrates two new modules (Emission and Air Quality modules). ✓ The service now integrates two new modules (Emission and Air Quality modules). ✓ The service now integrates two new modules (Emission and Air Quality modules). Figure 6. Screenshot of the ATMO-4CAST service, showing the input methods available for the service. Figure 6. Screenshot of the ATMO-4CAST service, showing the input methods available for the service. ✓ The Air Quality module was built as a system enabling the connection with the Emission module to gather local contributions; and the connection with CAMS (Copernicus Atmosphere Monitoring Service) through the ADAM API (another NEANIAS service) to gather background contributions. ✓ The Air Quality module was built as a system enabling the connection with the Emission module to gather local contributions; and the connection with CAMS (Copernicus Atmosphere Monitoring Service) through the ADAM API (another NEANIAS service) to gather background contributions. WP3 - Atmospheric Research Services Page 24 of 59 WP3 - Atmospheric Research Services Page 24 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) ✓ The input data for the three modules (based on WRF [17]; QTraffic [18] and AUSTAL2000 [19] models) can be stored and managed using NEANIAS Data storage/sharing core service (Nextcloud). ✓ The input data for the three modules (based on WRF [17]; QTraffic [18] and AUSTAL2000 [19] models) can be stored and managed using NEANIAS Data storage/sharing core service (Nextcloud). ✓ The service is also available through a REST API to enable integration with other services, namely the business cases of the project. ✓ The service is also available through a REST API to enable integration with other services, namely the business cases of the project. ✓ User feedback forms have been integrated in the service to enable a continuum improvement of the service by taking into consideration users' opinion. ✓ User feedback forms have been integrated in the service to enable a continuum improvement of the service by taking into consideration users' opinion. Figure 7. User’s feedback forms. Figure 7. User’s feedback forms. Figure 7. User’s feedback forms. ✓ Visualization of outputs are now possible in the webpage service besides of map images provided as PNG images (for both Weather and Air Quality modules). ✓ Visualization of outputs are now possible in the webpage service besides of map images provided as PNG images (for both Weather and Air Quality modules). Page 25 of 59 WP3 - Atmospheric Research Services WP3 - Atmospheric Research Services www.neanias. 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Figure 8. Screenshot of the ATMO-4CAST service, showing the results of a weather forecast in the webpage service. Figure 8. Screenshot of the ATMO-4CAST service, showing the results of a weather forecast in the webpage service. ✓ The documentation has been improved to help the user understand in a better way how to build the input data for the different modules. ✓ The documentation has been improved to help the user understand in a better way how to build the input data for the different modules. ✓ The documentation has been improved to help the user understand in a better way how to build the input data for the different modules. Figure 9. Screenshot of the ATMO-4CAST service, showing the updated description of the service. Figure 9. Screenshot of the ATMO-4CAST service, showing the updated description of the service. Page 26 of 59 WP3 - Atmospheric Research Services WP3 - Atmospheric Research Services www.neania 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services 5. Service Validation Summary Overall, the third release of WP3 services managed to fulfil the end-user requirements. In particular, the deployed Atmospheric services were compared against the table contained in D3.1, chapter 3 (Co-design and Service Specifications) [8]. We therefore keep track of the implementation of the most prominent user requirements assessed in the earlier stages of the project according to table 7 below. Regarding the service assessment performed by the end-users, all NEANIAS internal end-users thoroughly evaluated the third release of all WP3 services. Moreover, different researchers and personnel, from the internal end-users institutes/SMEs and external institutions, with different background and expertise evaluated the services in order to assess them in terms of the level of experience, technical or thematic expertise required for using smoothly and operationally the deployed in WP3 services. Based also on the specific application domain of each end-user, more emphasis was given on specific services that are addressing domain- related challenges. As stated in Section 4 the A2 service has been implemented in two separate instances. The first “Atmo-STRESS” produces maps of tectonic stress trajectories based on a series of local stress data while the second instance “ATMO-SEISM” targets users more experienced in programming to help them explore and discover possible correlations between the seismicity of a region and the variation of gas emission in the atmosphere. WP3 - Atmospheric Research Services NEANIAS is funded by European Union under Horizon 2020 research and innovation programme via grant agreement No. 863448. 5. Service Validation Summary Requirement  Specifications  3rd Release Validated Status for WP3 Services Req#  Requirement   Priority   Value  Spec  #  Description of the required functionalities, software specification  Address ed by Atmo-FLUD (A1) Atmo-STRESS (A2-1) Atmo-SEISM (A2-2) Atmo-4CAST (A3) R00 Common AAI High High  S00 Common SSO for all NEANIAS services A1, A2, A3  Y Y Y Y R01  Upload, store and possibly publish datasets of various formats  High  High S01  Interface with suitable Data Transfer NEANIAS/EOSC services for uploading / downloading data to medium / long storage locations through well-known protocols (FTP, WebDAV, etc.) and make data FAIR when applicable  A1, A2, A3  Y Y Y Y R02 Use datasets provided by the services Low Medium S01 Interface with suitable Data Transfer NEANIAS/EOSC services for accessing data made available by the service provider for testing and learning the functionalities of the service A1, A2, A3 Y Y Y Y R03  Visualize raw data, resulting products and reports  Medium High  S02 Each Atmospheric service implements task-specific reporting mechanisms  A1, A2, A3  Y Y Y Y S03  Interface with NEANIAS visualization services for visualizing raw data and results  A1, A2, A3  N/A N N N R04  To access data from suitable on-line repositories and data sources Medium High S04  Implement task-specific services for accessing data provided through custom APIs A1, A2, A3  Currently not applicable Currently not applicable Currently not applicable Currently not applicable S01 Interface with suitable Data Transfer NEANIAS/EOSC services for accessing data made available by external repositories when provided through standard data protocols A1, A2, A3  Y Y Y Y R05  Document the workflow  Medium Medium S05 Atmospheric services implement suitable logging mechanisms and store meta-data describing the service parametrization  A1, A2, A3  Y Y partly Y S06  Interface with AAI, auditing and other relevant NEANIAS services for collecting user-related information  A1, A2, A3  Y Y Y Y S01  Interface with NEANIAS data transfer and other relevant services for storing and publishing logs and the workflow related meta-data  A1, A2, A3  Y Y partly Y R06  Export the results in various file formats  Low Medium S07  Each service offers option to serialize the produced data in widely used file formats  A1, A2, A3  Y Y Y Y 3rd Release Validated Status for WP3 Services 3 Release Validated Status for WP3 Services NEANIAS is funded by European Union under Horizon 2020 research and innovation programme via grant agreement No. 863448. 5. Service Validation Summary www.neanias.eu www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 29 of 59 S01  Interfaces with NEANIAS data transfer services for storing the output data on suitable storage locations  A1, A2, A3  Y Y Y Y Requirement  Specifications  3rd Release Validated Status for WP3 Services Req#  Requirement   Priority   Value  Spec  #  Description of the required functionalities, software specification  Address ed by Atmo-FLUD (A1) Atmo-STRESS (A2-1) Atmo-SEISM (A2-2) Atmo-4CAST (A3) R07 Correlate gas emissions (e.g. Radon) with earthquake events and atmospheric conditions in active tectonic regions High High S08 Compute correlation and other statistical quantities of interest between a set of relevant variables defined by the user A2 - - Y - S01 Interface with NEANIAS/EOSC Data Transfer services for accessing data through standard data transfer protocols A2 - - Y - S04 Interface with task-specific services for accessing data through custom APIs A2 - - Y - R08 Produce maps of the regional stress field of a study area which are compatible with GIS environments High  High S09 Compute stress field maps based on well-known interpolation methods (e.g. inverse distance weighting or bivariate polynomial function) A2 - Y - - S07 Support export to data formats compatible with GIS environments (e.g. 5. Service Validation Summary geoTIFF, KML, geoJSON, etc.) A2 - Y - - S01 Interface with NEANIAS/EOSC Data Transfer services for storing data using standard data transfer protocols A2 - Y - - R09 Aggregate, harmonise and store cross-domain data from multiple sources (satellite, geospatial, in- situ) for air quality monitoring High  High S10 Integrate Smart Air Quality’s management platform as a SaaS platform A3 - - - Y S01 Interface with NEANIAS/EOSC Data Transfer services for accessing and storing data through standard data transfer protocols A3 - - - Y S04 Interface with task-specific services for accessing data through custom APIs A3 - - - Currently not applicable R10 Aggregate, harmonise and store GHG flux related High  High S11 Aggregate and harmonize cross-domain GHG flux data A1, A3 Y - - N www.neanias.eu www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 30 of 59 data on a regional / national scale S01 Interface with NEANIAS/EOSC Data Transfer services for accessing and storing data through standard data transfer protocols A1, A3 Y - - N S04 Interface with task-specific services for accessing data through custom APIs A1, A3 Y - - Currently not applicable Requirement  Specifications  3rd Release Validated Status for WP3 Services Req#  Requirement   Priority   Value  Spec  #  Description of the required functionalities, software specification  Address ed by Atmo-FLUD (A1) Atmo-STRESS (A2-1) Atmo-SEISM (A2-2) Atmo-4CAST (A3) R11 Calculate the flux densities of GHG emissions High High  S12 Compute flux densities of GHG emissions (dynamic gradient calculations, eddy covariance) A1 Y - - - R12 Filter the GHG emissions and energy fluxes per area or location Medium High  S14 EOSC service is able to filter data with geographies as input parameters A3 - - - N R13 To upload their own measurements for computing flux densities High  High  S01 Interface with NEANIAS/EOSC Data Transfer services for storing user- provided data using standard data transfer protocols A1 Y - - - R14 To employ available atmospheric data from NEANIAS for flux densities benchmarks and learning purposes Medium High S13 Define default parametrization and example scenarios for testing / learning each provided service A1, A2, A3 Y Y Y Y R15 To compare their own software with the NEANIAS developed one High  High S02 Each service reports suitable evaluation metrics which allow for comparison with other related methods / software A1, A2, A3 Y Y Y Y S01 Interface with NEANIAS/EOSC Data Transfer services for uploading user reference results for comparison A1, A2, A3 Y Y partly Y R16 Improve portability and reproducibility Medium High S05 Atmospheric services implement suitable logging mechanisms and store meta-data describing the service parametrization  A1, A2, A3 Y Y partly Y S01 Interface with NEANIAS data transfer and other relevant services for storing and publishing logs and the workflow related meta-data  A1, A2, A3 Y Y partly Y Table 7. WP3 - Atmospheric Research Services 5.1. Atmo-FLUD end-user evaluation summary The third release of the Atmo-FLUD service was validated mostly during the NΕΑΝΙΑS Atmospheric workshops that took place in Xanthi, Greece on June 3rd, 2022, and in Athens, Greece on June 28th-29th. The service was validated by 37 distinct users and received 13 new feedback responses for Eddy Covariance and 6 new feedback responses for the Gradient Method. Verbal communication with all validators was highly encouraging, and no negative comments were received. The users that did fill in our feedback forms also submitted strong positive assessment. 5.2. Atmo-STRESS end-user evaluation summary The users that have validated the third release of the service have in general expressed their satisfaction on the improvements done after the second release validation (Deliverable D3.6 [3]). The service has been thoroughly validate considering datasets which span a wide range of scales and locations. At the same time, the users highlight some problems regarding the results’ coordinate system for some of the validation datasets, which do not seem coherent with the input data. These recommendations will be taken into account to improve the user experience in the final release of the service. 5.3. Atmo-SEISM end-user evaluation summary The users validated the service by adapting the sample Jupyter notebook provided, introducing additional datasets besides the ones bundled with the service. Being the service implemented as a highly customisable Jupyter notebook powered by task-specific developed libraries, user feedback can be distinguished into two main categories. The first category regards feedback from users who are familiar with the read-eval-print loop (REPL) and other relevant programming environments. Feedback from this group of users states that the service can handle .csv data, whereas errors occurred when .xlsx files were imported into the service. The users are mostly satisfied regarding the results and the time required to obtain the outputs, but they think that some improvements can be done regarding the presentation of the results, and the download of them. The second feedback category comes from users who are less familiar with REPL environments. These users have found it very difficult to use the service and get results, and this is reflected in negative feedback in the validation form in Appendix 2, especially regarding the user-friendliness of the service. 5. Service Validation Summary User requirements addressed by WP3 service 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services 5.4. Atmo-4CAST end-user evaluation summary The service has been validated with students and researchers from different domains. First, we validated it with students from a Master Communication course in University of Aveiro in order to see what we could improve in terms of UI of the service and make it more user- friendly. They have indicated some useful tips to upgrade the service, although, given the complexity of the service, it won’t be as user-friendly for users out of the scope of the service. NEANIAS is funded by European Union under Horizon 2020 research and innovation programme via grant agreement No. 863448. www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Later, we validated the service in more workshops in University of Aveiro, Xanthi, Greece and Athens, Greece with students and researchers from the environment and atmospheric courses. Overall, the service is responsive and gives a lot of metrics, however, in the new modules, especially the emission module some improvements must be done in the representation of the outputs, which are currently under development. Page 32 of 59 www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 6. Summary and future steps This report documents the current status of NEANIAS Atmospheric services, as of the 3rd release milestone in the NEANIAS project. We successfully improved the four services belonging to the Atmospheric Thematic services. The Atmospheric Thematic services are: A1 - “ATMO-FLUD” (for flux densities) that provides a set of algorithms for the calculation of flux densities of Sensible Heat, Latent Heat, Turbulent Kinetic Energy and other scalars like GHGs. The input data to the service is user provided although validation “real data” are available for trial runs; A2 - that comprises two services that analyze the conditions that guide the uprising of gas through the Earth crust and its release in the atmosphere; these services are “ATMO-STRESS”, that calculates the trajectories of tectonic stress based on a series of local data of stress and presents them as a map (stress field), and “ATMO-SEISM”, that compares a series of parameters regarding the seismicity of a region and the variation of gas emission in the atmosphere, in order to find possible correlations; A3 - “ATMO-4CAST” that is responsible for air quality estimation, monitoring and forecasting, providing users with the ability to obtain and visualize meteorological and air quality forecasts based on their own datasets through a central server. This is done by processing the incoming data, passing it through popular models such as WRF-ARW, QTraffic and AUSTAL2000, and processing and creating visualizations based on their outputs. For each released service a point-of-access is provided, as well as an interface through which it interacts with other services and documentation explaining how to use it. Documentation of all NEANIAS atmospheric services is organized under NEANIAS docs service. Services also went through a third round of user validation tests (see Appendix 1 and 2), where users reported less error rates compared to the previous rounds of validations (see Appendix 2 of D3.4 [2] and Appendix 1 and 2 of D3.6 [3]). Compared to the second validation round, the number of surveyed users has increased from 12 up to 20 for the A1 service, from 12 to 57 for A2 services divided into 44 users for the Atmo-stress and 13 for the Atmo-seism and from 9 to 45 for the A3. For all the services they were mostly students and researchers from universities. Received users’ feedbacks will be important to improve services' user interface and the documentation. WP3 - Atmospheric Research Services 6. Summary and future steps Page 33 of 59 WP3 - Atmospheric Research Services WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services [19] Janicke, U. (2014). AUSTAL2000, Program Documentation of Version 2.6. 2014-02-24. Janicke Consulting, Dunum (Germany). [18] Dias D., Antunes A. P., Tchepel O., 2019. Modelling of emissions and energy use from biofuel fuelled vehicles at urban scale. Sustainability, 11(10), 2902. 7. References [1] NEANIAS WP3 Collaboration, “D3.7: Atmospheric Thematic Services Release #3,” NEANIAS, 2022. [1] NEANIAS WP3 Collaboration, “D3.7: Atmospheric Thematic Services Release #3,” NEANIAS, 2022. 2] NEANIAS WP3 Collaboration, “D3.4: Report on the Developed and Validated Atmospheric Thematic Services (Release #1),” NEANIAS, 2021. [3] NEANIAS WP3 Collaboration, “D3.6: Report on the Developed and Validated Atmospheric Thematic Services (Release #2),” NEANIAS, 2021. [4] NEANIAS WP6 Collaboration, “D6.2: Core services Release #1,” NEANIAS, 2020. [5] NEANIAS WP6 Collaboration, “D6.1: Core Services Architecture, Design Principles and Specifications,” NEANIAS, 2020. [6] NEANIAS WP3 Collaboration, “D3.3: Atmospheric Thematic Services Release #1,” NEANIAS, 2020. [7] NEANIAS WP3 Collaboration, “D3.5: Atmospheric Thematic Services Release #2,” NEANIAS, 2021. [8] NEANIAS WP3 Collaboration, “D3.1: Atmspheric Research Services Report on requirements, Specifications & Software development Plan,” NEANIAS, 2020. [9] Lee, J. C., & Angelier, J. (1994). Paleostress trajectory maps based on the results of local determinations: the “Lissage” program. Computers & Geosciences, 20(2), 161-191. [10] Antón, L., Muñoz-Martín, A., & De Vicente, G. (2010). Alpine paleostress reconstruction and active faulting in western Iberia. Central European Journal of Geosciences, 2(2), 152-164. [11] Tibaldi, A., Babayev, G., Bonali, F. L., Mariotto, F. P., Russo, E., Tsereteli, N., & Corti, N. (2021). Active Kinematics of the Greater Caucasus from Seismological and GPS Data: A Review. Building Knowledge for Geohazard Assessment and Management in the Caucasus and other Orogenic Regions, 33-57. [12] Faure, S., Tremblay, A., Malo, M., & Angelier, J. (2006). Paleostress analysis of Atlantic crustal extension in the Quebec Appalachians. The Journal of Geology, 114(4), 435-448. [13] De Vicente, G., Giner, J. L., Muñoz-Martín, A., González-Casado, J. M., & Lindo, R. (1996). Determination of present-day stress tensor and neotectonic interval in the Spanish Central System and Madrid Basin, central Spain. Tectonophysics, 266(1-4), 405-424. [14] Weil, A. B., Yonkee, A., & Kendall, J. (2014). Towards a better understanding of the influence of basement heterogeneities and lithospheric coupling on foreland deformation: A structural and paleomagnetic study of Laramide deformation in the southern Bighorn Arch, Wyoming. Bulletin, 126(3-4), 415-437. [15] Fernández, C., Casillas, R., Garcia Navarro, E., Gutiérrez, M., Camacho, M. A., & Ahijado, A. (2006). Miocene rifting of Fuerteventura (Canary islands). Tectonics, 25(6). [16] Neri, M., Ferrera, E., Giammanco, S., Currenti, G., Cirrincione, R., Patanè, G., & Zanon, V. (2016). Soil radon measurements as a potential tracer of tectonic and volcanic activity. Scientific reports, 6(1), 1-12. WP3 - Atmospheric Research Services 7. References Page 34 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) [17] UCAR (2019). Weather Research & Forecasting Model, Version 4 Modeling System User’s Guide. https://www2.mmm.ucar.edu/wrf/users/docs/user_guide_v4/ [17] UCAR (2019). Weather Research & Forecasting Model, Version 4 Modeling System User’s Guide. https://www2.mmm.ucar.edu/wrf/users/docs/user_guide_v4/ [18] Dias D., Antunes A. P., Tchepel O., 2019. Modelling of emissions and energy use from biofuel fuelled vehicles at urban scale. Sustainability, 11(10), 2902. [19] Janicke, U. (2014). AUSTAL2000, Program Documentation of Version 2.6. 2014-02-24. Janicke Consulting, Dunum (Germany). Page 35 of 59 www.neanias.eu List of acronyms List of acronyms Acronym Description ARW Advanced Research WRF CMAQ Community Multiscale Air Quality EOSC European Open Science Cloud FAIR Findable, Accessible, Interoperable, and Reusable GARR Gruppo per l'Armonizzazione delle Reti della Ricerca GFS Global Forecasting Service GHG Greenhouse Gases GIS Geographic Information System GPS Global Position System GRIB General Regularly-distributed Information in Binary form JSON JavaScript Object Notation MEEO Meteorological Environmental Earth Observation netCDF Network Common Data Form REPL Read-Eval-Print Loop TRL Technology Readiness Level WRF Weather Research and Forecasting QTraffic Traffic Emission and Energy Consumption Model WP3 - Atmospheric Research Services www.neanias 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Appendix 1 The following table summarizes the information on the validation of the Atmospheric Services including: the number of end users validating the service, their category (Student/Academic/Research/SMEs), their affiliation and their overall feedback. Atmospheric Service Number of Users Category Affiliation Overall Feedback ATMO-FLUD 37 Students and Researchers Mostly with Democritus University of Xanthi, NKUA, ATHENA, CERTH All executions were successful (100%), strong positive feedback. ATMO-STRESS 65 Students and Researchers University of Milan-Bicocca 88/90 successful executions (97.8%), in 84% of cases the result is consistent with input data ATMO-SEISM 13 Students and Researchers University of Milan-Bicocca 50/76 successful executions (66%) ATMO-4CAST 68 Students and Researchers University of Aveiro 96/122 successful executions (80%) ATMO-FLUD: No failures recorded, and even though feedback forms were not submitted, verbal communication with the audience of the workshop was highly positive. ATMO-FLUD: No failures recorded, and even though feedback forms were not submitted, erbal communication with the audience of the workshop was highly positive. ATMO-STRESS: The failed use cases related to the ATMO-STRESS service validation are mainly due to a system crash when the user clicks the “estimate parameters” button for the first validation after registering for the service. ATMO-SEISM: The failed Use Cases related to the ATMO-SEISM Service validation are mainly due to the complexity of the interface of the Jupyter Notebook, and with problems related with the use of .xlsx input files. However, also in the successful cases some improvements are proposed by the users, as indicated in paragraph 4.2.2. ATMO-4CAST: The failed Use Cases related to the ATMO-4CAST Service validation were due to incomplete input data, especially with the users that were not related to the weather or air quality domains. Although, some new users did not belong to these domains, they have Page 37 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) proposed some useful improvements related to the UI and how to improve the web service presentation. proposed some useful improvements related to the UI and how to improve the web service presentation. Page 38 of 59 www.neanias 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Appendix 2 The validation procedures have been prepared for the Atmospheric Services following the same template as the one prepared for the D3.4 and have been delivered from the Google Forms to simplify the collection of the responses and the analysis of the outcome. Following the list of the forms: - Atmo-Flud validation form: - Atmo-Stress validation form: https://files.neanias.eu/apps/forms/MCLjeFRHS7H6NGm - Atmo-Seism validation form: https://files.neanias.eu/apps/forms/M7N8bLZMgis6f - Atmo-4cast validation form: https://docs.google.com/forms/d/1sgS5C9ELXWte0qywgdhrgqIltI_imulwFiypKhadl3M/pr efill (used before creating forms in the service); Weather module - https://files.neanias.eu/apps/forms/imyooaaZkX7XKqWf ; Emission module - https://files.neanias.eu/apps/forms/LBXA9EnezsKGy3wi; Air Quality module - https://files.neanias.eu/apps/forms/SjYEaPYWTkFpeNnm - Atmo-4cast validation form: https://docs.google.com/forms/d/1sgS5C9ELXWte0qywgdhrgqIltI_imulwFiypKhadl3M/pr efill (used before creating forms in the service); Weather module - https://files.neanias.eu/apps/forms/imyooaaZkX7XKqWf ; Emission module - https://files.neanias.eu/apps/forms/LBXA9EnezsKGy3wi; Air Quality module - https://files.neanias.eu/apps/forms/SjYEaPYWTkFpeNnm ATMO-FLUD feedback Please note that reported feedback is aggregate for all validation rounds, as we could not obtain results for only the 3rd validation round. WP3 - Atmospheric Research Services Page 39 of 59 For Eddy Covariance: 78 responses What is your level of expertise in the micro-metorology domain? Multiple choice 1. 51 (65%): Pre-grad student 2. 17 (22%): Post-grad student 3. 5 (6%): Phd in the area 4. 4 (5%): Expert 5. 1 (1%): What is your level of expertise in the micro-metorology domain? 6. 0 (0%): No response How was your experience using our web interface? Multiple choice 1. 33 (42%): 5 (Excellent) 2. 31 (40%): 4 (Good) 3. 11 (14%): 3 (Average) 4. 3 (4%): 2 (Bad) 5. 0 (0%): No response 6. 0 (0%): 1 (Poor) For Eddy Covariance: Page 39 of 59 www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 40 of 59 Please provide your comments on the web interface (if any) Long text • 59 (76%): No response • Maybe lengthy form button actions (which result in long page loading) could directly lead to a new page, with the job submission being done in the background, communicating with the new page when job submission is done. • It would be helpful if a message were displayed while a job is being dispatched but hasn't yet started • please make it faster • There was a delay of showing the plots with the results • The web interface is intuative and easy to use. • I think is very nice and if you like to find the real results this is (NEANIAS) the best of all !!! • really well built • It's a good experience. • It should be a sign up button • Nice graphical presentation,easy to use and navigate into. • • it is a good program • Easy to use.Overall, ''catchy'' and with an aesthetic graphical presentation. • My experience using your wed, was very pleasant and easy to use. • good • /// • It was really easy to use by giving details on each step. • it is a nice program • good How satisfied are you with the results? Multiple choice 1. 38 (49%): 5 (Excellent) 2. 30 (38%): 4 (Good) 3. 9 (12%): 3 (Average) 4. 1 (1%): 2 (Bad) 5. 0 (0%): No response 6. 0 (0%): 1 (Poor) Please provide your comments on the results (if any) Long text • 64 (82%): No response • very nice diagrams • Results are well presented • is very clear and real • Please provide your comments on the web interface (if any) WP3 - Atmospheric Research Services Page 40 of 59 Please provide your comments on the web interface (if any) Long text • 59 (76%): No response • Maybe lengthy form button actions (which result in long page loading) could directly lead to a new page, with the job submission being done in the background, communicating with the new page when job submission is done. • It would be helpful if a message were displayed while a job is being dispatched but hasn't yet started • please make it faster • There was a delay of showing the plots with the results • The web interface is intuative and easy to use. • I think is very nice and if you like to find the real results this is (NEANIAS) the best of all !!! • really well built • It's a good experience. • It should be a sign up button • Nice graphical presentation,easy to use and navigate into. • • it is a good program • Easy to use.Overall, ''catchy'' and with an aesthetic graphical presentation. • My experience using your wed, was very pleasant and easy to use. • good • /// • It was really easy to use by giving details on each step. • it is a nice program • good How satisfied are you with the results? Multiple choice 1. 38 (49%): 5 (Excellent) 2. 30 (38%): 4 (Good) 3. 9 (12%): 3 (Average) 4. 1 (1%): 2 (Bad) 5. 0 (0%): No response 6. 0 (0%): 1 (Poor) Please provide your comments on the results (if any) Long text • 64 (82%): No response • very nice diagrams • Results are well presented • is very clear and real • • 59 (76%): No response Page 40 of 59 www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 41 of 59 • Excellent • Accurate,quick to show. • • it is a good program • Accurate. • Valid results and useful for the future. • bad • /// • it is a nice program • good Was the time to produce the results reasonable? Multiple choice 1. 32 (41%): 5 (Excellent) 2. 29 (37%): 4 (Good) 3. 14 (18%): 3 (Average) 4. 3 (4%): 2 (Bad) 5. 0 (0%): No response 6. Please provide your comments on the web interface (if any) 0 (0%): 1 (Poor) How satisfied are you with the presentation of the results? Multiple choice 1. 39 (50%): 5 (Excellent) 2. 26 (33%): 4 (Good) 3. 11 (14%): 3 (Average) 4. 1 (1%): No response 5. 1 (1%): 2 (Bad) 6. 0 (0%): 1 (Poor) Please provide your comments on the presentation of the results (if any) Long text • 67 (86%): No response • Results are presented with clarity and easily accessible • Very fast results at a little time unbelievable • • The presentation of the results is really good,as it is organized and nicely shown. • • Accurate and easy to keep track of. • Excellent • nice • /// • The results were well organized making it easier to understand it. • good 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 41 of 59 • Excellent • Accurate,quick to show. • • it is a good program • Accurate. • Valid results and useful for the future. • bad • /// • it is a nice program • good Was the time to produce the results reasonable? Multiple choice 1. 32 (41%): 5 (Excellent) 2. 29 (37%): 4 (Good) 3. 14 (18%): 3 (Average) 4. 3 (4%): 2 (Bad) 5. 0 (0%): No response 6. 0 (0%): 1 (Poor) How satisfied are you with the presentation of the results? Multiple choice 1. 39 (50%): 5 (Excellent) 2. 26 (33%): 4 (Good) 3. 11 (14%): 3 (Average) 4. 1 (1%): No response 5. 1 (1%): 2 (Bad) 6. 0 (0%): 1 (Poor) Please provide your comments on the presentation of the results (if any) Long text • 67 (86%): No response • Results are presented with clarity and easily accessible • Very fast results at a little time unbelievable • • The presentation of the results is really good,as it is organized and nicely shown. • • Accurate and easy to keep track of. • Excellent • nice • /// • The results were well organized making it easier to understand it. • good Page 41 of 59 www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 42 of 59 In what format would you like your results to be delivered? (e.g. WP3 - Atmospheric Research Services Please provide your comments on the web interface (if any) .dat, .csv, .txt) Short answer • 41 (53%): No response • CSV • .csv • .csv • .txt, .xlsx • csv • .csv and .txt • .txt • .txt • .txt • txt • .txt • .txt • .txt • .txt • .csv • • .dat • .txt • csv • csv • .txt • dat • dat • I would prefer the .csv format, as im more accustomed and used to it,due to my studies. • .csv • I would prefer a .csv format, as i am accustomed to it and much more comfortable with it. • .csv • - • .txt • beautifull • /// • .csv • .csv or .dat • .csv • • .dat • good WP3 - Atmospheric Research Services Page 42 of 59 In what format would you like your results to be delivered? (e.g. .dat, .csv, .txt) Short answer • 41 (53%): No response • CSV • .csv • .csv • .txt, .xlsx • csv • .csv and .txt • .txt • .txt • .txt • txt • .txt • .txt • .txt • .txt • .csv • • .dat • .txt • csv • csv • .txt • dat • dat • I would prefer the .csv format, as im more accustomed and used to it,due to my studies. • .csv • I would prefer a .csv format, as i am accustomed to it and much more comfortable with it. • .csv • - • .txt • beautifull • /// • .csv • .csv or .dat • .csv • • .dat • good www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 A h i R h S i P 43 f 59 Why did you choose to use our service? Long text • 0 (0%): No response • Workshop • research interest • Internal evaluation of the service • • for satisfied result • Demonstration at ATHENA RC • training service for validation and feedback • Seminar • as a part of a seminar • I wanted to evaluate the algorithms, and see how it works • I was curious about the results. • To get accustomed with tools used in this area of study • I was informed about it from a workshop. Please provide your comments on the web interface (if any) • workshop • validation • Because is the best service and they have very fast results in few minutes • Because it is the best service. • this service was used for a university project • i was curious • because of university studies • cause lupa told me to • I found it very interesting • Τo calculate energy flows in the Atmosphere • i found your site really interesting and i wanted to test it • because the site was very interesting and i wanted to try this experience • I found your site really interesting and i wanted to try it. • I found your site really interesting and i wanted to try it. • to do an exercise • School project • This service was chosen for a university project • It is interesting • I was assigned to make a presantation using the service. • Because it is a quick and reliable service to get great results • Για να κάνω την εργασία με βάση την ατμοσφαιρική ροή • to do an exercise • To do an assignment • they suggested me this service • Because my teacher told me that it is useful and I like it • the teacher in the univesrity told us aboyt thiw service and i find this very interesting Page 43 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) • our teacher in the uni told us about this service and i find this very interestring • For an excercise in the field of air pollution. • It was suggested by the professor • First, your service was very useful for an exercise-study for my university and also It could give many opportunities to students in the future • It was part of a lab project from my professor who highly recommended it. • !!!! • i choose it beacause i need it for my university studies • it is a good program • fffj • for a project • learn • because it is a trusted source of information • University exercises. • Because very ggod • it is a good program • For a job at university. • For my studies • University obligations and research. • Because I heard that it is very interesting. Please provide your comments on the web interface (if any) • Because of an assignment in my university • For academic research • I need it for a project • Because I heard that is good. • - • because it gives reliable results • my university professor recomended it • average • /// • Because i wanted to get feedback from users about atmospheric flow vortex fluctuation • It is an opportunity to interact in some way with all this data which are a part of a tool for my future • Highly suggested from my university. • I chose the service as a part of an exercise to better understand the facts about atmospheric flux densities. • i wanted to try out your program because i find it interesting • for a project at the university • it is a nice program • good • for e research • Because it's something new in the enviromental domain • I need it WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 45 of 59 Please provide your general comments you have on our service (if any) Long text • 65 (83%): No response • • Maybe include an option 'none' for Q1 of the evaluation form • Easy, clean UI, some delay was due to network load. • it is very usefull and easy to use it • The service is a great tool for beginners and experts alike,providing everyone with a great variety of data on the sets given by the user, • • Easy to use,accurate depiction of results,and with a nice user interface. • It is really easy to use. • - • • nice • /// • good For Gradient Method: 63 responses What is your level of expertise in the micro-metorology domain? Multiple choice 1. 46 (73%): Pre-grad student 2. 9 (14%): Post-grad student 3. 5 (8%): Phd in the area 4. 3 (5%): Expert 5. 0 (0%): No response How was your experience using our web interface? Multiple choice 1. 28 (44%): 5 (Excellent) 2. 23 (37%): 4 (Good) 3. 11 (17%): 3 (Average) 4. 1 (2%): 2 (Bad) 5. 0 (0%): No response 6. Please provide your comments on the web interface (if any) 0 (0%): 1 (Poor) Please provide your comments on the web interface (if any) Long text • 59 (94%): No response Please provide your general comments you have on our service (if any) Long text • 65 (83%): No response • • Maybe include an option 'none' for Q1 of the evaluation form • Easy, clean UI, some delay was due to network load. • it is very usefull and easy to use it • The service is a great tool for beginners and experts alike,providing everyone with a great variety of data on the sets given by the user, • • Easy to use,accurate depiction of results,and with a nice user interface. • It is really easy to use. • - • • nice • /// • good For Gradient Method: Page 45 of 59 www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 46 of 59 • A 'dispatching job' message after starting calculations would improve user experience • smooth navigation, user friendly • gg • good How satisfied are you with the results? Multiple choice 1. 32 (51%): 5 (Excellent) 2. 21 (33%): 4 (Good) 3. 7 (11%): 3 (Average) 4. 3 (5%): 2 (Bad) 5. 0 (0%): No response 6. 0 (0%): 1 (Poor) Please provide your comments on the results (if any) Long text • 60 (95%): No response • very good • gj • good Was the time to produce the results reasonable? Multiple choice 1. 33 (52%): 4 (Good) 2. 15 (24%): 3 (Average) 3. 14 (22%): 5 (Excellent) 4. 1 (2%): 2 (Bad) 5. 0 (0%): No response 6. 0 (0%): 1 (Poor) How satisfied are you with the presentation of the results? Multiple choice 1. 26 (41%): 5 (Excellent) 2. 23 (37%): 4 (Good) 3. 10 (16%): 3 (Average) 4. 2 (3%): No response 5. 2 (3%): 2 (Bad) 6. 0 (0%): 1 (Poor) Please provide your comments on the presentation of the results (if any) Long text • 58 (92%): No response • more view options could be provided (list, grid) WP3 - Atmospheric Research Services Page 46 of 59 • A 'dispatching job' message after starting calculations would improve user experience • smooth navigation, user friendly • gg • good How satisfied are you with the results? Multiple choice 1. 32 (51%): 5 (Excellent) 2. Please provide your comments on the web interface (if any) 21 (33%): 4 (Good) 3. 7 (11%): 3 (Average) 4. 3 (5%): 2 (Bad) 5. 0 (0%): No response 6. 0 (0%): 1 (Poor) Please provide your comments on the results (if any) Long text • 60 (95%): No response • very good • gj • good Was the time to produce the results reasonable? Multiple choice 1. 33 (52%): 4 (Good) 2. 15 (24%): 3 (Average) 3. 14 (22%): 5 (Excellent) 4. 1 (2%): 2 (Bad) 5. 0 (0%): No response 6. 0 (0%): 1 (Poor) How satisfied are you with the presentation of the results? Multiple choice 1. 26 (41%): 5 (Excellent) 2. 23 (37%): 4 (Good) 3. 10 (16%): 3 (Average) 4. 2 (3%): No response 5. 2 (3%): 2 (Bad) 6. 0 (0%): 1 (Poor) Please provide your comments on the presentation of the results (if any) Long text • 58 (92%): No response • more view options could be provided (list, grid) www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 47 of 59 • ηταν μια ωραια παρουσιση με εδνιαφερον • very good • ez • good In what format would you like your results to be delivered? (e.g. .dat, .csv, .txt) Short answer • 43 (68%): No response • CSV • csv • .csv • csv • .csv • dat • txt • I would like to receive me results in ".csv" format . • .csv • .csv , .pdf • csc,txt • no • .dat • dat • .txt • .txt • csv • .dat • lol • good Why did you choose to use our service? Long text • 0 (0%): No response • Internal evaluation • • Demonstration at ATHENA RC • satisfied • To evaluate its operation • I was curious about the results • validation • because i am gamatos • I used your service once before and it was great! • for an exercise • for my excercise • to do an excersize Page 47 of 59 www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 48 of 59 • For my project. • for academic purposes • cause you seem reliable • I need it for a project • highly suggested from my university! WP3 - Atmospheric Research Services Please provide your comments on the web interface (if any) • through the university • curiosity • Για να κάνω τις ασκήσεις για ρύπανση • for a class exercise !!! • for a university project • Needed for university research • for exam • Cause i heard that your service is excellent • i needed for my university studies • For a job at the university. • Through the university • Professor suggested it • Because your service is excellent!!! • Because it is a fast and easy way to calculate anything you wish about the atmospheric densities. • Because it's easy to use and very helpful for my school projects • Βecause it is a good experience. • Εργασια για το πανεπιστημιο • To familiarise with using the gradient method to derive atm flux densities graphs • για να μαθω νεα πραγματα που δεν γνωριζα • my professor recommended this service • it was part of a college assignment that required that kind of data. • for a project at my university • i heared from a friend that is good expireance • i heard from a friend that is good experiance • idk • Because I am doing a research and this programm is very useful and reliable • very good • The details of the results are incredible • for homework • the teacher told us about this service and i find this very interesting • universtity project • good • !!! • Due to a job on air pollution. • university projects • for my university • For my studies in university. • My teacher suggest me this Page 48 of 59 www.neanias.eu www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 49 of 59 • they suggested it to me • Becayse this service is the best of all with a clear and very fast results • I used the service for educational purposes. Please provide your comments on the web interface (if any) Whenever a process is running a sign should show to the user that something is happening - the bars of showing the progress of the process should be closer to the commands. The final message after or before the “Download” should be “successful running or execution of the A2 service” and then “Pls visit our Evaluation Form” • The web interface is easy and intuative to use • It could be more intuitive • It is intuitive • It is easy to use and intuitive How satisfied are you with the results? Multiple choice • 1. 20 (50%): 4 (Good) • 2. 10 (25%): 5 (Excellent) • 3. 9 (23%): 3 (Average) • 4. 1 (3%): 2 (Bad) • 5. 0 (0%): No response • 6. 0 (0%): 1 (Poor) Please provide your comments on the results (if any) Long text • 35 (88%): No response • Satisfactory - more information on what is presented is needed ( pls ask if the results on the scientific part are fine) • The results are interesting and presented with clarity • Sometimes the results are not geologically accurate compared to the input data • In some cases, the results of the interpolation are not correct Was the time to produce the results reasonable? Multiple choice • 1. 14 (35%): 3 (Average) • 2. 14 (35%): 4 (Good) • 3. 6 (15%): 5 (Excellent) • 4 5 (13%) 2 (Bad) • 1. 17 (43%): 4 (Good) • 2. 14 (35%): 5 (Excellent) • 3. 8 (20%): 3 (Average) • 4. 1 (3%): 1 (Poor) • 5. 0 (0%): No response • 6. 0 (0%): 2 (Bad) Please provide your comments on the web interface (if any) Long text Please provide your comments on the web interface (if any) Long text • 34 (85%): No response • 34 (85%): No response • the user should informed in every step what is going on and which is the next step. Whenever a process is running a sign should show to the user that something is happening - the bars of showing the progress of the process should be closer to the commands. Please provide your comments on the web interface (if any) • because they told me to • it's a good service • it is a good program • good • it was suggested by the university Please provide your general comments you have on our service (if any) Long text • 59 (94%): No response • • ειναι μια πολυ καλη εφαρμογη που σου μαθαινει συχνα νεα πραγματα • bad • good ATMO-STRESS feedback The results we obtained for Atmo-Stress are the following: User Feedback for Atmo-Stress 40 responses Summary Responses What is your level of expertise in stress field recognition domain? Multiple choice • 1. 16 (40%): None • 2. 12 (30%): Pre-grad student in this sector • 3. 7 (18%): PhD student in this sector • 4. 4 (10%): Expert • 5. 1 (3%): Post-grad student in this sector • 6. 0 (0%): No response On which browser did you use the service? Multiple choice • 1. 23 (58%): Chrome • 2. 8 (20%): Safari • 3. 6 (15%): No response • 4. 2 (5%): Firefox • 5. 1 (3%): Other • 6. 0 (0%): Edge How user-friendly was the interface? Multiple choice 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) • they suggested it to me • Becayse this service is the best of all with a clear and very fast results • I used the service for educational purposes. • because they told me to • it's a good service • it is a good program • good • it was suggested by the university Please provide your general comments you have on our service (if any) Long text • 59 (94%): No response • • ειναι μια πολυ καλη εφαρμογη που σου μαθαινει συχνα νεα πραγματα • bad • good Page 49 of 59 www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) • 1. 17 (43%): 4 (Good) • 2. 14 (35%): 5 (Excellent) • 3. 8 (20%): 3 (Average) • 4. 1 (3%): 1 (Poor) • 5. 0 (0%): No response • 6. 0 (0%): 2 (Bad) Please provide your comments on the web interface (if any) Long text • 34 (85%): No response • the user should informed in every step what is going on and which is the next step. Please provide your comments on the web interface (if any) • it is clear enough • The results are presented in a clear way How satisfied are you with the output formats of the results (e.g. .shp, .kml, .png, .json)? Multiple choice • 1. 19 (48%): 4 (Good) • 2. 16 (40%): 5 (Excellent) • 3. 4 (10%): 3 (Average) • 4. 1 (3%): 1 (Poor) • 5. 0 (0%): No response • 6. 0 (0%): 2 (Bad) If you are not satisfied, in what format would you like your results to be delivered? Long text • 36 (90%): No response • pdf,png • excell - world – pictures • .xlsx Would you recommend the service to your colleagues? Multiple choice • 1. 33 (83%): Yes • 2. 6 (15%): No response • 3. 1 (3%): No How did you find out about the service? Long text • 6 (15%): No response WP3 - Atmospheric Research Services Page 51 of 59 • 6. 0 (0%): No response How satisfied are you with the presentation of the results? Multiple choice • 1. 25 (63%): 4 (Good) • 2. 11 (28%): 5 (Excellent) • 3. 4 (10%): 3 (Average) • 4. 0 (0%): No response • 5. 0 (0%): 1 (Poor) • 6. 0 (0%): 2 (Bad) Please provide your comments on the presentation of the results (if any) Long text • 35 (88%): No response • more info are needed • The presentation is really easy to understand and use • It could be useful to integrate the coordinates on the resulting map (i.e. 45N is different than 45S) and to have an arrow pointing towards the north. • it is clear enough • The results are presented in a clear way How satisfied are you with the output formats of the results (e.g. .shp, .kml, .png, .json)? Multiple choice • 1. 19 (48%): 4 (Good) • 2. 16 (40%): 5 (Excellent) • 3. 4 (10%): 3 (Average) • 4. 1 (3%): 1 (Poor) • 5. 0 (0%): No response • 6. 0 (0%): 2 (Bad) If you are not satisfied, in what format would you like your results to be delivered? Long text • 36 (90%): No response • pdf,png • excell - world – pictures • .xlsx Would you recommend the service to your colleagues? Multiple choice • 1. 33 (83%): Yes • 2. 6 (15%): No response • 3. Please provide your comments on the web interface (if any) The final message after or before the “Download” should be “successful running or execution of the A2 service” and then “Pls visit our Evaluation Form” • 34 (85%): No response • the user should informed in every step what is going on and which is the next step. Whenever a process is running a sign should show to the user that something is happening - the bars of showing the progress of the process should be closer to the commands. The final message after or before the “Download” should be “successful running or execution of the A2 service” and then “Pls visit our Evaluation Form” • The web interface is easy and intuative to use • It is easy to use and intuitive How satisfied are you with the results? Multiple choice • 1. 20 (50%): 4 (Good) • 2. 10 (25%): 5 (Excellent) • 3. 9 (23%): 3 (Average) • 4. 1 (3%): 2 (Bad) • 5. 0 (0%): No response • 6. 0 (0%): 1 (Poor) Please provide your comments on the results (if any) Long text Please provide your comments on the results (if any) Long text • 35 (88%): No response • Satisfactory - more information on what is presented is needed ( pls ask if the results on the scientific part are fine) • The results are interesting and presented with clarity • Sometimes the results are not geologically accurate compared to the input data • In some cases, the results of the interpolation are not correct Page 50 of 59 www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) p p p ( ) WP3 - Atmospheric Research Services Page 51 of 59 • 6. 0 (0%): No response How satisfied are you with the presentation of the results? Multiple choice • 1. 25 (63%): 4 (Good) • 2. 11 (28%): 5 (Excellent) • 3. 4 (10%): 3 (Average) • 4. 0 (0%): No response • 5. 0 (0%): 1 (Poor) • 6. 0 (0%): 2 (Bad) Please provide your comments on the presentation of the results (if any) Long text • 35 (88%): No response • more info are needed • The presentation is really easy to understand and use • It could be useful to integrate the coordinates on the resulting map (i.e. 45N is different than 45S) and to have an arrow pointing towards the north. WP3 - Atmospheric Research Services Please provide your comments on the web interface (if any) 1 (3%): No How did you find out about the service? Long text • 6 (15%): No response If you are not satisfied, in what format would you like your results to be delivered? Long text If you are not satisfied, in what format would you like your results to be delivered Long text Would you recommend the service to your colleagues? Would you recommend the service to your colleagues? Page 51 of 59 www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) • Excellent service, and very user friendly interface ! • Workshop • by NEANIAS • ATMO-Workshop • Internal evaluation • Demonstration at ATHENA RC • from colleagues • from a seminar • During a relative workshop • From a webinar that the group carried out at the University of Athens • Seminar • testing and validating event • neanias workshop • I was informed during a workshop in NKUA. • Workshop • Through university • Colleagues • University • Colleagues at University • NGWM Congress • University • Lezione • Attending structural geology • We use it in our university • University • I was able to attend a lesson given by professor Tibaldi's collaborators during an ordinary lesson • University • On the class • Università • University • University course • Good • University Please provide your general comments on our service (if any) Long text • 34 (85%): No response • Very interesting with high potential • I really like the fact that the shapefiles are generated, this is really a useful format! • The service is a great tool for begginers and experts alike to evaluate their data • The service is easy to use but does not produce results quickly and if you want to switch to the grid map from the trajectory map it crashes Page 52 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) The service is user-friendly and intuitive, the problem is that with some datasets the results are not coherent with the input data. Sometimes the production of the results is slow and the service crashes, and the user has to refresh the page. WP3 - Atmospheric Research Services ATMO-SEISM feedback WP3 - Atmospheric Research Services Page 53 of 59 ATMO-SEISM feedback The results we obtained for Atmo-Seism are the following: User Feedback for Atmo-Seism 12 responses Summary Responses What is your level of expertise in the active tectonics domain? Multiple choice • 7 (58%): PhD student in this sector • 3 (25%): Expert • 2 (17%): post-grad student in this sector • 0 (0%): No response • 0 (0%): None • 0 (0%): Pre-grad student in this sector How user-friendly was the interface? Multiple choice • 9 (75%): 2 (Bad) • 3 (25%): 1 (Poor) • 0 (0%): No response • 0 (0%): 3 (Average) • 0 (0%): 4 (Good) • 0 (0%): 5 (Excellent) Please provide your comments on the web interface (if any) Long text • 4 (33%): No response • It's not intuitive • It's not user-friendly • It is quite difficult to understand how the service works • It is difficult to understand how to run the service • The software was not easy to use, since it is not very user-friendly. • The service is a Jupyter Notebook, so it is not user-friendly and it is difficult to use for people who are not expert with this language • The service does not work, it says there is an internal error The results we obtained for Atmo-Seism are the following: Page 53 of 59 www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 54 of 59 How satisfied are you with the results? Multiple choice • 4 (33%): 3 (Average) • 4 (33%): 2 (Bad) • 3 (25%): 4 (Good) • 1 (8%): 1 (Poor) • 0 (0%): No response • 0 (0%): 5 (Excellent) Please provide your comments on the results (if any) Long text • 6 (50%): No response • With .xlsx files I obtained an error and it was not possible to obtain the results. Everything was ok, on the other hand, with .csv files. • the radon predicted value graph is very complex and confusing: the variation of daily peaks does not allow the graph to be read clearly • Other graphs can be represented, like the filtered gas values vs seismic energy • I did not obtain any result since I was not able to use the software. ATMO-SEISM feedback • The results are ok, but a graph relating the released energy and the filtered gas values (not only measured gas values) should also be presented as a result. It could be better also to present not all the measured values in the graphs, but just the daily averages. • The service does not work, it says there is an internal error Was the time to produce the results reasonable? Multiple choice • 7 (58%): 4 (Good) • 2 (17%): 5 (Excellent) • 1 (8%): 1 (Poor) • 1 (8%): 2 (Bad) • 1 (8%): 3 (Average) • 0 (0%): No response How satisfied are you with the presentation of the results? Multiple choice • 8 (67%): 3 (Average) • 2 (17%): 2 (Bad) • 1 (8%): 1 (Poor) • 1 (8%): 4 (Good) • 0 (0%): No response • 0 (0%): 5 (Excellent) Please provide your comments on the presentation of the results (if any) 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) port on the Developed and Validated Atmospheric Thematic Services (Release #3) How satisfied are you with the results? Multiple choice • 4 (33%): 3 (Average) • 4 (33%): 2 (Bad) • 3 (25%): 4 (Good) • 1 (8%): 1 (Poor) • 0 (0%): No response • 0 (0%): 5 (Excellent) • 6 (50%): No response • With .xlsx files I obtained an error and it was not possible to obtain the results. Everything was ok, on the other hand, with .csv files. • the radon predicted value graph is very complex and confusing: the variation of daily peaks does not allow the graph to be read clearly • Other graphs can be represented, like the filtered gas values vs seismic energy • I did not obtain any result since I was not able to use the software. • The results are ok, but a graph relating the released energy and the filtered gas values (not only measured gas values) should also be presented as a result. It could be better also to present not all the measured values in the graphs, but just the daily averages. ATMO-SEISM feedback • The service does not work, it says there is an internal error www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) WP3 - Atmospheric Research Services Page 55 of 59 Long text • 6 (50%): No response • In some graphs the results are not easy to understand • Some graphs can be improved, some of them are not easily readable because there are too many peaks • Some graphs can be improved and are not very readable • I did not obtain any result since I was not able to use the software. • Some improvements can be made regarding the map of the hypocenters, where a scalebar should be added, and it would be better to add a basemap. In the graph relating longitude and latitude vs depth, the dots should be scaled based on magnitude. • The service does not work, it says there is an internal error How easy was the download of the results? Multiple choice • 7 (58%): 2 (Bad) • 2 (17%): 1 (Poor) • 2 (17%): 3 (Average) • 1 (8%): 4 (Good) • 0 (0%): No response • 0 (0%): 5 (Excellent) How satisfied are you with the output format of the results? Multiple choice • 5 (42%): 3 (Average) • 5 (42%): 2 (Bad) • 1 (8%): 1 (Poor) • 1 (8%): 4 (Good) • 0 (0%): No response • 0 (0%): 5 (Excellent) Please provide your comments on the output format of the results (if any) Long text • 4 (33%): No response • Other formats can be useful, like image files • Probably it could be useful to download the single graphs as images (.png or .jpg) • I didn't understand how I could download the results • How do I can download the results? • I did not manage to download the single images • I did not obtain any result since I was not able to use the software. • I managed just to download the pdf with all the results of the validation. ATMO-SEISM feedback I think that it would be useful to have the resulting graphs as image files (like .png or .jpg) • The service does not work, it says there is an internal error 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Long text • 6 (50%): No response • In some graphs the results are not easy to understand • Some graphs can be improved, some of them are not easily readable because there are too many peaks • Some graphs can be improved and are not very readable • I did not obtain any result since I was not able to use the software. • Some improvements can be made regarding the map of the hypocenters, where a scalebar should be added, and it would be better to add a basemap. In the graph relating longitude and latitude vs depth, the dots should be scaled based on magnitude. Th i d k i h i i l • 4 (33%): No response • Other formats can be useful, like image files • Probably it could be useful to download the single graphs as images (.png or .jpg) • I didn't understand how I could download the results • How do I can download the results? • I did not manage to download the single images • I did not obtain any result since I was not able to use the software. • I managed just to download the pdf with all the results of the validation. I think that it would be useful to have the resulting graphs as image files (like .png or .jpg) • The service does not work, it says there is an internal error Page 55 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Please provide your general comments on our service (if any) Long text • 7 (58%): No response • The service could have a more user-friendly interface and be more intuitive • The service is not user-friendly and some graphs can be added as results. It is not clear how to download the results. • In general, the service works, but it is not user-friendly. Some improvements can be done both regarding the results, and how they are presented. ATMO-SEISM feedback It is not easy to download the results, and it would be useful to download just the graphs as image files. • The service does not work, it says there is an internal error • The service does not work, it says there is an internal error WP3 - Atmospheric Research Services Page 56 of 59 ATMO-4CAST feedback For ATMO-4CAST, the following forms were used to obtain the user feedback. User Feedback for Atmo-4Cast 24 responses Summary Responses What is your level of expertise in the meteorology / air quality domain? Multiple choice • 18 (75%): None • 4 (17%): Pre-grad student in this sector • 1 (4%): Post-grad student in this sector • 1 (4%): Expert • 0 (0%): PhD student in this sector How was your experience using our web interface? Multiple choice • 3 (13%): 1 (Poor) • 2 (8%): 2 (Bad) • 2 (8%): 3 (Average) • 10 (42%): 4 (Good) • 7 (29%): 5 (Excellent) Please provide your comments on the web interface (if any) Long text • 14 (58%): No response • UI is easy to use, maybe showing estimated time or a % bar filling would be useful WP3 - Atmospheric Research Services Page 56 of 59 ATMO-4CAST feedback For ATMO-4CAST, the following forms were used to obtain the user feedback. User Feedback for Atmo-4Cast 24 responses Summary Responses What is your level of expertise in the meteorology / air quality domain? Multiple choice • 18 (75%): None • 4 (17%): Pre-grad student in this sector • 1 (4%): Post-grad student in this sector • 1 (4%): Expert • 0 (0%): PhD student in this sector How was your experience using our web interface? Multiple choice • 3 (13%): 1 (Poor) • 2 (8%): 2 (Bad) • 2 (8%): 3 (Average) • 10 (42%): 4 (Good) • 7 (29%): 5 (Excellent) Please provide your comments on the web interface (if any) Long text • 14 (58%): No response • UI is easy to use, maybe showing estimated time or a % bar filling would be useful ATMO-4CAST feedback www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) • It gives the necessary context and information to create knowledge and address different topics. p • Having a progress bar for the simulation would be nice. p • Having a progress bar for the simulation would be nice. ATMO-SEISM feedback • It is not possible of registering with a new email, only with existing hotmail or google accounts. After logging out -> interacting with the page, the login will be done automatically, without asking for credentials. y g • “NEANIAS”, “UNDERWATER”, “SPACE”, “GUIDE”, “HELP” buttons are not clickable; “My Account” is not clickable; When a simulation is "Running" it is necessary to refresh the page so that the state is up to date; “Here” which is in the description of “new weather forecast” redirects to login; by “Here” in “New emission simulator” and from “New Air quality” redirect directly to the documentation – it should be uniform; The right column must be enlarged, so that when hovering over these buttons they do not occupy two lines of text. When clicking on “Documentation”, the link must be opened in a new tab, so that the user loses the platform page - facilitate the reading of the documentation while the platform is not explored; It's not very intuitive on the first iteration - there should be pop ups on an interaction, or bring drafts from the documentation (which is in the lower left corner) to the upper left corner; so that the user knows better what to do first (read the documentation). • For someone with little to no prior knowledge on the subject the interface is a bit too technical and unfriendly. I tried to do a test report with the dummy information • A lot of refresing is needed - Ovelapping of the selections on the left part of the screen - Hard to be informed and use the Feedback Form - Wrong position of the form • Maybe the UI could converge a bit more among the services. For instance, user feedback was in a different place in each service. • The only problem I locate is that all different services have the feedback button at different positions in the website. Some homogenisation should be preferable. • The web interface is intuitive and easy to use. WP3 - Atmospheric Research Services Page 57 of 59 Which module did you use? Multiple choice • 3 (13%): 1 (Weather forecast) • 4 (17%): 2 (Emission simulation) • 14 (58%): 4 (Air quality simulation) • 3 (13%): 1 (All) How satisfied are you with the results? ATMO-SEISM feedback Multiple choice • 3 (13%): 1 (Poor) • 1 (4%): 2 (Bad) • 0 (0%): 3 (Average) • 10 (42%): 4 (Good) • 10 (42%): 5 (Excellent) Page 57 of 59 www.neanias.eu www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Please provide your comments on the results (if any) Long text • 18 (75%): No response • Alot of metrics to analyse, very complete • Simulation date and time is not correct - must be simulation time • Use of module was poorly done • Same as mentioned before, I wasn't able to understand the results so I can't say I was satisfied with them. Not sure what they were. • very interesting - more information is needed • The results are comprehensive. Was the time to produce the results reasonable? Multiple choice • 1 (4%): 1 (Poor) • 3 (13%): 2 (Bad) • 1 (4%): 3 (Average) • 9 (38%): 3 (Good) • 10 (42%): 4 (Excellent) How satisfied are you with the presentation of the results? Multiple choice • 3 (13%): 1 (Poor) • 2 (8%): 2 (Bad) • 1 (4%): 3 (Average) • 8 (33%): 3 (Good) • 10 (42%): 4 (Excellent) Please provide your comments on the presentation of the results (if any) Long text • 18 (75%): No response • The platform must be as interactive as possible - there are graphics resulting from the simulations, so that it can be adapted to a greater number of users; output format: it should be possible to open the output files (formatted) on the platform itself or to see the results directly on the platform (to make it more user friendly). • If possible to show the results in a more graphical way, would benefit the platform more. • Could not understand • Please include the name of the axes in the diagrams if possible • Do you mean the scientific aspect of the results? More info and explanations are needed • The presentation of the results is easy to understand and great quality. ATMO-SEISM feedback 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) Please provide your comments on the results (if any) Long text • 18 (75%): No response • Alot of metrics to analyse, very complete • Simulation date and time is not correct - must be simulation time • Use of module was poorly done • Same as mentioned before, I wasn't able to understand the results so I can't say I was satisfied with them. Not sure what they were. • very interesting - more information is needed • The results are comprehensive. Please provide your comments on the presentation of the results (if any) Long text • 18 (75%): No response • The platform must be as interactive as possible - there are graphics resulting from the simulations, so that it can be adapted to a greater number of users; output format: it should be possible to open the output files (formatted) on the platform itself or to see the results directly on the platform (to make it more user friendly). • If possible to show the results in a more graphical way, would benefit the platform the simulations, so that it can be adapted to a greater number of users; output format: it should be possible to open the output files (formatted) on the platform itself or to see the results directly on the platform (to make it more user friendly). • If possible to show the results in a more graphical way, would benefit the platform more. • Could not understand • Please include the name of the axes in the diagrams if possible • Please include the name of the axes in the diagrams if possible • Do you mean the scientific aspect of the results? More info and explanations are needed • The presentation of the results is easy to understand and great quality. Page 58 of 59 WP3 - Atmospheric Research Services www.neanias.eu 3.8 Report on the Developed and Validated Atmospheric Thematic Services (Release #3) How satisfied are you with the output formats of the results (e.g. .png, .json, .netCDF; .dmna, .txt)? Multiple choice • 1 (4%): 1 (Poor) • 3 (13%): 2 (Bad) • 0 (0%): 3 (Average) • 9 (38%): 3 (Good) • 10 (42%): 4 (Excellent) Why did you choose to use our service? WP3 - Atmospheric Research Services ATMO-SEISM feedback Long text • 6 (25%): No response • MCTW Workshop • I was asked to try • Easy to use • Researching and testing several similar services • Used in order to test it • research interest • Workshop • by NEANIAS • I wanted to try the service to understand better how to test air quality • Demonstration at ATHENA RC • Internal evaluation • For evaluation • I was curious about the results • Seminar • training and evaluation event • I was informed about it from a workshop in NKUA. • workshop • To get better acquainted with air quallity simulations Please provide your general comments on our service (if any) Long text • 20 (83%): No response • Good concept, could benefit from having a better result presentation. • Not the best person to use such thing. • more explanation on the process, scientifc explanations, running, use friendliness are needed. • The service is great for beginners and experts alike. Page 59 of 59 WP3 - Atmospheric Research Services
https://openalex.org/W2555453841	https://europepmc.org/articles/pmc5129340?pdf=render	English	null	Effects of Di-(2-ethylhexyl) Phthalate on the Hypothalamus–Uterus in Pubertal Female Rats	International journal of environmental research and public health/International journal of environmental research and public health	2,016	cc-by	11,194	Te Liu 1,2,†, Yiyang Jia 1,†, Liting Zhou 1, Qi Wang 1, Di Sun 1, Jin Xu 1, Juan Wu 3, Huaiji Chen 1, Feng Xu 1 and Lin Ye 1,* Te Liu 1,2,†, Yiyang Jia 1,†, Liting Zhou 1, Qi Wang 1, Di Sun 1, Jin Xu 1, Juan Wu 3, Huaiji Chen 1, Feng Xu 1 and Lin Ye 1,* Te Liu 1,2,†, Yiyang Jia 1,†, Liting Zhou 1, Qi Wang 1, Di Sun 1, Jin Xu 1, Juan Wu 3, Huaiji Chen Feng Xu 1 and Lin Ye 1,* 1 Department of Occupational and Environmental Health, School of Public Health, Jilin University, Changchun 130021, China; iamliute@126.com (T.L.); jiayy14@mails.jlu.edu.cn (Y.J.); zhoulttg@163.com (L.Z.) wangqi15@mails.jlu.edu.cn (Q.W.); s184349549@live.com (D.S.); xujin14@mails.jlu.edu.cn (J.X.); chenhj15@mails.jlu.edu.cn (H.C.); xufeng15@mails.jlu.edu.cn (F.X.) 1 Department of Occupational and Environmental Health, School of Public Health, Jilin University, Changchun 130021, China; iamliute@126.com (T.L.); jiayy14@mails.jlu.edu.cn (Y.J.); zhoulttg@163.com (L.Z.); wangqi15@mails.jlu.edu.cn (Q.W.); s184349549@live.com (D.S.); xujin14@mails.jlu.edu.cn (J.X.); chenhj15@mails.jlu.edu.cn (H.C.); xufeng15@mails.jlu.edu.cn (F.X.) 2 Scientiﬁc Research Center, China Japan Union Hospital, Jilin University, Changchun 130000, China 3 Prevention and Health Section, Jiangxi Provincial Cancer Hospital, Nanchang 330000, China; wujuan13@mails.jlu.edu.cn 2 Scientiﬁc Research Center, China Japan Union Hospital, Jilin University, Changchun 130000, China 3 Prevention and Health Section, Jiangxi Provincial Cancer Hospital, Nanchang 330000, China; wujuan13@mails.jlu.edu.cn j j * Correspondence: jlyelin@163.com; Tel.: +86-431-8561-9453; Fax: +86-431-8561-9431 † These authors equally contributed to this work. † These authors equally contributed to this work. Academic Editor: William A. Toscano Received: 19 September 2016; Accepted: 7 November 2016; Published: 12 November 2016 Abstract: The pollution of endocrine disruptors and its impact on human reproductive system have attracted much attention. Di-(2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor, is widely used in food packages, containers, medical supplies and children’s toys. It can cause diseases such as infertility, sexual precocity and uterine bleeding and thus arouse concerns from the society and scholars. The effect of DEHP on pubertal female reproductive system is still not well-studied. This study was to investigate the effects of DEHP on the hypothalamus–uterus in pubertal female rats, reveal the reproductive toxicity of DEHP on pubertal female rats and its mechanism, and provide scientiﬁc evidence for the evaluation of toxicity and toxic mechanism of DEHP on reproductive system. Forty-eight pubertal female rats were randomly divided into four groups and respectively administered via oral gavage 0, 250, 500, or 1000 mg/kg/d DEHP in 0.1 mL corn oil/20 g body weight for up to four weeks. Te Liu 1,2,†, Yiyang Jia 1,†, Liting Zhou 1, Qi Wang 1, Di Sun 1, Jin Xu 1, Juan Wu 3, Huaiji Chen 1, Feng Xu 1 and Lin Ye 1,* Compared with control rats, the DEHP-treated rats showed: (1) higher gonadotropin-releasing hormone (GnRH) level in the hypothalamus; (2) higher protein levels of GnRH in the hypothalamus; and (3) higher mRNA and protein levels of GnRH receptor (GnRHR) in the uterus. Our data reveal that DEHP exposure may lead to a disruption in pubertal female rats and an imbalance of hypothalamus–uterus. Meanwhile, DEHP may, through the GnRH in the hypothalamus and its receptor on the uterus, lead to diseases of the uterus. DEHP may impose a negative inﬂuence on the development and functioning of the reproductive system in pubertal female rats. Keywords: di-(2-ethylhcxyl) phthalate; hypothalamus; uterus; reproductive toxicity; pubertal International Journal of Environmental Research and Public Health 1. Introduction Di-(2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor, is one of the organic chemicals that is used in a large variety of industrial and consumer applications. It is the most commonly used plasticizer worldwide [1–3]. An endocrine disruptor alters the function of the endocrine systems and causes adverse effects in organisms [4]. DEHP has been approved for use and has also been detected in a wide range of consumer products [5,6], including personal care products [7], infant toys [8], food wraps [9], cosmetics, medical consumables, nutritional supplements, cleaning materials, lubricants, insecticides, solvents, Int. J. Environ. Res. Public Health 2016, 13, 1130; doi:10.3390/ijerph13111130 www.mdpi.com/journal/ijerph www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2016, 13, 1130 2 of 15 adhesives, paints, lacquers, etc. Unfortunately, DEHP may leach slowly from these plastic products into foods, beverages, and even directly into body ﬂuids [10,11]. It is also detected in indoor air [12–14], indoor dust [15–18], and air inside vehicles [19]. The general population may be exposed since DEHP is ubiquitous environmental contaminants [20–22]. Epidemiological investigation showed that the exposure dose of DEHP for the population was 5 mg/kg/d [23]. p p p g g Studies have shown that correlating DEHP levels with negative endocrine effects in females are increasing [24]. DEHP can cause the abortion of female animals, increase ovarian weight and the follicle developmental disorders, and gonadal synthesis of estradiol and gonadotropin receptor gene expression decreased pituitary gonadotropin upregulated genes [25–27]. DEHP can enter the fetus through the placental barrier and gather in the fetus. This may have an impact on the fetus directly [28,29]. y Until now, most of the research has focused on reproductive toxicity of adult females’ exposure to DEHP, and little is known regarding the mechanism of DEHP in pubertal females. With the development of society and economy, precocious puberty has been getting more and more attention. The rate of precocious puberty is increasing. Meanwhile, the incidents of reproductive system diseases are also rising. The average age of menarche of girls in Australia and Europe are three years earlier than before [30–32]. Epidemiology found that exposure to phthalates are associated with endometritis, earlier breast development, sexual precocity and earlier onset of puberty [33–36]. However, the effect of DEHP on pubertal female reproductive system is still not well studied. 1. Introduction Animal reproduction is regulated by a variety of factors both inside and outside the body, and the hypothalamic-pituitary-gonadal axis (HPGA) plays a major regulatory role in it. The central precocious puberty (CPP) is due to the hypothalamic-pituitary-gonadal axis function starting early, and because the level of gonadotropin-releasing hormone (GnRH) is premature increased [37]. GnRH is a hypothalamic neuronal secretory decapitate that plays an important regulatory role in the mammalian reproductive system. The present study supports the notion that GnRH-(1-5) is functionally capable of regulating the reproductive neuroendocrine system [38]. GnRH inﬂuences the reproductive processes mainly by regulating pituitary gonadotropin synthesis and release, which, in turn, modulate steroid genesis and gametogenesis [39,40]. Even if the hypothalamus is the principal source of GnRH, and the pituitary is the target site for it, several studies have reported that GnRH receptor (GnRHR) will be in many other tissues including reproductive organs, such as testes, prostates, ovaries, and uteruses [41–44]. Precocious puberty may cause polycystic ovary syndrome, and thus will cause heart disease, diabetes, endometrial cancer, infertility and other diseases [45,46]. This study investigated the effect of pubertal female rats’ exposure to DEHP on the expression of GnRH in hypothalamus and GnRHR in the uterus and to explore the role of DEHP in reproductive endocrine disruption. The DEHP doses were set from the human exposure dose (5 mg/kg/d) [23] and the LD50 (30 g/kg) in rats [47]. 2.4. Immunohistochemistry Tissue sections of hypothalamus and uterus were deparafﬁnized in xylene, rehydrated through graded ethanols, and ﬁnally rinsed in distilled water. The specimens were treated with antigen retrieval in 0.1 M citric acid solution by boiling for 15 min and cooling to room temperature. They were then incubated with 3% H2O2 (v/v) in methanol for 10 min to quench the endogenous peroxidase activity. The sections were blotted with normal rat serum (1:10 dilution) for 30 min at room temperature, and incubated with rabbit polyclonal primary antibody (GnRH and GnRHR, Proteintech, Rosemont, IL, USA) (1:1000 in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin) overnight at 4 ◦C in a moist chamber. The slides were incubated with peroxidase-conjugated anti-rabbit secondary antibody (Bioss Biotechnology Company, Shanghai, China) (1:200 dilution in PBS) for 30 min at room temperature after three washes in PBS. The staining was visualized using a 3,3′-diaminobenzidine (DAB) (Bioss Biotechnology Company, Shanghai, China) kit. The specimens were counterstained with hematoxylin (Bogoo Biotechnology Company, Shanghai, China), mounted, observed under a light microscope, and photographed [48]. 2.1. Animals and Treatment Female Wistar rats (15 days old, 60 ± 10 g) were purchased from the Experimental Animal Center of Jilin University. They were allowed at least a 7-day acclimatization-period and observed for signs of illness before starting experimental procedures, and their body weight was measured every day. The 48 rats were randomly apportioned into 4 groups (n = 12, each). The rats were housed in polypropylene cages (6 rats per cage) with sterilized bedding, and maintained under controlled conditions of temperature (23 ± 1 ◦C), humidity (55% ± 5%), and a 12:12 h light-dark cycle. The experiment lasted approximately 4 weeks including the period of adaptation to the feeding regime. Rats were administered via oral gavage 0, 250 mg/kg/d DEHP (1/120 LD50), 500 mg/kg/d DEHP (1/60 LD50), 1000 mg/kg/d DEHP (1/30 LD50) (Sinopharm Chemical Reagent, purity >99%, Shanghai, China) in 0.1 mL corn oil/20 g body weight for up to 4 weeks. The experimental protocol was subject Int. J. Environ. Res. Public Health 2016, 13, 1130 3 of 15 to approval by the Animal Use and Care Committee of Jilin University, and the ethical approval code of the animal experiment is 2015-03-14. to approval by the Animal Use and Care Committee of Jilin University, and the ethical approval code of the animal experiment is 2015-03-14. 2.2. Tissue Collection After 4 weeks, the rats were weighed and killed by decapitation. The hypothalamus and uterus of each rat were also dissected, weighed, and collected immediately after decapitation. 2.3. Quantiﬁcation of GnRH GnRH in the hypothalamus was quantiﬁed using enzyme-linked immunosorbent assay kits (R&D systems, Minneapolis, MN, USA). The total protein from the hypothalamus sample was extracted, and 0.1 mL diluted sample from each group was mixed with 10 µL GnRH antibody and 50 µL horseradish peroxidase-labeled streptavidin. This was incubated at 37 ◦C for 60 min. The wells were rinsed ﬁve times with distilled water and incubated with chromogen solution from the kit (50 µL A and 50 µL B) for 10 min at 37 ◦C in dark. The reaction was then stopped with 50 µL of stop solution, and the absorbance was read at 450 nm within 10 min. 2.5. RNA Extraction Total RNA was isolated using Trizol reagent (Invitrogen, Waltham, MA, USA). The quantity and the integrity of total RNA were determined by a UV spectrophotometer and evaluated by formaldehyde denaturing gel electrophoresis. 2.7. Western Blotting Isolated tissues were immediately put on ice and homogenized in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1% deoxycholate, 1 mM sodium ﬂuoride, 2 mM sodium orthovanadate, and complete protease inhibitor tablets). Homogenates were centrifuged at 12,000 g for 5 min at 4 ◦C. Protein in the supernatant was quantiﬁed using the BCA protein assay (Beyotime, Shanghai, China). Equal amounts of protein from each sample were mixed with sodium dodecyl sulfate (SDS) sample buffer (Beyotime, Shanghai, China). Samples were separated using pre-cast 10% Bis-Tris gel (Beyotime, Shanghai, China) in Tris-Glycine-SDS running buffer and transferred to nitrocellulose ﬁlter membrane (NC) (Phamacia, Goteborg, Sweden) in transfer buffer. Prior to immunoblotting, NC membranes were blocked with 5% nonfat milk in tris buffered saline tween (TBST) buffer (20 mmol/L Tris-HCl, 140 mmol/L NaCl, and 0.05% Tween 20, pH 7.5). Following incubation with the primary antibodies speciﬁc for each protein for 24 h at 4 ◦C—mouse anti-GnRH antibody (1:1000, 34 KDa, Abcam, Cambridge, UK), rabbit anti-GnRHR antibody (1:500, 36 KDa, Abcam, Cambridge, UK), and rabbit anti-beta Actin antibody (1:1000, 42 KDa, Abcam, Cambridge, UK)—the blots were washed with TBST and then incubated with the horseradish peroxidase-conjugated secondary antibodies (1:2000, Proteintech, Rosemont, IL, USA) for 1 h at room temperature. Immunolabeling was detected by enhanced chemiluminescence (Proteintech, Rosemont, IL, USA) according to the recommended conditions [50]. Western blot analysis was performed in duplicates for each sample and its average protein level was calculated for comparison. Digital images of the blots were created by scanning the blots on a scanner in transparency mode and optical density measurements of the bands were taken with Image-Pro software (Media Cybernetics Company, Bethesda, MD, USA). Each protein level was normalized to control samples from the same membrane and presented in percentage. 2.6. Real-Time Reverse Transcription-PCR Real-time reverse transcription-PCR was used to verify gene expression using Stratagene MX3000p (TaKaRa, Shiga, Japan). Reverse transcription was performed with 500 ng total RNA in a 10-ìL reaction, and 1 ìL of cDNA was then used for a 25-ìL PCR reaction mixture containing an optimal concentration of primers and SYBR-Green Supermix (SYBR premix Ex Tap II, TaKaRa, Shiga, Japan). The PCR reaction was carried out in 45 cycles of 95 ◦C for 20 s and 60 ◦C for 20 s. â-actin was used as an internal control [49]. The primers are listed in Table 1. 4 of 15 Int. J. Environ. Res. Public Health 2016, 13, 1130 Table 1. Gene-speciﬁc forward and reverse primer sequence. Gene Direction Sequence GNRH Forward TCCAGCCAGCACTGGTCCTA Reverse GGGTTCTGCCATTTGATCCTC GnRHR Forward TCACCTTCAGCTGCCTGTTCA Reverse CTCAGCCGTGCTCTTGGGATA β-actin Forward CCCATTGAACACGGCATTG Reverse GGTACGACCAGAGGCATACA Table 1. Gene-speciﬁc forward and reverse primer sequence. 2.8. Statistical Analysis Statistical evaluations were calculated using SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA). All data were tested for normal distribution and independence using the Normal Plots in SPSS. Shapiro–Wilk signiﬁcance was over 0.05, indicating that the assumptions were valid. Differences between the treatment and control groups were analyzed by analysis of Bonferroni’s test. Data are presented as mean ± standard error of the mean. p < 0.05 was considered signiﬁcantly signiﬁcant. 3.1. General Toxicity and Reproductive Toxicity of DEHP in Pubertal Female Rats 3. Results Groups n Time (Weeks) 1 2 3 4 Control 12 17.12 ± 3.08 25.62 ± 7.22 29.21 ± 7.82 25.10 ± 5.87 250 mg/kg/d 12 18.92 ± 4.21 26.04 ± 5.20 26.83 ± 8.20 32.85 ± 1.79 * 500 mg/kg/d 12 22.38 ± 4.25 * 27.30 ± 3.63 29.26 ± 6.67 35.48 ± 5.80 * 1000 mg/kg/d 12 17.98 ± 1.32 17.46 ± 3.34 ,▲, 22.80 ± 8.94 23.76 ± 3.35 ▲, p < 0.05, vs. Control group; ▲p < 0.05, vs. 250 mg/kg/d group; p < 0.05, vs. 500 mg/kg/d group. 3) The ovaries of the rats treated with DEHP were congestive and swelling, and the volume of the ovaries and the uterus became bigger (Figure 1). nt. J. Environ. Res. Public Health 2016, 13, 1130 5 of 14 Table 3. Body weights of pubertal female rats treated with DEHP during the administration period (mean ± SE, g). Groups n Time (Weeks) 1 2 3 4 Control 12 76.87 ± 8.43 106.73 ± 9.25 134.00 ± 6.85 152.12 ± 3.89 250 mg/kg/d 12 8 85.12 ± 9.31 116.58 ± 8.70 * 144.27 ± 7.40 * 164.69 ± 8.05 * 500 mg/kg/d 12 87.17 ± 9.75 117.50 ± 7.46 * 143.74 ± 8.89 * 168.52 ± 7.59 * 1000 mg/kg/d 12 90.43 ± 10.91 * 122.05 ± 7.88 * 149.02 ± 7.35 * 170.16 ± 6.46 * * p < 0.05, vs. Control group. Table 4. Water consumption by pubertal female rats treated with DEHP (mean ± SE, mL). Groups n Time (Weeks) 1 2 3 4 Control 12 17.12 ± 3.08 25.62 ± 7.22 29.21 ± 7.82 25.10 ± 5.87 250 mg/kg/d 12 18.92 ± 4.21 26.04 ± 5.20 26.83 ± 8.20 32.85 ± 1.79 * 500 mg/kg/d 12 22.38 ± 4.25 * 27.30 ± 3.63 29.26 ± 6.67 35.48 ± 5.80 * 1000 mg/kg/d 12 17.98 ± 1.32 ● 17.46 ± 3.34 ,▲,● 22.80 ± 8.94 23.76 ± 3.35 ▲,● p < 0.05, vs. Control group; ▲ p < 0.05, vs. 250 mg/kg/d group; ● p < 0.05, vs. 500 mg/kg/d group. 3) The ovaries of the rats treated with DEHP were congestive and swelling, and the volume of the ovaries and the uterus became bigger (Figure 1). Table 2. Food consumption by pubertal female rats treated with DEHP (mean ± SE, g). 3. Results Groups n Time (Weeks) 1 2 3 4 Control 12 13.33 ± 3.01 13.66 ± 4.20 14.60 ± 2.86 14.53 ± 3.69 250 mg/kg/d 12 17.00 ± 2.44 * 18.82 ± 3.10 * 17.77 ± 3.34 16.20 ± 3.95 500 mg/kg/d 12 14.25 ± 1.23 ▲ 15.48 ± 3.38 18.39 ± 2.97 17.15 ± 2.93 1000 mg/kg/d 12 16.55 ± 2.22 * 17.82 ± 4.32 22.28 ± 2.37 * 18.53 ± 1.74 * * p < 0.05, vs. Control group; ▲p < 0.05, vs. 250 mg/kg/d group. Table 3. Body weights of pubertal female rats treated with DEHP during the administration period (mean ± SE, g). J. Environ. Res. Public Health 2016, 13, 1130 5 o Table 2. Food consumption by pubertal female rats treated with DEHP (mean ± SE, g). Table 3. Body weights of pubertal female rats treated with DEHP during the administration period (mean ± SE, g). J. Environ. Res. Public Health 2016, 13, 1130 5 of (mean ± SE, g). Groups n Time (Weeks) 1 2 3 4 Control 12 76.87 ± 8.43 106.73 ± 9.25 134.00 ± 6.85 152.12 ± 3.89 250 mg/kg/d 12 8 85.12 ± 9.31 116.58 ± 8.70 * 144.27 ± 7.40 * 164.69 ± 8.05 * 500 mg/kg/d 12 87.17 ± 9.75 117.50 ± 7.46 * 143.74 ± 8.89 * 168.52 ± 7.59 * 1000 mg/kg/d 12 90.43 ± 10.91 * 122.05 ± 7.88 * 149.02 ± 7.35 * 170.16 ± 6.46 * * p < 0.05, vs. Control group. . J. Environ. Res. Public Health 2016, 13, 1130 5 of Table 3. Body weights of pubertal female rats treated with DEHP during the administration period (mean ± SE, g). Groups n Time (Weeks) 1 2 3 4 Control 12 76.87 ± 8.43 106.73 ± 9.25 134.00 ± 6.85 152.12 ± 3.89 250 mg/kg/d 12 8 85.12 ± 9.31 116.58 ± 8.70 * 144.27 ± 7.40 * 164.69 ± 8.05 * 500 mg/kg/d 12 87.17 ± 9.75 117.50 ± 7.46 * 143.74 ± 8.89 * 168.52 ± 7.59 * 1000 mg/kg/d 12 90.43 ± 10.91 * 122.05 ± 7.88 * 149.02 ± 7.35 * 170.16 ± 6.46 * Table 4. Water consumption by pubertal female rats treated with DEHP (mean ± SE, mL). 3. Results Groups n Time (Weeks) 1 2 3 4 Control 12 17.12 ± 3.08 25.62 ± 7.22 29.21 ± 7.82 25.10 ± 5.87 250 mg/kg/d 12 18.92 ± 4.21 26.04 ± 5.20 26.83 ± 8.20 32.85 ± 1.79 * 500 mg/kg/d 12 22.38 ± 4.25 * 27.30 ± 3.63 29.26 ± 6.67 35.48 ± 5.80 * 1000 mg/kg/d 12 17.98 ± 1.32 17.46 ± 3.34 ,▲, 22.80 ± 8.94 23.76 ± 3.35 ▲, p < 0.05, vs. Control group; ▲p < 0.05, vs. 250 mg/kg/d group; p < 0.05, vs. 500 mg/kg/d group. * p < 0.05, vs. Control group. Table 4. Water consumption by pubertal female rats treated with DEHP (mean ± SE, mL). Groups n Time (Weeks) 1 2 3 4 Control 12 17.12 ± 3.08 25.62 ± 7.22 29.21 ± 7.82 25.10 ± 5.87 250 mg/kg/d 12 18.92 ± 4.21 26.04 ± 5.20 26.83 ± 8.20 32.85 ± 1.79 * 500 mg/kg/d 12 22.38 ± 4.25 * 27.30 ± 3.63 29.26 ± 6.67 35.48 ± 5.80 * 1000 mg/kg/d 12 17.98 ± 1.32 ● 17.46 ± 3.34 ,▲,● 22.80 ± 8.94 23.76 ± 3.35 ▲,● p < 0 05 vs Control group; ▲p < 0 05 vs 250 mg/kg/d group; ●p < 0 05 vs 500 mg/kg/d group Table 4. Water consumption by pubertal female rats treated with DEHP (mean ± SE, mL). * p < 0.05, vs. Control group. (3) The ovaries of the rats treated with DEHP were congestive and swelling, and the volume of the ovaries and the uterus became bigger (Figure 1). (3) The ovaries of the rats treated with DEHP were congestive and swelling, and the volume of the ovaries and the uterus became bigger (Figure 1). (3) The ovaries of the rats treated with DEHP were congestive and swelling, and the volume of the ovaries and the uterus became bigger (Figure 1). (3) The ovaries of the rats treated with DEHP were congestive and swelling, and the volume of the ovaries and the uterus became bigger (Figure 1). Figure 1. Effects of DEHP on ovaries and the uterus in a pubertal female rat. Figure 1. Effects of DEHP on ovaries and the uterus in a pubertal female rat. Figure 1. Effects of DEHP on ovaries and the uterus in a pubertal female rat. Figure 1. Effects of DEHP on ovaries and the uterus in a pubertal female rat. Figure 1. (4) Compared with the control group, there were no significant differences of the coefficients in uteruses of the rats treated with DEHP (p > 0.05, Figure 2). (4) Compared with the control group, there were no signiﬁcant differences of the coefﬁcients in uteruses of the rats treated with DEHP (p > 0.05, Figure 2). 3. Results 3.1. General Toxicity and Reproductive Toxicity of DEHP in Pubertal Female Rats (1) The rats treated with DEHP appeared less active and less spirited, moved more slowly, had untidy and lusterless fur and lost hair. These changes were most obvious in the group of 1000 mg/kg/d DEHP. (1) The rats treated with DEHP appeared less active and less spirited, moved more slowly, had untidy and lusterless fur and lost hair. These changes were most obvious in the group of 1000 mg/kg/d DEHP. (2) Compared with the control group, the food consumption and body weight of rats treated with DEHP were signiﬁcantly higher (p < 0.05, Tables 2 and 3, while their water consumption signiﬁcantly decreased (p < 0.05, Table 4). (2) Compared with the control group, the food consumption and body weight of rats treated with DEHP were signiﬁcantly higher (p < 0.05, Tables 2 and 3, while their water consumption signiﬁcantly decreased (p < 0.05, Table 4). Int. J. Environ. Res. Public Health 2016, 13, 1130 5 of 15 Table 2. Food consumption by pubertal female rats treated with DEHP (mean ± SE, g). Groups n Time (Weeks) 1 2 3 4 Control 12 13.33 ± 3.01 13.66 ± 4.20 14.60 ± 2.86 14.53 ± 3.69 250 mg/kg/d 12 17.00 ± 2.44 * 18.82 ± 3.10 * 17.77 ± 3.34 16.20 ± 3.95 500 mg/kg/d 12 14.25 ± 1.23 ▲ 15.48 ± 3.38 18.39 ± 2.97 17.15 ± 2.93 1000 mg/kg/d 12 16.55 ± 2.22 * 17.82 ± 4.32 22.28 ± 2.37 * 18.53 ± 1.74 * * p < 0.05, vs. Control group; ▲p < 0.05, vs. 250 mg/kg/d group. Table 3. Body weights of pubertal female rats treated with DEHP during the administration period (mean ± SE, g). Groups n Time (Weeks) 1 2 3 4 Control 12 76.87 ± 8.43 106.73 ± 9.25 134.00 ± 6.85 152.12 ± 3.89 250 mg/kg/d 12 8 85.12 ± 9.31 116.58 ± 8.70 * 144.27 ± 7.40 * 164.69 ± 8.05 * 500 mg/kg/d 12 87.17 ± 9.75 117.50 ± 7.46 * 143.74 ± 8.89 * 168.52 ± 7.59 * 1000 mg/kg/d 12 90.43 ± 10.91 * 122.05 ± 7.88 * 149.02 ± 7.35 * 170.16 ± 6.46 * * p < 0.05, vs. Control group. Table 4. Water consumption by pubertal female rats treated with DEHP (mean ± SE, mL). 3. Results Effects of DEHP on ovaries and the uterus in a pubertal female rat. Figure 1. Effects of DEHP on ovaries and the uterus in a pubertal female rat. (4) Compared with the control group, there were no significant differences of the coefficients in uteruses of the rats treated with DEHP (p > 0.05, Figure 2). (4) Compared with the control group, there were no signiﬁcant differences of the coefﬁcients in uteruses of the rats treated with DEHP (p > 0.05, Figure 2). (4) Compared with the control group, there were no significant differences of the coefficients in uteruses of the rats treated with DEHP (p > 0.05, Figure 2). (4) Compared with the control group, there were no signiﬁcant differences of the coefﬁcients in uteruses of the rats treated with DEHP (p > 0.05, Figure 2). 6 of 15 ents in Int. J. Environ. Res. Public Health 2016, 13, 1130 ( ) Co pa e i e co o g ou uteruses of the rats treated with D Figure 2. Effects of DEHP on coefficients of uteruses in pubertal female rats; n = 12. The coefficient of uterus was expressed as the mean value ± standard error (SE). Figure 2. Effects of DEHP on coefﬁcients of uteruses in pubertal female rats; n = 12. The coefﬁcient of uterus was expressed as the mean value ± standard error (SE). t. J. Environ. Res. Public Health 2016, 13, 1130 6 of Figure 2. Effects of DEHP on coefficients of uteruses in pubertal female rats; n = 12. The coefficient of uterus was expressed as the mean value ± standard error (SE). Figure 2. Effects of DEHP on coefﬁcients of uteruses in pubertal female rats; n = 12. The coefﬁcient of uterus was expressed as the mean value ± standard error (SE). Environ. Res. Public Health 2016, 13, 1130 6 3.2. Effect of DEHP on GnRH Level 3.2. Effect of DEHP on GnRH Level 3.2. Effect of DEHP on GnRH Level 3.2. Effect of DEHP on GnRH Level The effect of DEHP on GnRH level in the hypothalamus was examined. We found here that GnRH level was signiﬁcantly higher in the hypothalamus of rats treated with 500 mg/kg/d DEHP compared with both the control rats and the rats treated with 250 mg/kg/d DEHP (p < 0.05, Figure 1). GnRH level was signiﬁcantly higher in the rats treated with 1000 mg/kg/d DEHP compared with the rats in all other groups (p < 0.05, Figure 3). The effect of DEHP on GnRH level in the hypothalamus was examined. We found here that GnRH level was significantly higher in the hypothalamus of rats treated with 500 mg/kg/d DEHP compared with both the control rats and the rats treated with 250 mg/kg/d DEHP (p < 0.05, Figure 1). GnRH level was significantly higher in the rats treated with 1000 mg/kg/d DEHP compared with the rats in all other groups (p < 0.05, Figure 3). Figure 3. Effect of DEHP on gonadotropin-releasing hormone (GnRH) level in the hypothalamus (n = 12). GnRH level in the hypothalamus was expressed as the mean value ± standard error (SE). * Significant difference compared with control (p < 0.05); ▲ Significant difference compared with 250 mg/kg/d (p <0.05); ● Significant difference compared with 500 mg/kg/d (p < 0.05). Figure 3. Effect of DEHP on gonadotropin-releasing hormone (GnRH) level in the hypothalamus (n = 12). GnRH level in the hypothalamus was expressed as the mean value ± standard error (SE). * Signiﬁcant difference compared with control (p < 0.05); ▲Signiﬁcant difference compared with 250 mg/kg/d (p <0.05); Signiﬁcant difference compared with 500 mg/kg/d (p < 0.05). Figure 3. Effect of DEHP on gonadotropin-releasing hormone (GnRH) level in the hypothalamus (n = 12). GnRH level in the hypothalamus was expressed as the mean value ± standard error (SE). * Significant difference compared with control (p < 0.05); ▲ Significant difference compared with 250 mg/kg/d (p <0.05); ● Significant difference compared with 500 mg/kg/d (p < 0.05). Figure 3. Effect of DEHP on gonadotropin-releasing hormone (GnRH) level in the hypothalamus (n = 12). GnRH level in the hypothalamus was expressed as the mean value ± standard error (SE). * Signiﬁcant difference compared with control (p < 0.05); ▲Signiﬁcant difference compared with 250 mg/kg/d (p <0.05); Signiﬁcant difference compared with 500 mg/kg/d (p < 0.05). Figure 3. Effect of DEHP on gonadotropin-releasing hormone (GnRH) level in the hypothalamus (n = 12). 3.2. Effect of DEHP on GnRH Level 3.2. Effect of DEHP on GnRH Level GnRH level in the hypothalamus was expressed as the mean value ± standard error (SE). * Significant difference compared with control (p < 0.05); ▲ Significant difference compared with 250 mg/kg/d (p <0.05); ● Significant difference compared with 500 mg/kg/d (p < 0.05). Figure 3. Effect of DEHP on gonadotropin-releasing hormone (GnRH) level in the hypothalamus (n = 12). GnRH level in the hypothalamus was expressed as the mean value ± standard error (SE). * Signiﬁcant difference compared with control (p < 0.05); ▲Signiﬁcant difference compared with 250 mg/kg/d (p <0.05); Signiﬁcant difference compared with 500 mg/kg/d (p < 0.05). 3.3. Gene and Protein Expression Levels of GnRH in the Hypothalamus 3.3. Gene and Protein Expression Levels of GnRH in the Hypothalamus 3.3. Gene and Protein Expression Levels of GnRH in the Hypothalamus 3.3. Gene and Protein Expression Levels of GnRH in the Hypothalamus The mRNA level of GnRH in the hypothalamus was measured by real-time RT-PCR (Figure 4). Compared with the control group, there was no significant difference in the mRNA level of GnRH in the hypothalamus of the rats treated with DEHP (p > 0.05). The mRNA level of GnRH in the hypothalamus was measured by real-time RT-PCR (Figure 4). Compared with the control group, there was no signiﬁcant difference in the mRNA level of GnRH in the hypothalamus of the rats treated with DEHP (p > 0.05). The mRNA level of GnRH in the hypothalamus was measured by real-time RT-PCR (Figure 4). Compared with the control group, there was no significant difference in the mRNA level of GnRH in the hypothalamus of the rats treated with DEHP (p > 0.05). The mRNA level of GnRH in the hypothalamus was measured by real-time RT-PCR (Figure 4). Compared with the control group, there was no signiﬁcant difference in the mRNA level of GnRH in the hypothalamus of the rats treated with DEHP (p > 0.05). 7 of 15 nRH in Int. J. Environ. Res. Public Health 2016, 13, 1130 Compared with the control group, the the hypothalamus of the rats treated w Figure 4. Effects of DEHP exposure on the level of GnRH mRNA in the hypothalamus; n = 12. The level of GnRHR mRNA was expressed as the mean value ± standard error (SE). Figure 4. Effects of DEHP exposure on the level of GnRH mRNA in the hypothalamus; n = 12. The level of GnRHR mRNA was expressed as the mean value ± standard error (SE). Figure 4. Effects of DEHP exposure on the level of GnRH mRNA in the hypothalamus; n = 12. The level of GnRHR mRNA was expressed as the mean value ± standard error (SE). Figure 4. Effects of DEHP exposure on the level of GnRH mRNA in the hypothalamus; n = 12. The level of GnRHR mRNA was expressed as the mean value ± standard error (SE). The protein level of GnRH in the hypothalamus was measured by Western blotting (Figure 5). Our results showed that the protein level of GnRH was significantly higher in the hypothalamus of rats treated with DEHP compared with the control rats (p < 0.05). 3.3. Gene and Protein Expression Levels of GnRH in the Hypothalamus 3.3. Gene and Protein Expression Levels of GnRH in the Hypothalamus The level of GnRHR protein was expressed as the mean value ± standard error (SE). * Signiﬁcant difference compared to control (p < 0.05); ▲Signiﬁcant difference compared to 250 mg/kg/d (p < 0.05); Signiﬁcant difference compared to 500 mg/kg/d (p < 0.05). 3.3. Gene and Protein Expression Levels of GnRH in the Hypothalamus 3.3. Gene and Protein Expression Levels of GnRH in the Hypothalamus However, the protein level of GnRH was significantly lower in rats administered 1000 mg/kg/d DEHP, compared with both of the rats treated with 250 and 500 mg/kg/d DEHP (p < 0.05). The protein level of GnRH in the hypothalamus was measured by Western blotting (Figure 5). Our results showed that the protein level of GnRH was signiﬁcantly higher in the hypothalamus of rats treated with DEHP compared with the control rats (p < 0.05). However, the protein level of GnRH was signiﬁcantly lower in rats administered 1000 mg/kg/d DEHP, compared with both of the rats treated with 250 and 500 mg/kg/d DEHP (p < 0.05). Int. J. Environ. Res. Public Health 2016, 13, 1130 7 of 14 Figure 5. (A) Protein bands of GnRH expression in the hypothalamus of pubertal female rats. (B) effects of DEHP exposure on the protein level of GnRHR in the hypothalamus; n = 12. The level of GnRHR protein was expressed as the mean value ± standard error (SE). * Significant difference compared to control (p < 0.05); ▲ Significant difference compared to 250 mg/kg/d (p < 0.05); ● Significant difference compared to 500 mg/kg/d (p < 0.05). 3 4 G d P i E i L l f G RHR i h U Figure 5. (A) Protein bands of GnRH expression in the hypothalamus of pubertal female rats. (B) effects of DEHP exposure on the protein level of GnRHR in the hypothalamus; n = 12. The level of GnRHR protein was expressed as the mean value ± standard error (SE). * Signiﬁcant difference compared to control (p < 0.05); ▲Signiﬁcant difference compared to 250 mg/kg/d (p < 0.05); Signiﬁcant difference compared to 500 mg/kg/d (p < 0.05). Figure 5. (A) Protein bands of GnRH expression in the hypothalamus of pubertal female rats. (B) effects of DEHP exposure on the protein level of GnRHR in the hypothalamus; n = 12. The level of GnRHR protein was expressed as the mean value ± standard error (SE). * Significant difference compared to control (p < 0.05); ▲ Significant difference compared to 250 mg/kg/d (p < 0.05); ● Significant difference compared to 500 mg/kg/d (p < 0.05). Figure 5. (A) Protein bands of GnRH expression in the hypothalamus of pubertal female rats. (B) effects of DEHP exposure on the protein level of GnRHR in the hypothalamus; n = 12. 3.4. Gene and Protein Expression Levels of GnRHR in the Uterus The mRNA level of GnRHR in the uterus was measured 3.4. Gene and Protein Expression Levels of GnRHR in the Uterus The mRNA level of GnRHR in the uterus was measured by real-time RT-PCR (Figure 6). The level in the uterus was significantly higher in the rats treated with 1000 mg·kg−1·d−1 DEHP compared with the rats in all other groups (p < 0.05). The mRNA level of GnRHR in the uterus was measured by real-time RT-PCR (Figure 6). The level in the uterus was signiﬁcantly higher in the rats treated with 1000 mg·kg−1·d−1 DEHP compared with the rats in all other groups (p < 0.05). The mRNA level of GnRHR in the uterus was measured by real time RT PCR (Figure 6). The level in the uterus was significantly higher in the rats treated with 1000 mg·kg−1·d−1 DEHP compared with the rats in all other groups (p < 0.05). The mRNA level of GnRHR in the uterus was measured by real-time RT-PCR (Figure 6). The level in the uterus was signiﬁcantly higher in the rats treated with 1000 mg·kg−1·d−1 DEHP compared with the rats in all other groups (p < 0.05). 8 of 15 ared Int. J. Environ. Res. Public Health 2016, 13, 1130 level in the uterus was significantly hi with the rats in all other groups (p < 0 n all other groups (p < 0.05). g p (p ) Figure 6. Effects of DEHP exposure on the level of GnRHR mRNA in the uterus; n = 12. The level of GnRHR mRNA was expressed as the mean value ± standard error (SE). * Significant difference compared to control (p < 0.05); ▲ Significant difference compared to 250 mg/kg/d (p < 0.05); ● Significant difference compared to 500 mg/kg/d (p < 0.05). Figure 6. Effects of DEHP exposure on the level of GnRHR mRNA in the uterus; n = 12. The level of GnRHR mRNA was expressed as the mean value ± standard error (SE). * Signiﬁcant difference compared to control (p < 0.05); ▲Signiﬁcant difference compared to 250 mg/kg/d (p < 0.05); Signiﬁcant difference compared to 500 mg/kg/d (p < 0.05). Figure 6. Effects of DEHP exposure on the level of GnRHR mRNA in the uterus; n = 12. The level of GnRHR mRNA was expressed as the mean value ± standard error (SE). * Significant difference compared to control (p < 0.05); ▲ Significant difference compared to 250 mg/kg/d (p < 0.05); ● Significant difference compared to 500 mg/kg/d (p < 0.05). Figure 6. 3.4. Gene and Protein Expression Levels of GnRHR in the Uterus The mRNA level of GnRHR in the uterus was measured 3.4. Gene and Protein Expression Levels of GnRHR in the Uterus The level of GnRHR protein was expressed as the mean value ± standard error (SE). * Significant difference compared to control (p < 0.05); ▲ Significant difference compared to 250 mg/kg/d (p < 0.05); ● Significant difference compared to 500 mg/kg/d (p < 0.05). Figure 7. (A) Protein bands of GnRHR expression in the uterus of pubertal female rats; (B) effects of DEHP exposure on the protein level of GnRHR in the uterus; n = 12. The level of GnRHR protein was expressed as the mean value ± standard error (SE). * Signiﬁcant difference compared to control (p < 0.05); ▲Signiﬁcant difference compared to 250 mg/kg/d (p < 0.05); Signiﬁcant difference compared to 500 mg/kg/d (p < 0.05). 3.4. Gene and Protein Expression Levels of GnRHR in the Uterus The mRNA level of GnRHR in the uterus was measured 3.4. Gene and Protein Expression Levels of GnRHR in the Uterus Effects of DEHP exposure on the level of GnRHR mRNA in the uterus; n = 12. The level of GnRHR mRNA was expressed as the mean value ± standard error (SE). * Signiﬁcant difference compared to control (p < 0.05); ▲Signiﬁcant difference compared to 250 mg/kg/d (p < 0.05); Signiﬁcant difference compared to 500 mg/kg/d (p < 0.05). The protein level of GnRHR in the uterus was measured by Western blotting (Figure 7). The level of GnRHR in the uterus was significantly higher in the rats treated with 1000 mg/kg/d DEHP compared with the rats in all of other groups (p < 0.05). Compared with the control rats, the protein level was significantly lower in rats treated with 250 and 500 mg/kg/d DEHP (p < 0.05). The protein level of GnRHR in the uterus was measured by Western blotting (Figure 7). The level of GnRHR in the uterus was signiﬁcantly higher in the rats treated with 1000 mg/kg/d DEHP compared with the rats in all of other groups (p < 0.05). Compared with the control rats, the protein level was signiﬁcantly lower in rats treated with 250 and 500 mg/kg/d DEHP (p < 0.05). Int. J. Environ. Res. Public Health 2016, 13, 1130 8 of 14 Figure 7. (A) Protein bands of GnRHR expression in the uterus of pubertal female rats. (B) effects of DEHP exposure on the protein level of GnRHR in the uterus; n = 12. The level of GnRHR protein was expressed as the mean value ± standard error (SE). * Significant difference compared to control (p < 0.05); ▲ Significant difference compared to 250 mg/kg/d (p < 0.05); ● Significant difference compared to 500 mg/kg/d (p < 0.05). Figure 7. (A) Protein bands of GnRHR expression in the uterus of pubertal female rats; (B) effects of DEHP exposure on the protein level of GnRHR in the uterus; n = 12. The level of GnRHR protein was expressed as the mean value ± standard error (SE). * Signiﬁcant difference compared to control (p < 0.05); ▲Signiﬁcant difference compared to 250 mg/kg/d (p < 0.05); Signiﬁcant difference compared to 500 mg/kg/d (p < 0.05). Figure 7. (A) Protein bands of GnRHR expression in the uterus of pubertal female rats. (B) effects of DEHP exposure on the protein level of GnRHR in the uterus; n = 12. 3.5. GnRH and GnRHR Immunohistochemistry 3.5. GnRH and GnRHR Immunohistochemistry The expression of GnRH in the hypothalamus of pubertal female rats was examined by immunohistochemistry (Figure 8, Table 5). Our results showed that positive staining for GnRH was observed in the cytomembrane, cytoplasm and nucleus of glial cells and neurons, and we could see the brown yellow granules. Compared with the control rats, the level of immunohistochemical The expression of GnRH in the hypothalamus of pubertal female rats was examined by immunohistochemistry (Figure 8, Table 5). Our results showed that positive staining for GnRH was observed in the cytomembrane, cytoplasm and nucleus of glial cells and neurons, and we could see the Int. J. Environ. Res. Public Health 2016, 13, 1130 p (p < 0.05); ▲ Significant difference compared to 500 mg/kg/d (p < 0 05) 9 of 15 brown yellow granules. Compared with the control rats, the level of immunohistochemical staining in the hypothalamus was signiﬁcantly higher in rats treated with DEHP. 3.5. GnRH and GnRHR Immunohistochemistry The expression of GnRH in the hypothalamus of pubertal female rats was examined by GnRHR localized in the uterus was examined by immunohistochemistry (Figure 9, Table 6). The result of this study shows that the cytomembrane, cytoplasm and nucleus of uterus epithelial cells and glandular epithelial cells are GnRHR-positive. The level of immunohistochemical staining in the uterus was signiﬁcantly higher in rats treated with DEHP compared with the control rats. immunohistochemistry (Figure 8, Table 5). Our results showed that positive staining for GnRH was observed in the cytomembrane, cytoplasm and nucleus of glial cells and neurons, and we could see the brown yellow granules. Compared with the control rats, the level of immunohistochemical staining in the hypothalamus was significantly higher in rats treated with DEHP. Figure 8. Immunohistochemical staining of GnRH expression in the hypothalamus of pubertal female rats (×400). Int. J. Environ. Res. Public Health 2016, 13, 1130 9 of 14 Figure 8. Immunohistochemical staining of GnRH expression in the hypothalamus of pubertal female rats (×400). Table 5. Effects of DEHP on mean optical density (MOD) and integral optical density (IOD) of GnRH staining in the hypothalamus a. Groups Dosage, mg/kg/d MOD (10−2) IOD (×104) H expression in the hypothalam MOD)a d i te al o ti al de ity P ti Figure 8. Immunohistochemical staining of GnRH expression in the hypothalamus of pubertal female rats (×400). p y g p y staining in the hypothalamus a. 4. Discussion The puberty of female rats is one of the key stages of the development of their reproductive system and sexual differentiation. During puberty, female rats are extremely sensitive to both endogenous and exogenous hormone changes, and any small change in their sex hormone levels could exert a persistent effect on the development of the reproductive system. Pubertal development is complicated and orderly, which is affected by heredity, nutrition, systemic diseases, endocrine hormone, psycho-mental factors, etc. It begins with the releasing of GnRH by the hypothalamus. By acting on the GnRH receptors on the luteinizing hormone (LH)- and follicle-stimulating hormone (FSH) secreting cells in the anterior pituitary, GnRH promotes the secretion of LH and FSH in the pituitary, which then act on the gonad and promotes the development of it, the generation of mature gametes and the secretion of sex hormones. GnRH plays a pivotal role in regulating hypothalamic-pituitary-gonadal axis function. HPG axis is temporarily activated in the late fetal period and early infancy, suppressed during childhood, and then activated again when puberty begins. During puberty, the secretion of GnRH in the hypothalamus increases, which enters the adenohypophysis through the portal system of the hypothalamus and pituitary. With its sensitivity to GnRH gradually increasing, the adenohypophysis secretes LH and FSH when stimulated by GnRH. DEHP may have negative impacts on the development and function of female reproductive system. According to previous studies, exposure to steroid hormones or environmental endocrine disruptors (EEDs) during pregnancy or neonatal period could affect the starting time of puberty. This proves that exposure to exogenous estrogenic chemicals in perinatal period could affect the development of the whole endocrine axis [51,52]. Exogenous estrogens, like estradiol estradiol benzoate (EB), can decrease the vaginal opening time of pubertal female rats, increase the level of GnRH in hypothalamus of rats, start the hypothalamus-pituitary-gonadal axis, and then lead to the onset of puberty [53]. Liu et al.’s study also said, compared with control rats, the DEHP-treated adult female rats showed higher GnRH levels in the hypothalamus [54]. In this study, DEHP is applied on female rats in puberty. We observed that DEHP treatment resulted in higher food consumption and body weight, and they were the same as previous results. Some studies have reported that the body weight, food intake and visceral fat content of female mice exposed to DEHP were signiﬁcantly increased [26]. 3.5. GnRH and GnRHR Immunohistochemistry 3.5. GnRH and GnRHR Immunohistochemistry Groups Dosage, mg/kg/d MOD (10−2) IOD (×104) Figure 8. Immunohistochemical staining of GnRH expression in the hypothalamus of pubertal female rats (×400). staining in the hypothalamus a. Groups Dosage, mg/kg/d MOD (10−2) IOD (×104) Table 5. Effects of DEHP on mean optical density (MOD) and integral optical density (IOD) of GnRH staining in the hypothalamus a. Control - 15.322 ± 0.853 66.195 ± 6.038 DEHP 250 17.081 ± 0.728 b 78.000 ± 4.794 b 500 16.242 ± 0.537 b 72.200 ± 6.123 b Table 5. Effects of DEHP on mean optical density (MOD) and integral optical density (IOD) of GnRH staining in the hypothalamus a. Control - 15.322 ± 0.853 66.195 ± 6.038 DEHP 250 17.081 ± 0.728 b 78.000 ± 4.794 b 500 16.242 ± 0.537 b 72.200 ± 6.123 b Table 5. Effects of DEHP on mean optical density (MOD) and integral optical density (IOD) of GnRH staining in the hypothalamus a. Control - 15.322 ± 0.853 66.195 ± 6.038 DEHP 250 17.081 ± 0.728 b 78.000 ± 4.794 b 500 16.242 ± 0.537 b 72.200 ± 6.123 b Groups Dosage, mg/kg/d MOD (10−2) IOD (×104) Control - 15.322 ± 0.853 66.195 ± 6.038 DEHP 250 17.081 ± 0.728 b 78.000 ± 4.794 b 500 16.242 ± 0.537 b 72.200 ± 6.123 b 1000 16.492 ± 0.521 b 74.340 ± 7.962 b a n = 12, each treatment group; b Signiﬁcant difference compared to control (p < 0.05). 1000 16.492 ± 0.521 b 74.340 ± 7.96 a n = 12, each treatment group; b Significant difference compared to control (p < 0.05). RHR localized in the uterus was examined by immunohistochemistry (Figure 9 ult of this study shows that the cytomembrane, cytoplasm and nucleus of uterus d glandular epithelial cells are GnRHR-positive. The level of immunohistochemic erus was significantly higher in rats treated with DEHP compared with the contr Figure 9. Immunohistochemical staining of GnRHR expression in the uterus of pubertal female rats (×400). Figure 9. Immunohistochemical staining of GnRHR expression in the uterus of pubertal female rats (×400). Figure 9. Immunohistochemical staining of GnRHR expression in the uterus of pubertal female rats (×400). Figure 9. Immunohistochemical staining of GnRHR expression in the uterus of pubertal female rats (×400). 10 of 15 Int. J. Environ. Res. Public Health 2016, 13, 1130 Table 6. Effects of DEHP on MOD and IOD of GnRHR staining in the uterus a. 3.5. GnRH and GnRHR Immunohistochemistry 3.5. GnRH and GnRHR Immunohistochemistry Groups Dosage, mg/kg/d MOD (10−2) IOD (×104) Control - 12.941 ± 1.024 44.028 ± 0.868 DEHP 250 16.096 ± 0.711 b 50.458 ± 4.719 b 500 14.893 ± 0.563 b,c 48.971 ± 1.825 b,c 1000 15.748 ± 0.160 b 52.083 ± 7.565 b a n = 12, each treatment group; b Signiﬁcant difference compared to control (p < 0.05); c Signiﬁcant difference compared to 250 mg/kg/d (p < 0.05). Table 6. Effects of DEHP on MOD and IOD of GnRHR staining in the uterus a. 4. Discussion The association between DEHP and higher body weight suggests its negative effects on the growth and development of pubertal female rats. In our study, we also ﬁnd that the ovaries of the rats treated with DEHP were congestive and swelling, and the volume of the ovaries and uterus became bigger. It suggests ovary and uterus might be the target organ of DEHP. The result shows that the secretion of GnRH in the hypothalamus of the exposure group signiﬁcantly increased. It indicates that the effect of DEHP is similar to that of GnRH agonists and estrogen. Sustained DEHP exposure might increase the sensitivity of GnRH in the hypothalamus and then raise the secretion of GnRH. Meanwhile, however, there is no signiﬁcant difference in the mRNA level of GnRH in the hypothalamus of the rats treated with DEHP. The protein level in the hypothalamus is signiﬁcantly higher in the rats treated with DEHP compared with the rats in control group, and the level was signiﬁcantly lower in the rats treated with 1000 mg·kg−1·d−1 DEHP compared with the rats in 250 and 11 of 15 Int. J. Environ. Res. Public Health 2016, 13, 1130 500 mg/kg/d DEHP. This means that DEHP could affect the regulation of GnRH after translation, and the protein expression of GnRH is much more stable. However, 1000 mg/kg/d DEHP may inhibit the protein level of GnRH in the hypothalamus. This suggests that GnRH of hypothalamus might be an important target of DEHP. Studies have found that EEDs, like bisphenol A and 4-nonylphenol, can have signiﬁcant adverse effects on the gene regulatory network related to the function of the pituitary-ovary axis. GnRHR mRNA, FSH β-subunit, mRNA of LH β-subunit, FSH-R, mRNA of LH-R, the receptor of E2α and mRNA of the receptor of E2α all increased signiﬁcantly. Those that also increased signiﬁcantly included the expression levels of the mRNA of GnRH-R pathway (phosphatidylinositol pathway), E2-R receptor pathway (including E2-R receptor signal transduction pathway and crosstalk pathway between IGF, EGF and TGF) and the estrogen-metabolizing enzymes [55]. Liu et al.’s study said, compared with control rats, the DEHP-treated adult female rats showed higher mRNA and protein levels of GnRHR in the pituitary [54]. Under the inﬂuence of GnRH in the hypothalamus, the pituitary releases FSH and LH that decide the secretion of sex hormones in the ovary. The cyclic variation of the endometrium is regulated by sex hormones. 4. Discussion Previous research suggested that GnRHR only exists in the pituitary. However, basic research on GnRH and GnRHR in recent years shows that GnRHR also exists in other organs, including the ovaries, testes, uteruses, etc. [41–44]. In addition, tissues of malignant tumors, such as breast cancer, ovarian cancer and endometrial cancer, experience high afﬁnity of GnRHR expression as well [56–58]. DEHP is an environmental endocrine disruptor, and it has estrogen-like activity. Estrogen plays a role in both normal uterine physiology and uterine pathologies, such as dysfunctional uterine bleeding or breakthrough bleeding, endometriosis, infertility, leiomyoma, and endometrial cancer [59]. Previous study investigated the in vitro and in vivo effects of DEHP and also the comparison of the urinary levels of several phthalate metabolites between women with and without endometriosis. These ﬁndings suggest that exposure to phthalate may lead to establishment of endometriosis by enhancing invasive and proliferative activities of endometrial cells [60]. A report showed a 31-year old woman in Italy who had a small uterus, sterility and secondary amenorrhea. Cytogenetic analysis demonstrated the complete loss of a copy of the GnRHR gene. GnRHR may associate with gonadal function [61]. The result of this study shows that the cytomembrane, cytoplasm and nucleus of uterus epithelial cells and glandular epithelial cells are GnRHR-positive. The immunoreactive substance is yellow or yellow brown. In addition, 250 and 500 mg/kg/d DEHP can decrease the protein expression level of GnRHR in uteruses, while high doses of DEHP may increase the GnRHR mRNA and protein expression levels in uteruses of pubertal female rats. This proves that DEHP may, through the toxic effect of GnRH in the hypothalamus and its receptor on the uterus, do damage to the regulatory function of autocrine or paracrine of the uterus and result in uterus related diseases. DEHP is an environmental endocrine disruptor, and it has estrogen-like activity. Estrogen plays a role in both normal uterine physiology and uterine pathologies, such as dysfunctional uterine bleeding or breakthrough bleeding, endometriosis, infertility, leiomyoma, and endometrial cancer [59]. Previous study investigated the in vitro and in vivo effects of DEHP and also the comparison of the urinary levels of several phthalate metabolites between women with and without endometriosis. Th ﬁdi t th t t hth l t l d t t bli h t f d t i i b Author Contributions: Te Liu, Yiyang Jia conceived and designed the experiments; Te Liu, Lin Ye, Qi Wang, Di Sun, Jin Xu, Huaiji Chen, Feng Xu performed the experiments; Te Liu collected the data; Liting Zhou, Qi Wang, Juan Wu analyzed the data; Te Liu wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. Acknowledgments: This study was supported by the Graduate Innovation Fund of Jilin University (Grant No. 2015100) and the National Natural Science Foundation of China (Grant No. 81573184). References 1. 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Conclusions In the present study, our data reveal that DEHP exposure may lead to a disruption in pubertal female rats and an imbalance of the hypothalamus–uterus. Meanwhile, DEHP might, through the GnRH in the hypothalamus and its receptor on the uterus, lead to diseases of the uterus. It may impose a negative inﬂuence on the development and function of the reproductive system in female rats. Acknowledgments: This study was supported by the Graduate Innovation Fund of Jilin University (Grant No. 2015100) and the National Natural Science Foundation of China (Grant No. 81573184). Author Contributions: Te Liu, Yiyang Jia conceived and designed the experiments; Te Liu, Lin Ye, Qi Wang, Di Sun, Jin Xu, Huaiji Chen, Feng Xu performed the experiments; Te Liu collected the data; Liting Zhou, Qi Wang, Juan Wu analyzed the data; Te Liu wrote the paper. 12 of 15 12 of 15 Int. J. Environ. Res. 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https://openalex.org/W4361298335	https://f1000research.com/articles/11-593/pdf	English	null	Whole genome sequencing data of Candida krusei, the pathogen causing Candidaemia, from Department of Parasitology Culture Collection, Faculty of Medicine Universitas Indonesia	F1000Research	2,023	cc-by	3,713	F1000Research 2023, 11:593 Last updated: 19 OCT 2023 GENOME NOTE Whole genome sequencing data of Candida krusei, the pathogen causing Candidaemia, from Department of Parasitology Culture Collection, Faculty of Medicine Universitas Indonesia [version 2; peer review: 2 approved] Fadilah Fadilah 1,2, Robiatul Adawiyah 3,4, Retno Wahyuningsih3,5, Anna Rozaliyani3,6, Ayu Eka Fatril3, Rafika Indah Paramita 1,2, Linda Erlina1,2, Khaerunissa Anbar Istiadi2 1Bioinformatics Core Facilities, Indonesian Medical Education and Research Institute (IMERI), Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia, 10430, Indonesia 2Department of Medical Chemistry, , Faculty of Medicine, Universitas Indonesia,, Jakarta, Indonesia, 10430, Indonesia 3Clinical Parasitology Study Programme, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia 4Department of Parasitology, Faculty of Medicine Universitas Indonesia, Jakarta, 10430, Indonesia 5Department of Parasitology, School of Medicine, Indonesian Christian University, Jakarta, 13630, Indonesia 6Pulmonary Mycosis Centre, Jakarta, 10430, Indonesia Open Peer Review Approval Status 1 2 version 2 (revision) 29 Mar 2023 view version 1 01 Jun 2022 view view Xuepeng Sun, Zhejiang A&F University, Hangzhou, China 1. Atanu Banerjee , Amity University Haryana, Manesar, India 2. Any reports and responses or comments on the article can be found at the end of the article. Open Peer Review Approval Status 1 2 version 2 (revision) 29 Mar 2023 view version 1 01 Jun 2022 view view Xuepeng Sun, Zhejiang A&F University, Hangzhou, China 1. Atanu Banerjee , Amity University Haryana, Manesar, India 2. Any reports and responses or comments on the article can be found at the end of the article. First published: 01 Jun 2022, 11:593 https://doi.org/10.12688/f1000research.121402.1 Latest published: 29 Mar 2023, 11:593 https://doi.org/10.12688/f1000research.121402.2 v2 F1000Research 2023, 11:593 Last updated: 19 OCT 2023 Keywords y Candida krusei, fluconazole, genomic profiling, resistance gene Abstract Candida krusei is a Candida non-albicans species with a high prevalence, which causes candidaemia. Current treatment guidelines include fluconazole as a primary therapeutic option for the treatment of these infections; however, it is only a fungistatic against Candida spp., and both inherent and acquired resistance to fluconazole have been reported. C. krusei species is also reported as the only Candida sp . which has an intrinsic resistance factor to fluconazole. Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by C. krusei. The purpose of this study was to investigate the genome of clinical C. krusei isolates and correlate the resistant phenotypes with mutations in resistance genes. A total of 16 samples of C. krusei from clinical samples from hospitals in Jakarta were used in the experiment. All colonies were extracted using the QIAamp DNA Mini Kit. The library was prepared using the Illumina DNA Prep Kit. The sequencing process was carried out on the Illumina MiSeq Platform using a 2x301 paired-end configuration. FASTQ raw files are available under the BioProject Accession Number PRJNA819536 and Sequence Read Archive Accession Numbers SRR18739949 and SRR18739964. Page 1 of 9 Introduction Candida krusei (teleomorph Pichia kudriavzevii) is an opportunistic fungal pathogen.1 It is the fourth most common non-albicans Candida species (NAC) causing high prevalence and invasive candidiasis and candidemia.2,3 C. krusei is among unique species with natural resistance towards fluconazole, a commonly used antifungal for Candida infections.4 Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by C. krusei One approach in examining the drug resistance of C. krusei is by sequencing the genome with potential roles in resistance. Here we report the whole genome sequencing data of Candida krusei from Department of Parasitology Culture Collection, Faculty of Medicine Universitas Indonesia. The sequencing data obtained from this study will help to understand the correlation between phenotypes with mutations in resistance genes and the development of appropriate treatments and medication for the infection. Sample collection and DNA extraction p The C. krusei clinical sample were retrieved from the Department of Parasitology Culture Collection, Faculty of Medicine, Universitas Indonesia. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, catalog number: 51304) with bead-beating methods.5 100 μL culture of C. krusei were added into 1.5 ml microcentrifuge tube containing 200 μL ATL buffer (Qiagen). Three spoon full of sterilized sand were added into the mixture for bead-beating, which was performed for 10 minutes. 20 μL Proteinase K (Qiagen) were added and the mixture was incubated at 56°C until completely lysed (three hours), and agitated every 10 minutes during incubation time, until homogeneous. 200 μL Buffer AL (Qiagen) were added and the mixture was incubated at 70°C for 10 minutes. 200 μL pure ethanol were added and the mixture was transferred into the QIAamp Mini spin column (Qiagen). Column were centrifugated at 6000 g for one minute and the flow-through were discarded. QIAamp Mini spin columns were placed innew 2 mL collection tubes and 500 μL Buffer AW1 were added into the column. Columns were centrifugated at 6000 g for one minute and the flow- through was discarded. QIAamp Mini spin columns were placed into new 2 mL collection tubes and 500 μL Buffer AW2 (Qiagen) were added. Columns were centrifugated at 17000 g for three minutes and the flow-through was discarded. QIAamp Mini spin columns were transferred into new 1.5 mL microcentrifuge tubes, and 200 μl distilled water were added. Columns were incubated at room temperature for one minute and centrifugated at 6000 g for one minute to elute the DNA. The quality of extracted DNA (A260/280) was measured using a spectrophotometer (NanoDrop ND-1000). The quantity of extracted DNA was measured with Qubit 4.0 Fluorometer using the dsDNA HS Assay kit. Methods Sample collection and DNA extraction Tagmentation 100 ng DNA were added into a 96-well plate and mixed with tagmentation master mix from Illumina Nextera Kit (Illumina). The mix were then incubated at 55°C in a thermal cycler (The Applied Biosystems ProFlex PCR System) for 15 minutes. Post tagmentation clean-up and amplification of tagmented DNA Illumina Nextera Kit tagment stop buffer (Illumina) were added to the tagmentation reaction and incubated at 37°C for 15 minutes. The mixture was washed with Illumina Nextera Tagment wash buffer (Illumina) on the magnetic stand. The tagment Wash Buffer was discarded and Nextera PCR master mixes (Illumina) were added onto the beads. Index adapters were added as sample barcoding. The mixture was amplified in thermal cycler. Libraries clean-up Library preparation A lib i DNA libraries were prepared using the Illumina DNA Preparation Kit. The index used for the library preparation was Integrated DNA Technologies, Inc (IDT) for Illumina Nextera Indexes for ligation step. The library construction steps were as follows: Tagmentation Corresponding author: Robiatul Adawiyah (bundaadah@gmail.com) Author roles: Fadilah F: Conceptualization, Formal Analysis, Supervision, Writing – Original Draft Preparation; Adawiyah R: Conceptualization, Funding Acquisition, Resources, Supervision, Writing – Original Draft Preparation; Wahyuningsih R: Methodology, Project Administration, Resources, Supervision; Rozaliyani A: Methodology, Resources, Supervision; Fatril AE: Data Curation, Methodology, Resources; Paramita RI: Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; Erlina L: Data Curation, Formal Analysis, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing; Istiadi KA: Data Curation, Formal Analysis, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Page 2 of 9 F1000Research 2023, 11:593 Last updated: 19 OCT 2023 REVISED Amendments from Version 1 Revisions were made to the Introduction section within the manuscript. Any further responses from the reviewers can be found at the end of the article Libraries clean-up The beads were added into the supernatant of the mixture and washed twice using 80% ethanol on a magnetic stand. Nextera resuspension buffer (Illumina) reagents were added onto beads and final libraries were retrieved from the supernatant. The libraries of each sample were pooled.6 Page 3 of 9 F1000Research 2023, 11:593 Last updated: 19 OCT 2023 F1000Research 2023, 11:593 Last updated: 19 OCT 2023 Whole genome sequencing The barcoded DNA libraries were sequenced using an Illumina Miseq Platform on the v3-Flow Cell. The DNA library was denatured according to the manufacturer’s protocol.7 For quality control, the library was spiked with 1% PhiX Control v3. The sequencing run produced 2 301 bp paired-end libraries. The data were deposited to the NCBI Sequence Read Archive (SRA) under BioProject. Total raw reads were obtained using FastQC software, and the total raw bases and percentage of Q30 were evaluated using q30 python scripts.8 g q g The barcoded DNA libraries were sequenced using an Illumina Miseq Platform on the v3-Flow Cell. The DNA library was denatured according to the manufacturer’s protocol.7 For quality control, the library was spiked with 1% PhiX Control v3. The sequencing run produced 2 301 bp paired-end libraries. The data were deposited to the NCBI Sequence Read Archive (SRA) under BioProject. Total raw reads were obtained using FastQC software, and the total raw bases and percentage of Q30 were evaluated using q30 python scripts.8 Table 1. DNA quantity and quality. Sample Concentration (ng/μL) Purity (A260/A280) CK1_2021 13.4 2.1 CK2_2021 39.3 2.03 CK3_2021 42.3 2.08 CK4_2021 38.3 2.02 CK5_2021 34.3 1.95 CK6_2021 45.4 1.93 CK7_2021 37 2.07 CK8_2021 23 2.03 CK9_2021 34.3 2.09 CK10_2021 16.7 2.1 CK11_2021 18.9 2.05 CK12_2021 10.3 1.91 CK13_2021 14.2 1.97 CK14_2021 10.1 1.86 CK15_2021 17.9 2 CK16_2021 8.95 1.98 Table 1. DNA quantity and quality. Table 2. Information of raw sequencing data of C. krusei clinical isolates. Libraries clean-up Sample BioSample accession number SRA accession number Q30 (%) GC content (%) CK1_2021 SAMN26901813 SRR18739964 89.75 36 CK2_2021 SAMN26901814 SRR18739963 88.98 38 CK3_2021 SAMN26901815 SRR18739956 89.66 38 CK4_2021 SAMN26901816 SRR18739955 90.00 37 CK5_2021 SAMN26901817 SRR18739954 88.83 37 CK6_2021 SAMN26901818 SRR18739953 89.67 37 CK7_2021 SAMN26901819 SRR18739952 89.57 38 CK8_2021 SAMN26901820 SRR18739951 90.40 37 CK9_2021 SAMN26901821 SRR18739950 92.39 37 CK10_2021 SAMN26901822 SRR18739949 89.34 37 CK11_2021 SAMN26901823 SRR18739962 90.65 37 CK12_2021 SAMN26901824 SRR18739961 90.42 37 CK13_2021 SAMN26901825 SRR18739960 93.18 34 CK14_2021 SAMN26901826 SRR18739959 89.01 37 CK15_2021 SAMN26901827 SRR18739958 88.69 37 CK16_2021 SAMN26901828 SRR18739957 89.57 37 Page 4 of 9 Table 2. Information of raw sequencing data of C. krusei clinical isolates. Page 4 of 9 F1000Research 2023, 11:593 Last updated: 19 OCT 2023 Ethical approval This research was approved by the Faculty of Medicine Universitas Indonesia Ethical Committee (approval number: 970/UN2.F1/ETIK/PPM.00.02/2021). Ethical approval This research was approved by the Faculty of Medicine Universitas Indonesia Ethical Committee (approval number: 970/UN2.F1/ETIK/PPM.00.02/2021). Results The whole raw genome sequence data of 16 clinical isolates of C. krusei from the Department of Parasitology, Faculty of Medicine of Universitas Indonesia were deposited into NCBI data under BioProject accession number PRJNA819536 and SRA SRR18739949-SRR18739964. The DNA quality and quantity of the samples are shown in Table 1. Information regarding the raw data is described in Table 2. These raw data of C. krusei genome are useful for genome profiling and correlating the resistant phenotypes with mutations in resistance genes. 2. Pfaller MA, Jones RN, Castanheira M: Regional data analysis of Candida non- albicans strains collected in United States medical sites over a 6-year period, 2006-2011. Mycoses. Oct. 2014; 57(10): 602–611. Underlying data y g a (FASTQ) files have been deposited into National Center for Biotechnology Information (NCBI, https://www. .nih.gov). BioProject: Raw sequence reads of Candida krusei from clinical samples, Accession Number: PRJNA819536; https:// www.ncbi.nlm.nih.gov/bioproject/PRJNA819536 BioSample: Pathogen: clinical or host-associated sample from Pichia kudriavzevii, Accession Number: SAMN26901813; https://identifiers.org/ncbiprotein:SAMN26901813 BioSample: Pathogen: clinical or host-associated sample from Pichia kudriavzevii, Accession Number: SAMN26901828; https://identifiers.org/ncbiprotein:SAMN26901828 BioSample: Pathogen: clinical or host-associated sample from Pichia kudriavzevii, Accession Number: SAMN26901828; https://identifiers.org/ncbiprotein:SAMN26901828 Raw sequence reads of Candida krusei from clinical samples: Sequence Read Archive: Raw sequence reads of Candida krusei from clinical samples, Accession Number: SRR18739949; https://identifiers.org/insdc.sra:SRR18739949 Sequence Read Archive: Raw sequence reads of Candida krusei from clinical samples, Accession Number: SRR18739949; https://identifiers.org/insdc.sra:SRR18739949 Sequence Read Archive: Raw sequence reads of Candida krusei from clinical samples, Accession Number: SRR18739964; https://identifiers.org/insdc.sra:SRR18739964 Sequence Read Archive: Raw sequence reads of Candida krusei from clinical samples, Accession Number: SRR18739964; https://identifiers.org/insdc.sra:SRR18739964 Acknowledgement This research is funded by Directorate of Research and Development, Universitas Indonesia under Hibah PUTI 2020 (Grant No. NKB-1302/UN2.RST/HKP.05.00/2020). . Cooper CR: Yeasts pathogenic to humans. Elsevier B.V; 2011; vol. 1. 5. Scharf S, Bartels A, Kondakci M, et al.: Introduction of a bead beating step improves fungal DNA extraction from selected patient specimens. Int. J. Med. Microbiol. Sep. 2020; 310(6): 151443. PubMed Abstract\|Publisher Full Text 6. Illumina: Illumina DNA Prep Reference Guide. 2020. Reference Source 7. Illumina: MiSeq System Denature and DIlute Librariers Guide. 2018. Reference Source 8. Chen S: q30 python script. 2016. Reference Source 1. Cooper CR: Yeasts pathogenic to humans. Elsevier B.V; 2011; vol. 1. 2. Pfaller MA, Jones RN, Castanheira M: Regional data analysis of Candida non- albicans strains collected in United States medical sites over a 6-year period, 2006-2011. Mycoses. Oct. 2014; 57(10): 602–611. PubMed Abstract\|Publisher Full Text 3. Lamping E, et al.: Abc1p is a multidrug efflux transporter that tips the balance in favor of innate azole resistance in Candida krusei. Antimicrob. Agents Chemother. Feb. 2009; 53(2): 354–369. PubMed Abstract\|Publisher Full Text 4. Guinea J, Sánchez-Somolinos M, Cuevas O, et al.: Fluconazole resistance mechanisms in Candida krusei: the contribution of efflux-pumps. Med. Mycol. Sep. 2006; 44(6): 575–578. 5. Scharf S, Bartels A, Kondakci M, et al.: Introduction of a bead beating step improves fungal DNA extraction from selected patient specimens. Int. J. Med. Microbiol. Sep. 2020; 310(6): 151443. PubMed Abstract\|Publisher Full Text 6. Illumina: Illumina DNA Prep Reference Guide. 2020. Reference Source 7. Illumina: MiSeq System Denature and DIlute Librariers Guide. 2018. Reference Source 8. Chen S: q30 python script. 2016. Reference Source Data availability Data availability Underlying data Raw data (FASTQ) files have been deposited into National Center for Biotechnology Information (NCBI, https://www. ncbi.nlm.nih.gov). 1. Cooper CR: Yeasts pathogenic to humans. Elsevier B.V; 2011; vol. 1. 2. Pfaller MA, Jones RN, Castanheira M: Regional data analysis of Candida non- albicans strains collected in United States medical sites over a 6-year period, 2006-2011. Mycoses. Oct. 2014; 57(10): 602–611. PubMed Abstract\|Publisher Full Text 3. Lamping E, et al.: Abc1p is a multidrug efflux transporter that tips the balance in favor of innate azole resistance in Candida krusei. Antimicrob. Agents Chemother. Feb. 2009; 53(2): 354–369. PubMed Abstract\|Publisher Full Text 4. Guinea J, Sánchez-Somolinos M, Cuevas O, et al.: Fluconazole resistance mechanisms in Candida krusei: the contribution of efflux-pumps. Med. Mycol. Sep. 2006; 44(6): 575–578. PubMed Abstract\|Publisher Full Text 3. Lamping E, et al.: Abc1p is a multidrug efflux transporter that tips the balance in favor of innate azole resistance in Candida krusei. Antimicrob. Agents Chemother. Feb. 2009; 53(2): 354–369. PubMed Abstract\|Publisher Full Text References Page 5 of 9 https://doi.org/10.5256/f1000research.133265.r159413 © 2023 Banerjee A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Atanu Banerjee 1 Amity Institute of Biotechnology,, Amity University Haryana, Manesar, Haryana, India 2 Amity Institute of Biotechnology,, Amity University Haryana, Manesar, Haryana, India The article by Fadilah et al, presents the whole genome sequence data of clinical isolates of C. krusei. While the genome sequence data generated could be useful to advance our knowledge Open Peer Review Current Peer Review Status: Atanu Banerjee Competing Interests: No competing interests were disclosed. F1000Research 2023, 11:593 Last updated: 19 OCT 2023 Open Peer Review Competing Interests: No competing interests were disclosed. Reviewer Expertise: Mycology. Antifungal resistance, Candida species, Efflux pumps, structure- function studies, sequence, and structural bioinformatics Reviewer Expertise: Mycology. Antifungal resistance, Candida species, Efflux pumps, structure- function studies, sequence, and structural bioinformatics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Reviewer Report 09 January 2023 https://doi.org/10.5256/f1000research.133265.r159413 Atanu Banerjee 1 Amity Institute of Biotechnology,, Amity University Haryana, Manesar, Haryana, Ind 2 Amity Institute of Biotechnology,, Amity University Haryana, Manesar, Haryana, India Amity Institute of Biotechnology,, Amity University Haryana, Manesar, Haryana, India Amity Institute of Biotechnology,, Amity University Haryana, Manesar, Haryana, Ind 2 Amity Institute of Biotechnology,, Amity University Haryana, Manesar, Haryana, Ind The article by Fadilah et al, presents the whole genome sequence data of clinical isolates of C. krusei. While the genome sequence data generated could be useful to advance our knowledge Page 6 of 9 F1000Research 2023, 11:593 Last updated: 19 OCT 2023 regarding factors that might be responsible for the intrinsic azole resistance exhibited quite often by C. krusei isolates, there are some significant gaps that need to be filled to make the manuscript more useful for readers. I elaborate on some of them: regarding factors that might be responsible for the intrinsic azole resistance exhibited quite often by C. krusei isolates, there are some significant gaps that need to be filled to make the manuscript more useful for readers. I elaborate on some of them: regarding factors that might be responsible for the intrinsic azole resistance exhibited quite often by C. krusei isolates, there are some significant gaps that need to be filled to make the manuscript more useful for readers. I elaborate on some of them: The introduction seems extremely brief and does not bring the importance of the work into the proper context. For instance, the authors write " Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by C. krusei" and then directly mention "The purpose of this study was to investigate the genome of clinical isolates of C. krusei and correlate the resistant phenotypes with mutations in resistance genes.". The two statements do not bring the perspective and importance into place appropriately. 1. Also, there isn't much background literature provided in this section. Also, there isn't much background literature provided in this section. The authors mention non-albicans Candida (NAC) species incorrectly in the abstract and introduction. It should be corrected. 2. The authors mention non-albicans Candida (NAC) species incorrectly in the abstract and introduction. It should be corrected. 2. The authors do not provide any background information related to the clinical isolates, except the center. Atanu Banerjee It is extremely important to at least briefly provide some background information on the isolates. 3. While the authors state that "The purpose of this study was to investigate the genome of clinical isolates of C. krusei and correlate the resistant phenotypes with mutations in resistance genes", there is no information provided regarding the same. It limits the importance of the paper. 4. In the keywords, "Candida krusei" is non-italicized. "Resistance gene" does not make much sense and should be replaced with something more meaningful. The same should also be replaced elsewhere in the MS. 5. In the keywords, "Candida krusei" is non-italicized. "Resistance gene" does not make much sense and should be replaced with something more meaningful. The same should also be replaced elsewhere in the MS. 5. Xuepeng Sun 1 Collaborative Innovation Center for Efficient and Green Production of Agriculture in Mountainous Areas of Zhejiang Province, College of Horticulture Science, Zhejiang A&F University, Hangzhou, China 2 Collaborative Innovation Center for Efficient and Green Production of Agriculture in Mountainous Areas of Zhejiang Province, College of Horticulture Science, Zhejiang A&F University, Hangzhou, China Drug resistance is a serious problem for human disease. As the human pathogen C. krusei has natural resistance to the commonly used azole fungicides, it is of particular importance to explore the molecular basis underlying the resistance. This study provides high-depth sequence data for 16 clinic isolates of C. krusei, which in combination with published C. krusei genome will provides new information for correlating the mutations with the resistance phenotypes. Due to the potential interest of the data to scientific community and the quality of the manuscript, I would suggest an acceptance of this manuscript in F1000Reseach. https://doi.org/10.5256/f1000research.133265.r147383 https://doi.org/10.5256/f1000research.133265.r147383 © 2022 Sun X. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Partly Reviewer Expertise: Mycology. Antifungal resistance, Candida species, Efflux pumps, structure- function studies, sequence, and structural bioinformatics Page 7 of 9 F1000Research 2023, 11:593 Last updated: 19 OCT 2023 I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Reviewer Report 30 August 2022 https://doi.org/10.5256/f1000research.133265.r147383 Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Page 8 of 9 F1000Research 2023, 11:593 Last updated: 19 OCT 2023 I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The benefits of publishing with F1000Research: Page 9 of 9
https://openalex.org/W2520852586	https://www.indianjournals.com/ijor.aspx?target=ijor:ijfcm&volume=3&issue=2&article=010&type=pdf	English	null	Analysis of postmortem findings of asphyxial deaths due to Hanging in urban region of Karnataka	Indian journal of forensic and community medicine	2,016	cc-by	1,734	Original Research Article Original Research Article Key Words: Violent asphyxia, Hanging, Autopsy, Salivary stains, Hyoid bone, Thyroid cartilage. www.IndianJournals.com Members Copy, Not for Commercial Sale Access this article online Quick Response Code: Website: www.innovativepublication.com DOI: 10.5958/2394-6776.2016.00027.8 Necessary information for the study was gathered from Police, inquest report and hospital treatment records. The relatives, friends, and neighbors of the victims were also taken separately for data collection. Necessary information for the study was gathered from Police, inquest report and hospital treatment records. The relatives, friends, and neighbors of the victims were also taken separately for data collection. In few cases additional information was gathered by a visit to the scene of crime or by reviewing the photographs. Downloaded From IP - 195.242.1.98 on da p y In few cases additional information was gathered by a visit to the scene of crime or by reviewing the photographs. A detailed proforma for recording the details of hanging was prepared for filling the observation of the present study. The information thus collected, was statistical analyzed. In certain cases of deaths resulting from hanging, the ligature mark may create uncertainty. All the characteristic features maybe present infrequently due to the variable amount of pressure applied on the neck. Hence it mandates a proper assessment of various post-mortem findings under such circumstances. Fig 1: Profile of hanging cases In this study an attempt has been undertaken in the view to gain, further knowledge and insight into the gross post mortem features of asphyxial deaths due to hanging in Bangalore South region. Introduction Hanging, a form of violent asphyxia, is due to constriction of neck as a result of suspension in such a manner that the weight of the body or a part of the victim's body pulls upon the ligature. Analysis of postmortem findings of asphyxial deaths due to Hanging in urban region of Karnataka V. Dekal1, Shruthi P2,* 1Associate Professor, 2Assistant Professor, Dept. of Forensic Medicine & Toxicology, Saveetha Medical College & Hospital, Chennai 1Associate Professor, 2Assistant Professor, Dept. of Forensic Medicine & Toxicology, Saveetha Medical College & Hospital, Chennai *Corresponding Author: Email: drshruths@gmail.com Abstract Hanging, a form of deliberate self-harm accounts for a large proportion of autopsies which forensic experts come across in their clinical tenure at various hospitals in almost all parts of India. A retrospective study was conducted in the Department of Forensic Medicine and Toxicology, Kempegowda Institute of Medical Sciences, Bangalore from April 2004 to March 2006, with an objective to study the post mortem findings in autopsy cases of hanging. Out of 810 autopsies, 27.9% cases were that of hanging. Most cases were atypical (95.6%) and complete hanging (90.3%) with the ligature mark situated above the thyroid cartilage. 34.1% of cases had dried salivary stain at the angle of mouth and 1.3% cases showed involuntary discharge. The incidence of fracture of hyoid bone was 15.49% of cases. This study depicts the possible findings in a suspected case of hanging which ought to be anticipated to avoid any flawed opinion. Indian Journal of Forensic and Community Medicine, April-June 2016;3(2):121-123 Materials and Methods Fig 1: Profile of hanging cases The study consisted of 810 medico-legal autopsy performed in the Department of Forensic Medicine, Kempegowda Institute of Medical Sciences, Bangalore, Karnataka during the period of 2 years (from April 2004 to March 2006). Out of total 810 medico-legal autopsies, 226 were hanging victims (27.9%). During this study period, 810 cases were brought for post-mortem examination out of which 226 (27.9%) deaths were due to hanging. (Fig. 1) During this study period, 810 cases were brought for post-mortem examination out of which 226 (27.9%) deaths were due to hanging. (Fig. 1) 121 Indian Journal of Forensic and Community Medicine, April-June 2016;3(2):121-123 Analysis of postmortem findings of asphyxial deaths due to Hanging in urban region of…. V. Dekal et al. Table 1: Typical and Atypical hanging Typical/ Atypical Position of knot Back Left Right Total Typical 10 (100.0%) - - 10(4.4%) Atypical - 93(43.1) 123(56.9%) 216(95.6%) 10(4.4) 93(41.2) 123(54.4) 226 Out of 226 cases, typical hanging was found only in 10 cases (4.4%) whereas most of the cases 216 (95.6%) were atypical hanging, with the position of knot on left or right side. Knot was on the right side of neck in 123 cases (56.9%) (Table 1). Similar findings were observed in the studies conducted by other authors.[1,2] Table 2: Type of Hanging (Complete/ Partial) Type of Hanging Number Percentage Complete 204 90.3 Partial 22 9.7 Total 226 100.0 In the present study complete hanging was seen in 90.3% deaths. Partial hanging was taking lives mostly, accounted for 22(9.7%) deaths (Table 2). This is in accordance with studies by few authors[3,4] but not in agreement with others.[1,2] Table 3: According to the Level of the Ligature Mark Level of ligature mark Cases Percentage Above the thyroid cartilage 192 84.95 Overriding the thyroid cartilage 19 8.41 Below the thyroid cartilage 15 6.64 Total 226 100 Downloaded From IP - 195.242.1.98 on In our study, it was observed that in 192 cases, the level of ligature mark was above the thyroid cartilage, below the thyroid cartilage in 15 cases and overriding the thyroid cartilage in 19 cases (Table 3). Materials and Methods This was also observed in various other authors’ studies.[1,4] Table 4: Salivary Stains Salivary Stain Number % Present 77 34.1% a) Left (n=77) 40 51.9% b) Right (n=77) 37 48.1% Absent 149 65.9% Total 226 100.0% Dribbling of saliva, surest sign of antemortem hanging, was found in 77 cases (34.1%) of hanging. The findings are consistent with those of Ashok Kumar Samanta et al[5] who observed 32.31% cases with dribbling of saliva.(Table 4) In 175 (77.43%) cases, postmortem staining was present over the back, indicating the body was noticed by the relatives within 2 hours of suspension, removed and placed in a prone position. Involuntary discharge of feces was Dribbling of saliva, surest sign of antemortem hanging, was found in 77 cases (34.1%) of hanging. The findings are consistent with those of Ashok Kumar Samanta et al[5] who observed 32.31% cases with dribbling of saliva.(Table 4) In 175 (77.43%) cases, postmortem staining was present over the back, indicating the body was noticed by the relatives within 2 hours of suspension, removed and placed in a prone position. Involuntary discharge of feces was present in 1 cases and semen on glans penis present in 2 cases of hanging. In 226 cases of hanging, hyoid bone was fractured in 35 cases (15.49 %). This is in agreement with a study by various authors from Gujrat.[6,7] In 175 (77.43%) cases, postmortem staining was present over the back, indicating the body was noticed by the relatives within 2 hours of suspension, removed and placed in a prone position. Involuntary discharge of feces was present in 1 cases and semen on glans penis present in 2 cases of hanging. In 226 cases of hanging, hyoid bone was fractured in 35 cases (15.49 %). This is in agreement with a study by various authors from Gujrat.[6,7] Indian Journal of Forensic and Community Medicine, April-June 2016;3(2):121-123 122 Analysis of postmortem findings of asphyxial deaths due to Hanging in urban region of…. V. Dekal et al. www.IndianJournals.com Members Copy, Not for Commercial Sale Downloaded From IP - 195.242.1.98 on dated 24-Oct-2024 7. Sarangi M. P. “Ligature marks” – In Forensic pathologist’s perspective. Journal of Forensic Medicine and Toxicology. 1998Jan – June;15(1):99–102. Conclusion gy 8. S. Nikolic, J. Micic, T. Atanasijevic, V. Djokic, D. Djonic. Analysis of neck injuries in hanging. Am. J. Forensic Med. Pathol. 2003;24(1):179–182. Age is doubtlessly one of the most important variable contribute to the fracture of the neck structure in hanging. The fracture of hyoid bone should preferably be confirmed by radiography and histology before cataloging it as an ante-mortem fracture. Dribbling of saliva present in case of hanging is a sure sign of antemortem hanging. Other post mortem findings like involuntary discharge of urine, fecal matter, semen on glans penis, postmortem staining etc. will help in the diagnosis when ligature marks are not clear. 9. B. Knight, P. Saukko, Fatal Pressure on the Neck in: Knight’s Forensic Pathology, 3th ed., Arnold Publishers, London, England, 2004:368–394. 10. H.M. Garvin. Ossification of laryngeal structures as indicators of age. J. Forensic Sci. 2008;53(1):1023–1027. 5. Ashok Kumar Samanta, Soumya Rajan Nayak. Newer trends in hanging death. Journal of Indian Academy of Forensic Medicine. 2012;34(1):37–39. Materials and Methods Table 5: Hyoid Bone Fracture Age in Years Hyoid Bone Fracture Present (n=35) Absent (n=191) Total (n=226) 11-20 1 2.9 41 21.5 42 18.6 21-30 1 2.9 100 52.4 101 44.7 31-40 6 17.1 40 20.9 46 20.4 41-50 14 40.0 9 4.7 23 10.2 51-60 8 22.9 1 0.5 9 3.9 >60 5 14.3 - - 5 2.2 Inference The incidence of fracture of hyoid bone in hanging is significantly more (61 times) in age group above 40 years in comparison to the age group below 40 years (X2 = 111.17, p< 0.001). Table 5: Hyoid Bone Fracture erence The incidence of fracture of hyoid bone in hanging is significantly more (61 times) in age group above 40 years in comparison to the age group below 40 years (X2 = 111.17, p< 0.001). 27 cases out of the 35 were above the age group of 40 years. The incidence of fracture of hyoid bone is significantly more in the age group above 40years when compared to that below 40 years (Table 5). It has been demonstrated in numerous studies that the incidence of fractures increases with age[2,8] because neck structures become calcified and more brittle in middle and later life[9,10]. 6. M.M.M Shaikh, H. J. Chotaliya, A.D. Modi, A. P. Parmar, S. D. Kalele. A study of gross postmortem findings in cases of Hanging and Ligature Strangulation. Journal of Indian Academy of Forensic Medicine.2013 Jan-March;35(1):63-65. References 1. Sharma BR, Harish D, Sharma S, Singh H. Injuries to structures in deaths due to constriction of neck, with special reference to hanging. J Forensic Leg Med. 2008 July;15(5):298-305. y 2. Sharma BR, Singh VP, Harish D. Neck structure injuries in Hanging-comparing retrospective and prospective studies. Med Sci. Law. 2005;45(4):321-330. 3. M Ahmad, MZ Hossain. Hanging as a Method of Suicide: Retrospective Analysis of Postmortem Cases. JAFMC Bangladesh. 2010;6(2):37-39. 4. T. Saisudheer, T. V. Nagaraja. A study of ligature mark in cases of hanging deaths. Int. J Pharm Biomed Sci. 2012;3(3):80-84. 123 Indian Journal of Forensic and Community Medicine, April-June 2016;3(2):121-123
https://openalex.org/W2966105081	https://europepmc.org/articles/pmc6676525?pdf=render	English	null	Precopulatory acoustic interactions of the New World malaria vector Anopheles albimanus (Diptera: Culicidae)	Parasites & vectors	2,019	cc-by	11,721	Abstract Background: Anopheles albimanus is a malaria vector in Central America, northern South America and the Carib‑ bean. Although a public health threat, An. albimanus precopulatory mating behaviors are unknown. Acoustics play important roles in mosquito communication, where flight tones allow males to detect and attract potential mates. The importance of sound in precopulatory interactions has been demonstrated in Toxorhynchites brevipalpis, Aedes aegypti, Culex quinquefasciatus and Anopheles gambiae; convergence in a shared harmonic of the wing beat frequency (WBF) during courtship is thought to increase the chance of copulation. To our knowledge, An. albimanus precopula‑ tory acoustic behaviors have not been described to date. Here, we characterized An. albimanus (i) male and female flight tones; (ii) male–female precopulatory acoustic interactions under tethered and free flight conditions; and (iii) male-male acoustic interactions during free flight. Results: We found significant increases in the WBFs of both sexes in free flight compared to when tethered. We observed harmonic convergence between 79% of tethered couples. In free flight, we identified a female-specific behavior that predicts mate rejection during male mating attempts: females increase their WBFs significantly faster during mate rejection compared to a successful copulation. This behavior consistently occurred during mate rejec‑ tion regardless of prior mating attempts (from the same or differing male). During group flight, males of An. albimanus displayed two distinct flying behaviors: random flight and a swarm-like, patterned flight, each associated with distinct acoustic characteristics. In the transition from random to patterned flight, males converged their WBFs and signifi‑ cantly decreased flight area, male-male proximity and the periodicity of their trajectories. Conclusions: We show that tethering of An. albimanus results in major acoustic differences compared to free flight. We identify a female-specific behavior that predicts mate rejection during male mating attempts in this species and show that male groups in free flight display distinct flying patterns with unique audio and visual characteristics. This study shows that An. albimanus display acoustic features identified in other mosquito species, further suggesting that acoustic interactions provide worthwhile targets for mosquito intervention strategies. Our results provide compelling evidence for swarming in this species and suggests that acoustic signaling is important for this behavior. Keywords: Mosquito, Malaria, Harmonic convergence, Swarming, Mating © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Parasites & Vectors Parasites & Vectors Parasites & Vectors Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 https://doi.org/10.1186/s13071-019-3648-8 Background Despite its status as an important vector in the Americas, there exists a large gap in our knowledge of the behavior, ecology and biology of this species, making the development and/or improvement of control methods difficult. Much of our understanding of anopheline reproduction is inferred from studies in An. gambiae (subgenus Cellia), whose post-mating reproduc- tive biology differs substantially from An. albimanus [8]. Mosquito vectorial capacity is dependent on biologi- cal factors that include population density, female blood- feeding rate and female longevity [9], each related to successful reproduction [8, 10, 11]. Thus, processes of reproduction offer promising targets to control mosquito vectors and, by extension, the diseases they spread [12– 14]. Contemporary mosquito control methods include the mass release of transgenic or sterile males to sup- press native mosquito populations [15, 16]. Therefore, mass-released males need to compete with wild males for mating opportunities, which require males to detect and successfully interact with females. In Aedes [17, 18], Culex [19] and Anopheles species [20, 21], acoustic sig- nals are used by males to locate and recognize females. Males detect nearby flying females of the same species by the sound of their wing beat frequency (WBF) [20, 22– 24], pursue them, and appear to acoustically interact with them at a local scale [25, 26] prior to copulation. It has also been suggested that females use acoustic signals as an indicator of male fitness [27, 28]. In several Anopheles species the initiation of reproduc Anopheles albimanus (subgenus Nyssorhynchus) is a vector of malaria throughout Central America, the northern portion of South America and the Caribbean [7]. In Colombia, An. albimanus is one of three primary vectors of malaria [7]. Despite its status as an important vector in the Americas, there exists a large gap in our knowledge of the behavior, ecology and biology of this species, making the development and/or improvement of control methods difficult. Much of our understanding of anopheline reproduction is inferred from studies in An. gambiae (subgenus Cellia), whose post-mating reproduc- tive biology differs substantially from An. albimanus [8]. Studies describing acoustic interactions between oppo- site-sex mosquitoes are more numerous. In several spe- cies, female flight tones act as a mating call that attracts males of the same species [23, 38, 39]. In experiments with tethered Ae. aegypti [17], Cx. quinquefasciatus [19] and An. Background gambiae [20, 21], males and females modulate their WBFs to match each other during courtship, a phe- nomenon known as harmonic convergence. Such con- vergence does not happen in the WBF of the flight tones but instead in a shared harmonic [17, 21]. An additional acoustic behavior, characterized by the rapid frequency modulation (RFM) of the WBF, was first detected in Cx. quinquefasciatus [25] and later described in An. coluzzi [40], An. gambiae [40] and Ae. aegypti [26]. RFM is observed after a steep increase of the male WBF when in close proximity to a tethered female or an artificial tone source [23, 38, 39]. In all species studied, RFM occurs prior to copulation. In Ae. aegypti, RFM coincides with initial contact of a female by a courting male and ends prior to copulation or separation of the pair [26]. Moreo- ver, RFM and harmonic convergence appear to be inde- pendent events [25, 26, 40].l f Mosquito vectorial capacity is dependent on biologi- cal factors that include population density, female blood- feeding rate and female longevity [9], each related to successful reproduction [8, 10, 11]. Thus, processes of reproduction offer promising targets to control mosquito vectors and, by extension, the diseases they spread [12– 14]. Contemporary mosquito control methods include the mass release of transgenic or sterile males to sup- press native mosquito populations [15, 16]. Therefore, mass-released males need to compete with wild males for mating opportunities, which require males to detect and successfully interact with females. In Aedes [17, 18], Culex [19] and Anopheles species [20, 21], acoustic sig- nals are used by males to locate and recognize females. Males detect nearby flying females of the same species by the sound of their wing beat frequency (WBF) [20, 22– 24], pursue them, and appear to acoustically interact with them at a local scale [25, 26] prior to copulation. It has also been suggested that females use acoustic signals as an indicator of male fitness [27, 28]. p To date, flight acoustics and male–female pre-mating acoustic interactions have not been investigated in An. albimanus. Furthermore, it is unknown if males of this species swarm in the wild. To gain insight into An. albi- manus precopulatory acoustic behaviors, we first char- acterized flight acoustics in this species by determining male and female WBFs when tethered and in free flight. Background vectors and represents a major disease that afflicts the inhabitants of tropical countries. Although most lethal malaria cases are concentrated in sub-Saharan Africa [1], numerous cases of the disease occur in South America [2]. In Colombia, an estimated 12 million people live in malaria endemic areas and are at risk of infection [3]. In 2017, ~53,000 cases of malaria were reported in Colom- bia [4], with the majority originating in areas near the Malaria is caused by Plasmodium parasites that are trans- mitted through the bites of infected Anopheles mosquito Correspondence: grupotandem.mosquito@udea.edu.co; catalfonso@gmail.com 4 Max Planck Tandem Group in Mosquito Reproductive Biology, Universidad de Antioquia, Medellín, Antioquia 050010, Colombia Full list of author information is available at the end of the article 4 Max Planck Tandem Group in Mosquito Reproductive Biology, Universidad de Antioquia, Medellín, Antioquia 050010, Colombia Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 12 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Atlantic and Pacific coasts. Despite the implementation of various control methods, which include indoor and outdoor insecticide spraying and the use of insecticide impregnated bednets, malaria continues to be a major public health issue in Colombia [5, 6]. evident in larger groups [36], although both frequency convergence and divergence behaviors are observed between pairs of tethered Ae. aegypti males in close prox- imity [37], showing how variable such interactions can be. Frequency divergence has also been reported between tethered Cx. quinquefasciatus males [19]. Characteriza- tion of male-male acoustic interactions may be key in understanding swarm formation and swarm cohesion. p Anopheles albimanus (subgenus Nyssorhynchus) is a vector of malaria throughout Central America, the northern portion of South America and the Caribbean [7]. In Colombia, An. albimanus is one of three primary vectors of malaria [7]. Background We observed a significant increase in the WBF of each sex when in free flight compared to when tethered. We next characterized male–female acoustic interactions when tethered and in free flight during courtship. We found that 79% of tethered couples display harmonic convergence. Using criteria established for tethered cou- ples, we did not detect harmonic convergence in free fight, possibly due to the brevity of mating attempts (1.38 ± 0.32 s) and the absence of paired flight after cou- ples made contact (when harmonic convergence likely occurs; [26]). However, we observed that during mate rejection, females increase their WBFs significantly faster than those that mate. Finally, using audio and visual anal- ysis, we show that male groups in free flight change flight trajectories and match flight tones during a stereotypic, patterned flight, which could be suggestive of swarm-like i In several Anopheles species, the initiation of reproduc- tive behavior begins when males form a swarm [29]. In African vectors such as An. funestus, An. gambiae and An. arabiensis, swarm formation begins when a few males begin to fly simultaneously [30–33]; additional males rap- idly join the flight, increasing the swarm size and density [29, 34]. At the initiation of swarm formation, males fly in a non-specific circular motion before forming a more tightly patterned, cohesive group [35]. Once formed, males either mate with females that penetrate the swarm or depart the swarm to mate with a female flying nearby [35]. To date, acoustic interactions between swarming males have not been described for Anopheles species. However, cohesive acoustic behavior between multiple tethered Ae. aegypti males has been observed [36]: male groups exhibit similar flight tones, a phenomenon more Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Page 3 of 12 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 behavior in An. albimanus. Our results represent impor- tant first steps toward dissecting the subtle, pre-mating behavioral interactions that occur in this important malaria vector. if An. albimanus display the precopulatory behavior of harmonic convergence [17]. One mosquito was placed 1 cm above one microphone and kept stationary. A teth- ered individual of the opposite sex, placed ~1 cm above a second microphone, was brought in and out of the near field of the first, stationary mosquito for intervals of 10 s with 5 s of rest between intervals. After repeating 3 times, the positions of the individuals were switched. Signal analysis Si i Since mosquito flight tone has been described as a har- monic sound [4], a sinusoidal-harmonic model was used to extract the WBF information of the recorded signals. From the spectrogram [fast Fourier transform (FFT) length of 4096 points, hamming window of 80 ms and 50% overlapping], we extracted the WBF by tracking the lower frequency peak of the spectrogram that persisted for at least 1 s and fulfilled two conditions: between consecutive windows, the frequency and power did not change more than ± 20 Hz and ± 10 dB FS, respectively. For individual flights, we analyzed WBF mean and WBF range of both sexes. For male–female interactions, we identified harmonic convergence as in Aldersley et al. [37]. Comparing the extent of frequency modulation and frequency distribution prior to and during interactions, we identified events where there was an active modula- tion of the WBF in response to the conspecific flight tone. To label convergence events as positive or nega- tive, we used a time-varying fundamental frequency ratio between females and males (♀WBF/♂WBF). For instance, a ratio of 0.667 means that the female’s third harmonic is converging with the males second. We set a positive convergence as an event of duration greater than 1 s, in which the convergence ratio was within an interval of tolerance ± 1.5%. The time between individual introduc- tion and the onset of convergence was noted as latency [21]. Analysis was performed using Matlab (R2016a, The Mathworks Inc, Natick, USA) Background Wing length of males and females were measured as previously described [41] to estimate individual size. All experiments were performed at 26 ± 2 °C. Tone analysis was performed using recordings that had > 10 s of con- tinuous flight. Mosquito rearing Anopheles albimanus were obtained from the col- ony maintained at Programa de Estudios y Control de Enfermedades Tropicales (PECET), Universidad de Antioquia, Colombia. These mosquitoes were originally collected in Santa Rosa, Bolivar, Colombia, and have been kept in colony since 1995. Mosquitoes were reared and maintained in a walk-in climate chamber at 27 °C and 80% relative humidity (RH) under a 12:12 h light:dark photoperiod. Eggs were collected in 473-ml cups filled with dH2O. Upon hatching, larvae were individually transferred to trays (~60 per tray) containing 0.5 l of dH2O. Larvae were fed daily with 20 mg of fish food (Tet- racolor; Tetra, Melle, Germany). Pupae were individually transferred to 15-ml vials to ensure virginity. Upon eclo- sion, adults were separated by sex, transferred to 4-l plas- tic cages, and held in same-sex groups until experiments commenced. Mosquitoes had access to 20% sucrose ad libitum. We used 4–6-day-old adults in all experiments. Tethered mosquitoes Audio recording setup Recordings were performed in a soundproof chamber that contained two particle velocity microphones (NR- 23158-000, Knowles; Itasca, IL, USA). Microphone sig- nals were amplified and digitalized by an USB audio interface (M-Track Quad Four Channel Audio; M-Audio, Cumberland, USA). Mosquito flight tones were processed using Matlab (R2016a, The Mathworks Inc, Natick, USA) at a sample rate of 11025 Hz/24 bits. Experimental procedures Males and females were recorded to determine their individual WBF distribution. Virgin mosquitoes were anesthetized on ice and tethered to a human hair using a cyanoacrylate-based adhesive (Super Pega Infinita, Medellin, Colombia). The hair was attached on one end to an insect pin while the opposite end was attached to the pronotum (dorsal section of the thorax) as in [27]. Before recording, the position of the hair was checked to ensure natural wing movement. To record flight tones, mosquitoes were placed ~1 cm above a single particle velocity microphone. When necessary, flight was trig- gered by blowing or by gently moving the legs of the individual. Audio signal analysis Signals from each of the four microphones were used to extract frequency components of the flight tones. By obtaining the cross spectrum among the microphones (FFT length of 4096, window length 0.08 s, 50% overlap- ping) [42], we generated a high order spectrogram which was used to analyze the signals continuously, regardless of the distance between mosquitoes and microphones. Depending on the interaction analyzed, we extracted dif- ferent frequency features from the spectrograms. (i) For Video analysis A di di Audio recording from the microphone integrated to the camera was used to synchronize audio and video. We used a 1 kHz pure tone to synchronize the camera and electret microphones. Once audio and video were syn- chronized, we extracted 5 s video fragments from record- ings in which the specific behavior was observed. Each fragment was segmented into individual frames and each frame converted to grayscale and adjusted through the equalization of its histogram to improve the contrast of the image. Finally, for each frame, X and Y coordinates of each mosquito were obtained manually to reconstruct flight trajectories using Matlab (R2016a, The Mathworks Inc, Natick, USA). Trajectories were represented by ellipse due to the natural flight of the individuals, follow- ing previous methodologies [43]. In brief, ellipse area was calculated to represent the area covered by the movement of each mosquito. The center of the ellipse corresponded to the mean position of each mosquito trajectory (the X and Y coordinates) during the 5 s analyzed. The horizon- tal and vertical diameters of each ellipse were calculated as 4 times the standard deviation of each trajectory point of each individual. The average distance of the centroids of each ellipse in relation to the others was calculated to assess the extent of the aggregation of the group. Finally, the loop period of each trajectory was calculated to assess the periodicity of the movement, as in Gibson [43]. l Audio and visual recording setupl Audio and video of free-flying mosquitoes were simulta- neously recorded in an experimental arena (20 × 20 × 25 cm transparent plastic box) placed within a sound-proof chamber. The experimental arena contained four electret microphones (Knowles FG-23329-C05), three placed on the side and back walls and one on the bottom of the box. Using tethered individuals as described above, male– female interactions (couples) were recorded to determine Page 4 of 12 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 male and female individual flight, the WBF distribution and range was determined. (ii) For male–female interac- tions, we visually selected relevant steps of mating and analyzed the WBF ratio (♀WBF/♂WBF) 1–1.5 s prior to a mating attempt, the rate of WBF increase during a mating interaction and the response latency of the WBF increase of the female following that of the male. The change in frequency (ΔF = Fmax − F0) was measured as the difference between the initial frequency at the onset of the increase (F0) and the maximum frequency reached (Fmax). For females, however, the change in frequency was divided by two, as measurements where extracted from the second harmonic (ΔF = (Fmax − F0)/2). The duration of the increase was measured by using the time at the same two points (Δt = tmax − t0). Finally, we calculated the rate of increase (ΔF/Δt) of each sex. (iii) For male groups, we measured the average difference between the high- est and the lowest WBFs, referred hereafter as frequency difference. Microphone signals were amplified by an operational amplifier (INA128; Texas Instruments, Dallas, USA) and digitalized by a 16-channel digital-analog converter (779676-01; National Instruments, Austin, USA) at a sample rate of 11025/16 bits. For video recording, a web- cam (HD Pro Webcam C920; Logitech, Lausanne, Swit- zerland) was placed 30 cm in front of the arena to follow mosquito movement. Experimental proceduresh Three types of recordings were performed. (i) We recorded individual males and females inside the arena (n = 30 for each sex). (ii) Male–female acoustic interac- tions were recorded by introducing two males into the arena followed by one female. Flight tones were continu- ously recorded until copulation was observed or the indi- viduals stopped flying. Mosquitoes were removed after a successful copulation. Successful copulations were veri- fied by dissecting females to determine insemination sta- tus (i.e. presence/absence of sperm in the spermathecae). A mating attempt was counted as successful if a couple locked genitalia for more than 5 s and sperm was trans- ferred to the female. An attempt was considered a rejec- tion when the male and female genitalia did not come into contact or the female did not allow the male to lock genitalia. As females often rejected courting males, we recorded 167 total mating attempts from 42 different females. Recordings from 9 females were discarded, as it was not possible to clearly identify the frequency com- ponents of each sex. (iii) Acoustic interactions of male groups were recorded by introducing 8 mosquitoes into the arena 5 h before recording started. Although 8 mos- quitoes were used in every experiment, only groups of 2, 3 and 4 mosquitoes flying simultaneously were analyzed. For each experiment, recordings (2.5 h in length) were segmented into 10 min sections. When a specific flying behavior was observed visually, unique fragments of 5 s were used in our analysis (for the respective number of males flying). All recordings were performed at 26 ± 2 °C between 16:00 and 18:30, corresponding to the 2 h prior to sunset. Statistical analysis For tethered mosquitoes, we performed a linear regres- sion analysis to determine the relationship between wing length (as a size index) and WBF. To compare frequency Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Page 5 of 12 females were individually introduced into a soundproof box and their flight tones recorded. During free flight, we observed a WBF of 713.76 ± 43.16 Hz (n = 30) for males, and 430.34 ± 28.48 Hz (n = 30) for females (Fig. 1a). A significant increase in flight tone was observed for individuals in free flight compared to tethered indi- viduals (t-test; males: t(106) = − 9.03, P < 0.01; females: t(144) = − 15.00, P < 0.01). Similar to tethered individuals, we did not observe a strong relationship between mos- quito size (males: 2.83 ± 2.02 mm; females: 3.05 ± 220.53 mm) and WBF (Linear regression; males: R2 = 0.12, Pear- son’s r(45) = 0.35, P = 0.02; females: R2 < 0.01, Pearson’s r(54) = 0.03, P = 0.81; Additional file 1: Figure S1b). distributions and ranges of tethered and free-flying individ- uals, we performed a t-test and a factor analysis. As factors, we evaluated sex and recording method. Residuals of t-tests and factorial models were tested for normality, homoge- neity of variance and independence using Shapiro-Wilks, Bartlett and Durbin-Watson tests, respectively. Aspects influencing male–female interactions during free flight tri- als were assessed using the non-parametric Mann–Whit- ney U-test. The frequency difference broadcast by male groups and the differences between the characteristics of flight trajectories were assessed using a Mann–Whitney-U test. Results are presented as mean ± SEM. Sample sizes are indicated for each experiment. ( )i When analyzing each spectrogram, we noted that the tones of individuals were characterized by larger mod- ulations of the WBF when in free flight compared to when tethered (Fig. 1b). To evaluate the effect of teth- ering on frequency modulation, we randomly selected a sample of 30 tethered males and 30 tethered females to compare them with 30 males and 30 females in free flight by performing a factorial ANOVA, using the recording method and sex as factors. Flight tones of tethered and free‑flying An. albimanus males and femalesl To characterize An. albimanus flight tones, virgin males and females were individually recorded while tethered or in free flight. When tethered, we observed a WBF of 524.11 ± 63.73 Hz (mean ± SEM, n = 78) for males and 368.91 ± 34.28 Hz (n = 116) for females (Fig. 1a). There was a significant difference in the WBF between males and females (t-test: t(192) = − 21.94, P < 0.01). By meas- uring the wing length of males (2.78 ± 0.02 mm) and females (2.89 ± 0.02 mm), we did not find evidence of a relationship between individual size and WBF (Linear regression; males: R2 < 0.01, Pearson’s r(78) = 0.06, P = 0.58; females: R2 = 0.03, Pearson’s r(116) = − 0.20, P = 0.05; Additional file 1: Figure S1a). Thus, size did not appear to significantly influence the WBF in tethered individuals of either sex.l Because various studies of insect flight have reported that WBFs of tethered individuals differ compared to individuals in free flight [4, 25, 44, 45], we sought to determine how An. albimanus acoustics might differ in this regard by examining mosquitoes in flight. Males and Statistical analysis Sex (factorial ANOVA: F(1, 116)=13.1, P < 0.01) and tethering (facto- rial ANOVA: F(1, 116) = 37.57, P < 0.01) each affected the range of modulation, and an interaction between these two factors was observed (factorial ANOVA: F(1, 116) = 4.66, P = 0.03). Thus, when males and females are tethered, the range of their WBF modulation is similar (males: 53.33 ± 7.89 Hz; females: 45.25 ± 3.14 Hz) but is significantly increased (males: 99.10 ± 5.75 Hz; females: 67.17 ± 4.67 Hz) during free flight (Fig. 1c). Harmonic convergence between tethered An. albimanus males and females We did not detect a relation- ship between the ratio of couple size (♂size/♀size) and the ratio of convergence (t-test: t(40) = 0.68, P = 0.49): couples where the female’s third harmonic converged with the male’s second had a size ratio of 0.954 ± 0.015; couples where the female’s fourth harmonic converged with the male’s third had a size ratio of 0.942 ± 0.007. first mating attempt. All mated females copulated within 1–5 min after introduction to the arena. Females that did not mate in our assays rejected males 1–9 times, with females that rejected once (n = 3) not flying the remain- der of the assay. To compare mating attempts that ended in a successful copulation or a rejection, we examined audio recordings of the 12 interactions that resulted in a successful copulation and randomly selected one mating attempt from each of the 21 females that did not mate in order to identify male- or female-specific acoustic behav- iors that occur immediately prior to An. albimanus copu- lation or mate rejection. We consistently observed that all mating attempts, regardless of outcome, were characterized by an increase in male WBF followed by an increase in female WBF (Fig. 3a; Additional file 2: Video S1). The increase in the WBF of the male coincided with the onset of the female chase, while that of the female coincided with her fly- ing away from the approaching male. After increasing their WBFs, males and females rapidly modulated their frequency until couples departed or stopped flying. Although these oscillations appear to be similar to those reported in other species [25, 26, 40], it was not always possible to observe this behavior in every mating attempt as 25% of the couples that copulated landed upon making contact. The time interval of rapid frequency oscillation after reaching maximum WBF ranged between 0.34–1.38 s and 0.18–1.56 s for males and females, respectively. Harmonic convergence between tethered An. albimanus males and females Since harmonic convergence has been previously described using tethered mosquitoes of other species Fig. 1 Flight tones of tethered and free-flying An. albimanus individuals. a Top: WBF distribution of tethered males (n = 78, blue) and females (n = 116, red). Bottom: WBF distribution of free-flying males (n = 30, blue) and females (n = 30, red). b Spectrograms of a representative tethered (top) and free-flying (bottom) male. c WBF range analysis of males (n = 30) and females (n = 30) when tethered or in free flight. There were significant effects for both sex and treatment. Vertical bars denote 0.95 confidence intervals and letters indicate significant differences for a post-hoc Tukey test with 0.95 confidence Fig. 1 Flight tones of tethered and free-flying An. albimanus individuals. a Top: WBF distribution of tethered males (n = 78, blue) and females (n = 116, red). Bottom: WBF distribution of free-flying males (n = 30, blue) and females (n = 30, red). b Spectrograms of a representative tethered (top) and free-flying (bottom) male. c WBF range analysis of males (n = 30) and females (n = 30) when tethered or in free flight. There were significant effects for both sex and treatment. Vertical bars denote 0.95 confidence intervals and letters indicate significant differences for a post-hoc Tukey test with 0.95 confidence Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Page 6 of 12 Page 6 of 12 [17, 19, 20], we characterized harmonic convergence using tethered male and female An. albimanus. We observed harmonic convergence in 41 of 52 couples (78.8%). By analyzing the ratio of the female WBF with respect to the males’, we observed two distinct groups where convergence was observed: in 21 cou- ples (40.4%), the female’s third harmonic converged with the male’s second (Fig. 2, left panel), while in 20 cases (38.5%), the female’s fourth harmonic converged with the male’s third (Fig. 2, right panel). The average time of convergence was 2.00 ± 0.81 s and the response latency ranged from 4 to 13 s. The remaining couples did not match harmonics. Male–female precopulatory acoustic behaviors during free flightl To characterize mosquito flight tones prior to copulation, we examined male–female acoustic behaviors during mating attempts. Male mating attempts were determined by visual and audio analysis: a male “chasing” a female [25, 40] was classified as the onset of a mating attempt. From 33 different females, 134 mating attempts were analyzed. Mating attempts resulted in either a copulation (i.e. the female was inseminated) or a mating rejection; 12 of 33 females mated. The duration of a mating interaction was brief (1.38 ± 0.32 s; n = 33). Ten of the mated females rejected a male at least once, while two mated after the Using the criteria for tethered pairs in Aldersley et al. [37], we did not detect harmonic convergence in free- flying pairs. The brevity of the mating interaction, the WBF range of individuals in free flight, and that sev- eral couples ceased flying upon contact suggest that our methods were not sufficient to detect this phenomenon. However, we analyzed the latency of the response of the female, the female/male WBF ratio prior the interac- tion and the WBF increase during the mating attempt to determine if these characteristics influenced the out- come of a mating attempt. The latency of the females’ WBF increase in response to that of the male did not significantly differ between interactions that resulted in a copulation or a rejection (copulation: 0.375 ± 0.074 s; rejection: 0.542 ± 0.047 s; Mann Whitney U-test: U(12, 21) = 78, Z = − 1.79, P = 0.07). The female/male frequency ratio average (♀WBF/♂WBF) 1–1.5 s prior to a copula- tion, before any observable male–female interaction occurred, was similar for mating attempts that resulted in copulation (♀WBF/♂WBF: 0.595 ± 0.024) or rejection (♀WBF/♂WBF: 0.596 ± 0.024; Mann–Whitney U-test: U(12, 21) = 125, Z = − 0.03, P = 0.97) and did not influence mating outcome. Finally, to analyze the WBF increase during the mating attempt, we measured the change in WBF (ΔF) and the time interval between the initial WBF Fig. 2 Harmonic convergence between tethered couples. Spectrograms of males and females are shown, displaying the WBF and harmonics. Pink labels indicate female harmonics, blue labels indicate male harmonics. Black rectangles highlight harmonic convergence between female f3 and male f2 (left) and female f4 and male f3 (right) Fig. 2 Harmonic convergence between tethered couples. Spectrograms of males and females are shown, displaying the WBF and harmonics. Male‑male acoustic behaviors during free flightl We next analyzed acoustic and flying behaviors among groups of males. We introduced eight males into a sound- proof chamber and made an audio record of their flight tones and a visual record of their flight characteristics. As only a portion of the introduced males flew at any given time, we have differing numbers of recordings of two, three and four males flying simultaneously. For our analy- sis, we selected 5 s intervals in which flying behavior was clear to analyze male flight trajectories using an ellipse [43], determining the distance of each individual in rela- tion to the rest of the group and following the X and Y coordinates of each individual over this time frame (see “Methods”). To assess flight acoustics of male groups, we analyzed the average frequency difference of each group; the difference between the highest and lowest WBFs observed in the spectrogram represented the frequency bandwidth of the interaction among males during the 5 s segments analyzed. increase and the maximum frequency reached (Δt), allowing us to calculate the WBF rate of increase (ΔF/Δt) of both sexes during this interaction (Table 1, Fig. 3a). In males, we found that the rate of WBF increase was simi- lar for mating attempts that resulted in a copulation or rejection (Mann–Whitney U-test: U(12, 21) = 109, Z = 0.63, P = 0.52) (Table 1, Fig. 3b). While females also increased their WBFs regardless of mating outcome, the rate of the frequency increase was significantly greater prior to a mating rejection compared to a copulation (Table 1, Fig. 3b) (Mann–Whitney U-test: U(12, 21) = 24, Z = − 3.81, P < 0.01), owing to differences in the extent of the fre- quency increase (Mann–Whitney U-test: U(12, 21) = 58, Z = − 2.54, P = 0.01) and the time interval of the increase (Mann–Whitney U-test: U(12, 21) = 40.5, Z = 3.19, P < 0.01) (Table 1).f As we detected differences in the female rate of WBF increase between mate rejection or copulation subse- quent to a mating attempt, we further analyzed this behavior. In females that rejected a male more than once (n = 18), we performed a paired comparison between two different mating attempts of the same female and Two distinct flying behaviors were apparent among groups of males flying simultaneously: random flight and a swarm-like, patterned flight (Fig. Male–female precopulatory acoustic behaviors during free flightl The spectrograms show the males fundamental frequency (WBF) and the females’ second harmonic (analysis was performed using the WBFs of each sex). The change in WBF (ΔF) and the time interval between the initial WBF increase and the maximum frequency reached (Δt) is shown for each sex. b Comparison of the rate of frequency increase (ΔF/Δt) for male– female interactions that ended in a rejection (n = 21) or copulation (n = 12). Signifies a significant difference for a Mann–Whitney U-test (P < 0.01) Fig. 3 Acoustic analysis of free-flying mosquito during courtship. a Representative spectrograms of a mating attempt that resulted in a mating rejection (left) or a copulation (right). The spectrograms show the males fundamental frequency (WBF) and the females’ second harmonic (analysis was performed using the WBFs of each sex). The change in WBF (ΔF) and the time interval between the initial WBF increase and the maximum frequency reached (Δt) is shown for each sex. b Comparison of the rate of frequency increase (ΔF/Δt) for male– female interactions that ended in a rejection (n = 21) or copulation (n = 12). Signifies a significant difference for a Mann–Whitney U-test (P < 0.01) Male–female precopulatory acoustic behaviors during free flightl However, in females that rejected and then copulated with a male (n = 10), we per- formed a paired comparison between the copulation and a rejection of the same female and found significant dif- ferences in the rate of WBF increase between outcomes (Wilcoxon matched pairs test: Z = 2.84, P < 0.01) (Addi- tional file 1: Figure S2b): the rate of increase was lower for mating attempts that resulted in copulation compared with a rejection. Thus, in our assays, the increasing rate of the female WBF predicted copulation or rejection. However, there are likely additional behavioral and physi- ological factors that influence mating. found no differences in the rate of WBF increase (Wil- coxon matched pairs test: Z = 0.97, P = 0.32) (Additional file 1: Figure S2a). Thus, females consistently increased their WBFs during rejections. However, in females that rejected and then copulated with a male (n = 10), we per- formed a paired comparison between the copulation and a rejection of the same female and found significant dif- ferences in the rate of WBF increase between outcomes (Wilcoxon matched pairs test: Z = 2.84, P < 0.01) (Addi- tional file 1: Figure S2b): the rate of increase was lower for mating attempts that resulted in copulation compared with a rejection. Thus, in our assays, the increasing rate of the female WBF predicted copulation or rejection. However, there are likely additional behavioral and physi- ological factors that influence mating. Fig. 3 Acoustic analysis of free-flying mosquito during courtship. a Representative spectrograms of a mating attempt that resulted in a mating rejection (left) or a copulation (right). The spectrograms show the males fundamental frequency (WBF) and the females’ second harmonic (analysis was performed using the WBFs of each sex). The change in WBF (ΔF) and the time interval between the initial WBF increase and the maximum frequency reached (Δt) is shown for each sex. b Comparison of the rate of frequency increase (ΔF/Δt) for male– female interactions that ended in a rejection (n = 21) or copulation (n = 12). Signifies a significant difference for a Mann–Whitney U-test (P < 0.01) Fig. 3 Acoustic analysis of free-flying mosquito during courtship. a Representative spectrograms of a mating attempt that resulted in a mating rejection (left) or a copulation (right). Male–female precopulatory acoustic behaviors during free flightl Pink labels indicate female harmonics, blue labels indicate male harmonics. Black rectangles highlight harmonic convergence between female f3 and male f2 (left) and female f4 and male f3 (right) Fig. 2 Harmonic convergence between tethered couples. Spectrograms of males and females are shown, displaying the WBF and harmonics. Pink labels indicate female harmonics, blue labels indicate male harmonics. Black rectangles highlight harmonic convergence between female f3 and male f2 (left) and female f4 and male f3 (right) Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Pantoja‑Sánchez et al. Parasites Vectors Page 7 of 12 Fig. 3 Acoustic analysis of free-flying mosquito during courtship. a Representative spectrograms of a mating attempt that resulted in a mating rejection (left) or a copulation (right). The spectrograms show the males fundamental frequency (WBF) and the females’ second harmonic (analysis was performed using the WBFs of each sex). The change in WBF (ΔF) and the time interval between the initial WBF increase and the maximum frequency reached (Δt) is shown for each sex. b Comparison of the rate of frequency increase (ΔF/Δt) for male– female interactions that ended in a rejection (n = 21) or copulation (n = 12). *Signifies a significant difference for a Mann–Whitney U-test (P < 0.01) Table 1 Values of frequency interval (ΔF), time interval (Δt) and the rate of frequency increase (ΔF/Δt) during mating attempts Table 1 Values of frequency interval (ΔF), time interval (Δt) and the rate of frequency increase (ΔF/Δt) during mating attempts Table 1 Values of frequency interval (ΔF), time interval (Δt) and the rate of frequency increase (ΔF/Δt) during mating attempts Copulation (n = 12) Mating rejection (n = 21) ΔF (Hz) ♂: 91.2 ± 6.3 ♂: 90.9 ± 4.9 ♀: 81.1 ± 10.3 ♀: 111.6 ± 5.3 Δt (s) ♂: 0.325 ± 0.034 ♂: 0.364 ± 0.025 ♀: 0.393 ± 0.048 ♀: 0.220 ± 0.029 ΔF/Δt (Hz s−1) ♂: 314.68 ± 36.60 ♂: 282.69 ± 27.35 ♀: 218.32 ± 29.59 ♀: 655.52 ± 67.71 found no differences in the rate of WBF increase (Wil- coxon matched pairs test: Z = 0.97, P = 0.32) (Additional file 1: Figure S2a). Thus, females consistently increased their WBFs during rejections. Discussionl Mosquito flight tones differ by species and are used to develop novel trapping tools [46] and recognition algo- rithms [47], highlighting the importance of flight tone characterization of disease vectors. While there is ample information about Ae. aegypti, Cx. quinquefasciatus and An. gambiae acoustic behavior prior to and during mat- ing, there is no information about acoustic behavior of one the most important malarial vectors in the Ameri- cas, An albimanus. Furthermore, most acoustic charac- terization of mosquito vectors has been performed using immobilized specimens. Here, we characterized acoustic behavior of An. albimanus. We observed significant dif- ferences in the flight acoustics between free-flying and tethered An. albimanus, describe acoustic interactions between free-flying and tethered male–female couples, and identify a relationship between male-male acous- tic interactions and the coordination of movement that occurs when males transition from random flight to pat- terned flight. l Tethered An. albimanus exhibit lower WBFs than other reported mosquito species (Ae. aegypti [4, 48], An coluzzi [40], An. gambiae [21, 22]). While WBFs vary among mosquito species [49], they can be affected by factors that include ambient temperature [4, 50], humidity and age [51]. We observed major acoustic dif- ferences between tethered and free-flying mosquitoes: (i) the mean WBF of free-flying An. albimanus is higher for both sexes; (ii) the mean WBF ratio between females and males (♀WBF/♂WBF) changes (tethered = 0.71, free flight = 0.61); and (iii) free-flying mosquitoes modulate their WBF to a greater extent than tethered individuals. Tethered insects have been used to study locomotion [52, 53], migration [52, 53] and wing move- ment [54]; these studies have demonstrated that teth- ering leads to unnatural flight behaviors and distorted wing strokes. Tethered individuals are restricted to a horizontal flight path, do not produce the equivalent lift required to achieve normal flight, and do not support their own body mass, factors that may give an inaccu- rate view of natural flight [55]. As flight tones are linked to motor function [56], our results show that restricting motion alters two flight tone characteristics: the WBF and the ability to modulate it. Although a decrease in the WBF linked to tethering has been reported for Ae. aegypti [4], Cx. quinquefasciatus [25], midges [44] and locusts [45], Villareal et al. [50] found no difference in WBF of tethered and free-flying Ae. aegypti females when assays were performed at the same temperature. Male‑male acoustic behaviors during free flightl 4a, b; Additional file 3: Video S2), referred to hereafter as “patterned flight”. Page 8 of 12 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 During random flight, male WBFs diverged or were dis- tinct (Fig. 4c, d, bottom panels), but converged once patterned flight was initiated, reducing the frequency dif- ferences of the male flight tones (two males: Fig. 4c; four males: Fig. 4d; Additional file 3: Video S2). By examining flight behavior in 5 s intervals, we observed a significant change in the frequency difference between males in ran- dom flight and those in patterned flight. This change was consistent among groups of two (Mann–Whitney U-test: U(8, 8) = 0, Z = − 3.36, P < 0.01), three (Mann–Whitney U-test: U(5, 5) = 0, Z = − 2.61, P < 0.01) and four (Mann– Whitney U-test: U(5, 5) = 0, Z = − 2.61, P < 0.01). The dif- ference in WBF of groups in patterned flight was always smaller than groups in random flight (Fig. 4e). The mean frequency difference of groups of two, three and four males in patterned flight was 38.20 ± 3.33, 52.60 ± 4.01 and 68.21 ± 7.11 Hz, respectively. On the other hand, the mean frequency difference of groups of two, three and four males in random flight was 110.88 ± 19.91, 177.05 ± 31.16 and 167.65 ± 19.88 Hz, respectively.l Discussionl Although our assays were performed at the same tem- perature, we found that tethering impacted An. albi- manus WBFs. During patterned flight, we observed that An. albi- manus fly in an ellipsoid pattern around a central position, similar to what has been reported in Cx. quinquefasciatus [43]. Therefore, we quantified visual differences between random and patterned flight (where we observed acoustic interactions) by examining the trajectory of individual mosquitoes and analyzing the ellipsoid shape formed while flying [43]. We found sig- nificant differences in the characteristics of male tra- jectories between the two types of flight observed: the ellipse area representing random flight is significantly larger than the area representing patterned flight [ran- dom flight: (1.55 ± 0.29) × 105 px2; patterned flight: (0.49 ± 0.06) × 105 px2; Mann–Whitney U-test: U(18, 18) = 58, Z = 3.24, P < 0.01]. We further measured the dis- tance (in pixels) of each mosquito in relation to the rest of the group by calculating the average distance among the centroids of each ellipse in comparison with the oth- ers. We found that mosquitoes flying randomly are sig- nificantly less aggregated than males in patterned flight (random flight: 244.66 ± 21.03 px; patterned flight: 95.79 ± 11.15 px; Mann–Whitney U-test: U(18, 18) = 69, Z = 2.89, P < 0.01). Finally, by separately analyzing the X and Y trajectories of males in flight, we observed char- acteristic loops (i.e. periodic movement) similar to those defined in previous studies [43]. Mosquitoes in patterned flight display loops with significantly shorter periods (random flight: 2.63 ± 0.39 s; patterned flight: 0.90 ± 0.08 s; Mann–Whitney U-test: U(18, 18) = 33.5, Z = 4.03, P < 0.01) (Fig. 4f). Taken together, these data show that patterned flight is associated with distinctive acoustic behaviors. Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Page 9 of 12 Fig. 4 Acoustic and visual analysis of free-flying males. Two (a) and four (b) males in random flight (left panels) and patterned flight (right panels). Grey boxes represent the spatial distribution of the experimental arena. Green squares within the boxes indicate microphone location. Colored lines show the 2D trajectory for each male during 5 s recordings. The X (top) and Y (middle) coordinates of the flight trajectories and the high order spectrogram (bottom) of groups of two (c) and four (d) mosquitoes during random and patterned flight. Discussionl Colored rectangles indicate the 5 s interval analyzed to associate flying pattern with acoustic behavior (green: random flight; blue: patterned flight). e Box and whiskers plots of the frequency difference of males flying randomly (green) and in patterns (blue). The box ranges from the first quartile to the third quartile of the distribution. A horizontal line across the box indicates the median. The whiskers extend from the quartiles to the extreme data points. Males flying randomly exhibit a higher frequency difference than males in patterned flight. f Differences in the characteristics of the flight trajectories of mosquitoes in random (green) or patterned flight (green) Fig. 4 Acoustic and visual analysis of free-flying males. Two (a) and four (b) males in random flight ( Grey boxes represent the spatial distribution of the experimental arena. Green squares within the bo lines show the 2D trajectory for each male during 5 s recordings. The X (top) and Y (middle) coordina spectrogram (bottom) of groups of two (c) and four (d) mosquitoes during random and patterned f analyzed to associate flying pattern with acoustic behavior (green: random flight; blue: patterned flig difference of males flying randomly (green) and in patterns (blue). The box ranges from the first quar A horizontal line across the box indicates the median. The whiskers extend from the quartiles to the exhibit a higher frequency difference than males in patterned flight. f Differences in the characterist random (green) or patterned flight (green) Fig. 4 Acoustic and visual analysis of free-flying males. Two (a) and four (b) males in random flight (left panels) and patterned flight (right panels). Grey boxes represent the spatial distribution of the experimental arena. Green squares within the boxes indicate microphone location. Colored lines show the 2D trajectory for each male during 5 s recordings. The X (top) and Y (middle) coordinates of the flight trajectories and the high order spectrogram (bottom) of groups of two (c) and four (d) mosquitoes during random and patterned flight. Colored rectangles indicate the 5 s interval analyzed to associate flying pattern with acoustic behavior (green: random flight; blue: patterned flight). e Box and whiskers plots of the frequency difference of males flying randomly (green) and in patterns (blue). The box ranges from the first quartile to the third quartile of the distribution. A horizontal line across the box indicates the median. Discussionl The whiskers extend from the quartiles to the extreme data points. Males flying randomly exhibit a higher frequency difference than males in patterned flight. f Differences in the characteristics of the flight trajectories of mosquitoes in random (green) or patterned flight (green) Fig. 4 Acoustic and visual analysis of free-flying males. Two (a) and four (b) males in random flight (left panels) and patterned flight (right panels). Grey boxes represent the spatial distribution of the experimental arena. Green squares within the boxes indicate microphone location. Colored lines show the 2D trajectory for each male during 5 s recordings. The X (top) and Y (middle) coordinates of the flight trajectories and the high order spectrogram (bottom) of groups of two (c) and four (d) mosquitoes during random and patterned flight. Colored rectangles indicate the 5 s interval analyzed to associate flying pattern with acoustic behavior (green: random flight; blue: patterned flight). e Box and whiskers plots of the frequency difference of males flying randomly (green) and in patterns (blue). The box ranges from the first quartile to the third quartile of the distribution. A horizontal line across the box indicates the median. The whiskers extend from the quartiles to the extreme data points. Males flying randomly exhibit a higher frequency difference than males in patterned flight. f Differences in the characteristics of the flight trajectories of mosquitoes in random (green) or patterned flight (green) Page 10 of 12 Page 10 of 12 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Harmonic convergence, or the synchronization of fre- quencies between two specific harmonics during male– female interactions, has been described in Ae. aegypti [17, 18], Cx. quinquefasciatus [19] and An. gambiae [20, 21] using tethered individuals. This behavior appears to be related to the male auditory strategy used to track females [57] and, more recently, has been suggested to be a manifestation of male–female coordination prior to formation of the copula position [26]. We observed harmonic convergence in tethered An. albimanus. The time of convergence and the latency of the response were similar to An. gambiae [21] and the convergence events we detected have been observed in other species [18, 21, 37]. When examining male–female acoustic interactions during a mating attempt in free flight, we were unable to observe harmonic convergence using the criteria of Aldersley et al. [37]. Conclusions Linking acoustic and flying behaviors under natural, unrestricted conditions will provide important informa- tion regarding behaviors during courtship and ultimately, female mate selection. Many of the male–female acoustic interactions we observed during mating attempts in free flight have been reported in other mosquito species, sug- gesting that such interactions are common during court- ship in mosquito species and supporting the idea that acoustic interactions are crucial for successful copula- tion. This study also reveals a connection between male acoustics and flight characteristics, giving insight into the importance of male-male acoustic interaction dur- ing patterned flight and potentially giving clues about swarm formation and cohesion. Furthermore, An. albi- manus males modulate their flight tones in the presence of other males. While this behavior likely entails addi- tional sensory cues, targeting acoustic male interactions may disrupt essential reproductive behaviors of this, and potentially other non-swarming species. Control programmes that rely on the mass release of laboratory- reared mosquitoes [60–63] need to consider acoustic behavior, since the results presented here, as well as those of previous reports, demonstrate that different mosquito species display similar precopulatory acoustic behaviors, highlighting the importance of acoustics in mating suc- cess [27, 28]. Characterization of An. albimanus acoustic flight behaviors, an important Latin American malaria vector, is a step toward understanding male-male and male–female interactions prior to copulation and will aid in the improvement of vector surveillance and control programmes in areas affected by this species. l However, we observed a common precopulatory behavior in free flight: a rapid increase in male WBF immediately followed by a rapid increase in female WBF. A similar behavior is seen in Cx. quinquefasciatus [25], An. coluzzii and An. gambiae [40], where males increase their WBF in response to female flight tone. The rate of frequency increase in males was similar regardless of mating outcome, while a more rapid increase in female frequency resulted in mate rejection. It is possible that the frequency increase results from controlled flight to reach or escape a potential mate. Results from the Culex [25] and Anopheles [40] auditory system suggest that to locate females, males use the difference between their own frequency and that of the female [57] rather than the female WBF itself. The rapid change in female WBF dur- ing mate rejection immediately modifies the male–female WBF difference. Discussionl Harmonic convergence presumably occurs rapidly during a mating attempt, resembling the early stages of Ae. aegypti precopulatory behavior (i.e. from the male approach to the end of RFM) [26]. It is possible that convergence similar to what we observed under tethered conditions occurs after male–female con- tact, as has been demonstrated in Ae. aegypti [26]. Thus, we might not have detected convergence in our assays as An. albimanus couples often did not exhibit long paired flight after making contact. interactions, although studies of male-male and male– female interactions in free flight are few. In our assays, An. albimanus males performed two identifiable types of flying behavior regardless of the number of males assessed: random and patterned flight. During random flight, males flew in large trajectories within the entire experimental arena. Once patterned flight was initiated, the large trajectories immediately gave way to a form of flight composed of small loops in a specific region of the arena. Interestingly, males in patterned flight acoustically interacted within a narrower band of frequency, linking flight pattern and acoustic behavior. This result is likely related to the clustering of male flight tones of closely located, tethered Ae. aegypti males [36]. During swarm- ing, males must coordinate their flight patterns and recognize females that enter the swarm. It has been pro- posed that flight tones are used to coordinate movement during group flight [36], dividing into frequency clusters to reduce acoustic interference [59]. Conclusions Remaining within the optimal auditory sensitivity range may be a critical characteristic to reach and copulate with a female. Male swarming behavior appears to be an obligatory feature of copulation for some Anopheles species [35, 58]. However, swarming behaviors have not been described for Latin American species. Furthermore, male-male interactions during swarming, and female mate selec- tion within a swarm, are not well understood. Acous- tic signaling might be an important factor during these Page 11 of 12 Page 11 of 12 Page 11 of 12 Pantoja‑Sánchez et al. Parasites Vectors (2019) 12:386 Pantoja‑Sánchez et al. Parasites Vectors Funding Thi d 13. Mitchell SN, Catteruccia F. Anopheline reproductive biology: Impacts on vectorial capacity and potential avenues for malaria control. Cold Spring Harb Perspect Med. 2017;7:a025593. This study was supported by COLCIENCIAS Grants CD 325671250804 (CT 409- 2016) (to CAP), 647 (to support HPS), COLCIENCIAS, Universidad de Antioquia and the Max Planck Society cooperation Grant 566-1 (2014) (to FWA) and Universidad de Antioquia Grant CPT1410 (to support VV). p 14. Diabate A, Tripet F. Targeting male mosquito mating behaviour for malaria control. Parasit Vectors. 2015;8:347. 15. Alphey L, Benedict M, Bellini R, Clark GG, Dame DA, Service MW, et al. Sterile-insect methods for control of mosquito-borne diseases: an analy‑ sis. Vector Borne Zoonotic Dis. 2010;10:295–311. Acknowledgements 8. Mitchell SN, Kakani EG, South A, Howell PI, Waterhouse RM, Catteruccia F. Mosquito biology. Evolution of sexual traits influencing vectorial capacity in anopheline mosquitoes. Science. 2015;347:985–8. We would like to thank the Programa de Estudio y Control de Enfermedades Tropicales (PECET, Universidad de Antioquia) for generously providing the mosquitoes used in this study and technical support; SISTEMIC for computing support; Laura Harrington, Ethan Degner, Garrett League and Miguel Toro for helpful comments on the manuscript; and J. Francisco Vargas B. and Freddy Ruiz-Lopez for technical advice. in anopheline mosquitoes. Science. 2015;347:985–8. 9. Kramer LD, Ciota AT. Dissecting vectorial capacity for mosquito-borne viruses. Curr Opin Virol. 2015;15:112–8. 10. Villarreal SM, Pitcher S, Helinski MEH, Johnson L, Wolfner MF, Harrington LC. Male contributions during mating increase female survival in the disease vector mosquito Aedes aegypti. J Insect Physiol. 2018;108:1–9. Author details 1 Departamento de Ingeniería Electrónica, SISTEMIC, Universidad de Antio‑ quia, Medellín, Antioquia 050010, Colombia. 2 Programa de Estudio y Control de Enfermedades Tropicales, PECET, Universidad de Antioquia, Medellín, Antioquia 050010, Colombia. 3 Instituto Colombiano de Medicina Tropical, Universidad CES, Sabaneta, Antioquia 055450, Colombia. 4 Max Planck Tandem Group in Mosquito Reproductive Biology, Universidad de Antioquia, Medellín, Antioquia 050010, Colombia. Additional file 1: Figure S1. The relationship between size and wing‑ beat frequency of a tethered and b free-flying mosquitoes recorded at 26 ± 2 °C. Linear regression between wing length and frequency of tethered males (blue, tethered: R2 < 0.01, Pearson’s r = 0.06, P = 0.58; free flight: R2 = 0.12, Pearson’s r = 0.35, P = 0.02) and females (red, tethered: R2 = 0.03, Pearson’s r = − 0.20, P = 0.05; free flight: R2 < 0.01, Pearson’s r = 0.03, P = 0.81). Although our rearing method did not intend to gener‑ ate individuals of different sizes, the determination coefficient (R2) and Pearson’s correlation coefficient (r) shows that in males and females there is a weak or a non-significant relationship between size and wingbeat frequency. Figure S2. Paired comparison between a two rejections and b a rejection and a successful copulation of the same female. While there is no significant difference between rejections (Wilcoxon matched pairs test: Z = 0.97, P = 0.32), there is a significant decrease of the WBF rate of increase (Wilcoxon matched pairs test: Z = 2.84, P < 0.01) between rejec‑ tions and successful copulations. Received: 17 February 2019 Accepted: 25 July 2019 Consent for publication Not applicable. Authors’ contributions 11. Helinski MEH, Harrington LC. Male mating history and body size influence female fecundity and longevity of the dengue vector Aedes aegypti. J Med Entomol. 2011;48:202–11. HPS, SG and CAP designed the experiments. HPS, SG, VV and CAP performed the experiments. HPS and SG analyzed the data. FWA, CAP and HPS wrote the manuscript. HPS and SG prepared the figures. All authors read and approved the final manuscript. 12. Childs LM, Cai FY, Kakani EG, Mitchell SN, Paton D, Gabrieli P, et al. Disrupt‑ ing mosquito reproduction and parasite development for malaria control. PLoS Pathog. 2016;12:e1006060. References 1. Hay SI, Guerra CA, Tatem AJ, Noor AM, Snow RW. The global distribution and population at risk of malaria: past, present, and future. Lancet Infect Dis. 2004;4:327–36. Additional file 2: Video S1. Video of male–female mating attempts: rejections and successful copulation. The left panel shows the experimen‑ tal arena and the reconstruction of the flight trajectories of the male (in blue) and the female (in red). Right panels indicate the time variation of X (top) and Y (middle) coordinates of the flight trajectories and the spectro‑ gram (bottom) of the fundamental frequency. 2. Recht J, Siqueira AM, Monteiro WM, Herrera SM, Herrera S, Lacerda MVG. Malaria in Brazil, Colombia, Peru and Venezuela: current challenges in malaria control and elimination. Malar J. 2017;16:273. 3. Padilla JC, Lizarazo FE, Murillo OL, Mendigana FA, Pachon E, Vera MJ. Transmission scenarios of major vector-borne diseases in Colombia, 1990–2016. Biomedica. 2017;37:27–40. 3. Padilla JC, Lizarazo FE, Murillo OL, Mendigana FA, Pachon E, Vera MJ. Transmission scenarios of major vector-borne diseases in Colombia, 1990–2016. Biomedica. 2017;37:27–40. 4. Arthur BJ, Emr KS, Wyttenbach RA, Hoy RR. Mosquito (Aedes aegypti) flight tones: frequency, harmonicity, spherical spreading, and phase relation‑ ships. J Acoust Soc Am. 2014;135:933–41. 4. Arthur BJ, Emr KS, Wyttenbach RA, Hoy RR. 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The use of Wolbachia by the World Mosquito Program to inter‑ rupt transmission of Aedes aegypti transmitted viruses. Adv Exp Med Biol. 2018;1062:355–60. 38. Charlwood JD, Jones MDR. Mating behaviour in the mosquito, Anopheles gambiae s.l. save. I. Close range and contact behaviour. Physiol Entomol. 1979;4:111–20. 63. Servick K. Winged warriors. Science. 2016;354:164–7. 63. Servick K. Winged warriors. Science. 2016;354:164–7. 39. Gibson G, Russell I. Flying in tune: sexual recognition in mosquitoes. Curr Biol. 2006;16:1311–6. Publisher’s Note S i N i 40. Simoes PMV, Gibson G, Russell IJ. Pre-copula acoustic behaviour of males in the malarial mosquitoes Anopheles coluzzii and Anopheles gambiae s.s. does not contribute to reproductive isolation. J Exp Biol. 2017;220:379–85. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 41. Heuvel MJ. The effect of rearing temperature on the wing length, thorax length and ovariole number of the adult mosquito, Aedes aegypti (L.). Trans R Entomol Soc Lond. 2009;115:197–216. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? Choose BMC and benefit from: 42. Swami A, Mendel JM, Nikias C. Higher-order spectral analysis toolbox: MATLAB user’s guide. Natick: The Math Works Inc.; 2001. 43. Gibson G. Swarming behaviour of the mosquito Culex pipiens quinquefas- ciatus: a quantitative analysis. Physiol Entomol. 1985;10:283–96. 44. de Silva P, Nutter B, Bernal XE. Use of acoustic signals in mating in an eavesdropping frog-biting midge. Anim Behav. 2015;103:45–51. 45. Kutsch W, Stevenson P. Time-correlated flights of juvenile and mature locusts: a comparison between free and tethered animals. J Insect Physiol. 1981;27:455–9. 45. Kutsch W, Stevenson P. Time-correlated flights of juvenile and mature locusts: a comparison between free and tethered animals. J Insect Physiol. 1981;27:455–9.
https://openalex.org/W1998771962	https://jcheminf.biomedcentral.com/track/pdf/10.1186/1758-2946-6-S1-P61	English	null	QM quality atomic charges for proteins	Journal of cheminformatics	2,014	cc-by	1,056	* Correspondence: standag@chemi.muni.cz National Centre for Biomolecular Research, Faculty of Science and CEITEC - Central European Institute of Technology, Masaryk University, Brno, 625 00, CZ, Czech Republic POSTER PRESENTATION Open Access QM quality atomic charges for proteins Stanislav Geidl*, Crina-Maria Ionescu, Radka Svobodová Vařeková, Jaroslav Koča From 9th German Conference on Chemoinformatics Fulda, Germany. 10-12 November 2013 From 9th German Conference on Chemoinformatics Fulda, Germany. 10-12 November 2013 Published: 11 March 2014 Geidl et al. Journal of Cheminformatics 2014, 6(Suppl 1):P61 http://www.jcheminf.com/content/6/S1/P61 Geidl et al. Journal of Cheminformatics 2014, 6(Suppl 1):P61 http://www.jcheminf.com/content/6/S1/P61 References Mortier WJ, Vangenechten K, Gasteiger J: Electronegativity Equalization - Application and Parametrization. J Am Chem Soc 1985, 107:829-835. 3. Bultinck P, Van Alsenoy C, Ayers PW, Carbó-Dorca R: Critical analysis and extension of the Hirshfeld atoms in molecules. J Chem Phys 2007, 126:144111. gy 2. Mortier WJ, Vangenechten K, Gasteiger J: Electronegativity Equalization - Application and Parametrization. J Am Chem Soc 1985, 107:829-835. pp 3. Bultinck P, Van Alsenoy C, Ayers PW, Carbó-Dorca R: Critical analysis and extension of the Hirshfeld atoms in molecules. J Chem Phys 2007, 126:144111. 4. Ionescu CM, Geidl S, Svobodová Vařeková R, Koča J: Rapid calculation of accurate atomic charges for proteins via the electronegativity equalization method. J Chem Inf Model 2013. doi:10.1186/1758-2946-6-S1-P61 Cite this article as: Geidl et al.: QM quality atomic charges for proteins. Journal of Cheminformatics 2014 6(Suppl 1):P61. doi:10.1186/1758-2946-6-S1-P61 Cite this article as: Geidl et al.: QM quality atomic charges for proteins. Journal of Cheminformatics 2014 6(Suppl 1):P61. In our work, we present the workflow of the EEM cali- bration process. Afterwards, we calibrate and validate EEM models for 12 types of QM charges, including the newest approaches like iterative Hirshfeld [3]. The accu- racy of the obtained EEM models is evaluated on insulin and ubiquitin. We also show two case studies demonstrat- ing the applicability of atomic charges computed via EEM: a small docking study, and the calculation of electrostatic potential based on the EEM charges [4]. Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. Publish with ChemistryCentral and every scientist can read your work free of charge W. Jeffery Hurst, The Hershey Company. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright Submit your manuscript here: http://www.chemistrycentral.com/manuscript/ Ltd. This is an Open Access article distributed under the terms of the Creative Commons org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in properly cited. The Creative Commons Public Domain Dedication waiver (http:// /) applies to the data made a ailable in this article nless other ise stated Publish with ChemistryCentral and every scientist can read your work free of charge Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. W. © 2014 Geidl et al; licensee Chemistry Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. References The concept of atomic point charges is well established in theoretical chemistry. Atomic point charges have played an important role in understanding and modeling chemical behavior by allowing to extract and quantify information stored in the molecular electron distribution of chemical compounds. Thus, atomic point charges have been used to estimate reactivity indices, dissocia- tion constants, partition coefficients, the electrostatic contribution in molecular dynamics or docking studies. It is therefore desirable to have knowledge of the values of atomic charges in proteins (see, e.g., [1]). Unfortu- nately, accurate and universally applicable approaches for atomic charge calculation based on quantum mechanics (QM) are very time consuming and thus can- not be employed for large biomolecules like proteins. An alternative is to use empirical charge calculation methods, such as the electronegativity equalization method (EEM) [2], which is very fast and has accuracy comparable to QM. The challenge is to calibrate (i.e., parametrize) this method for proteins. This parameteri- zation can be done using atomic charges calculated by different types of QM approaches. EEM can be as accu- rate as the QM approach for which EEM was calibrated. References 1. Ionescu CM, Svobodová Vařeková R, Prehn JH, Huber HJ, Koča J: Charge profile analysis reveals that activation of pro-apoptotic regulators Bax and Bak relies on charge transfer mediated allosteric regulation. PLoS computational biology 2012, 8(6):e1002565. 2. Mortier WJ, Vangenechten K, Gasteiger J: Electronegativity Equalization - Application and Parametrization. J Am Chem Soc 1985, 107:829-835. 3. Bultinck P, Van Alsenoy C, Ayers PW, Carbó-Dorca R: Critical analysis and extension of the Hirshfeld atoms in molecules. J Chem Phys 2007, 126:144111. 4. Ionescu CM, Geidl S, Svobodová Vařeková R, Koča J: Rapid calculation of accurate atomic charges for proteins via the electronegativity equalization method. J Chem Inf Model 2013. doi:10.1186/1758-2946-6-S1-P61 Cite this article as: Geidl et al.: QM quality atomic charges for proteins. Journal of Cheminformatics 2014 6(Suppl 1):P61. References 1. Ionescu CM, Svobodová Vařeková R, Prehn JH, Huber HJ, Koča J: Charge profile analysis reveals that activation of pro-apoptotic regulators Bax and Bak relies on charge transfer mediated allosteric regulation. PLoS computational biology 2012, 8(6):e1002565. p gy 2. Mortier WJ, Vangenechten K, Gasteiger J: Electronegativity Equalization - Application and Parametrization. J Am Chem Soc 1985, 107:829-835. 3. Bultinck P, Van Alsenoy C, Ayers PW, Carbó-Dorca R: Critical analysis and extension of the Hirshfeld atoms in molecules. J Chem Phys 2007, 126:144111. p gy 2. References Jeffery Hurst, The Hershey Company. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright © 2014 Geidl et al; licensee Chemistry Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
https://openalex.org/W3104121561	https://biblio.ugent.be/publication/8711224/file/8711225	English	null	Light-fueled dynamic covalent crosslinking of single polymer chains in non-equilibrium states	Chemical science	2,021	cc-by	7,993	Light-fueled dynamic covalent crosslinking of single polymer chains in non-equilibrium states† Cite this: Chem. Sci., 2021, 12, 1302 All publication charges for this article have been paid for by the Royal Society of Chemistry Cite this: Chem. Sci., 2021, 12, 1302 Daniel Kodura,ab Hannes A. Houck, abc Fabian R. Bloesser,ab Anja S. Goldmann, ab Filip E. Du Prez, c Hendrik Frisch ab and Christopher Barner-Kowollik ab All publication charges for this article have been paid for by the Royal Society of Chemistry While polymer synthesis proceeds predominantly towards the thermodynamic minimum, living systems operate on the reverse principle – consuming fuel to maintain a non-equilibrium state. Herein, we report the controlled formation of 3D macromolecular architectures based on light-fueled covalent non- equilibrium chemistry. In the presence of green light (525 nm) and a bivalent triazolinedione (TAD) crosslinker, naphthalene-containing polymers can be folded into single chain nanoparticles (SCNPs). At ambient temperature, the cycloaddition product of TAD with naphthalene reverts and the SCNP unfolds into its linear parent polymer. The reported SCNP is the ﬁrst example of a reversible light triggered folding of single polymer chains and can readily be repeated for several cycles. The folded state of the SCNP can either be preserved through a constant supply of light fuel, kinetic trapping or through a chemical modiﬁcation that makes the folded state thermodynamically favored. Whereas small molecule bivalent TAD/naphthalene cycloaddition products largely degraded after 3 days in solution, even in the presence of fuel, the SCNP entities were found to remain intact, thereby indicating the light- fueled stabilization of the SCNP to be an inherent feature of the conﬁned macromolecular environment. Received 22nd October 2020 Accepted 17th November 2020 DOI: 10.1039/d0sc05818a rsc.li/chemical-science The teams of Walther,7,8 Hermans9 or Bhatia10 for instance, developed such life-like systems based on reactions of competing enzymes. However, the design of articial systems is not constrained to the exclusive use of natural building blocks, yet can access the entire tool box of synthetic chemistry.11 Thus far, methylations,12,13 thiol–disulde exchange,14 oxidation of aldehydes into a-hydroxy sulfonates,15 thiol–enone chemistry,16 alkene metathesis,17 metal coordination,18 imine bond chem- istry,19 thiol–ester exchange20 and esterications21,22 have been explored in synthetic reaction cycles, which consume fuel to generate a chemical product that in its absence dissipates back into its ground state. In such examples, the resulting non- equilibrium molecules have altered propensities for supramo- lecular interactions, aﬀording higher order supramolecular architectures that are exclusively formed in the presence of fuel. aSchool of Chemistry and Physics, Queensland University of Technology (QUT), 2 George Street, Brisbane, QLD 4000, Australia. E-mail: a.goldmann@qut.edu.au; h. frisch@qut.edu.au; christopher.barnerkowollik@qut.edu.au bCentre for Materials Science, Queensland University of Technology (QUT), 2 George Street, Brisbane, QLD 4000, Australia cPolymer Chemistry Research Group, Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4-bis, 9000 Gent, Belgium. E-mail: Filip.DuPrez@UGent.be † Electronic supplementary information (ESI) available. See DOI: 10.1039/d0sc05818a Light-fueled dynamic covalent crosslinking of single polymer chains in non-equilibrium states† For the same purpose, the fabrication of covalently connected macromolecular architectures, however, is far less explored.7 Especially in the absence of catalysts, dynamic covalent non- equilibrium nanostructures remain elusive. Herein, we report synthetic polymer chains that consume a bivalent crosslinker in the presence of light to fold into a covalently bonded macro- molecular architecture, namely a single chain nanoparticle (SCNP, Scheme 1a).23–26 Whereas the natural inspiration of SCNPs, i.e. the folding of peptides into proteins, strives towards the minimum of the energetic landscape to create dened 3D architectures,27 our current synthetic system folds away from its minimum energy. In the absence of light, these non- Chemical Science View Article Online View Journal \| View Issue © 2021 The Author(s). Published by the Royal Society of Chemistry EDGE ARTICLE Light-fueled dynamic covalent crosslinking of single polymer chains in non-equilibrium states† Daniel Kodura,ab Hannes A. Houck, abc Fabian R. Bloesser,ab Anja S. Goldmann, ab Filip E. Du Prez, c Hendrik Frisch ab and Christopher Barner-Kowollik ab Introduction During the last century, the majority of chemical trans- formations was covalently driven, aiming to approach a ther- modynamic minimum.1 Chemistry at equilibrium has eminent advantages, such as facile synthesis of materials, straightfor- ward analysis and long lasting stability, yet limits the ability of a material to reform – leading to challenging repair and expensive recycling.2 In contrast to equilibrium driven chem- istry, all living organisms consume fuel to operate far from equilibrium states – otherwise life could not exist.3,4 The vast potential of dynamic non-equilibrium materials has inspired a wide range of synthetic systems over the last decade comprising life-like properties.5 To design chemical systems that consume fuel to acquire a higher energetic state and dissipate back into the original state in the absence of fuel, the use of natural building blocks provides an attractive avenue.6 © 2021 The Author(s). Published by the Royal Society of Chemistry 1302 \| Chem. Sci., 2021, 12, 1302–1310 Chemical Science View Article Online Scheme 1 (a) Schematic illustration of the reversible folding process of a linear polymer into a single-chain nanoparticle, using green light as a fuel. As long as the light source is switched on, fuel is consumed and the architecture is in a folded non-equilibrium state. If the light source is turned oﬀ, the architecture unfolds again and reaches the equilibrium point. (b) Chemical structures of the statistical RAFT-copolymers P1 and P2 of functional naphthalene monomers and methyl methacrylate, used in the folding/unfolding process with BisTAD crosslinkers. Edge Article Chemical Science View Article Online Edge Article Open Access Article. Published on 17 November 2020. Downloaded on 6/8/2021 3:18:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Scheme 1 (a) Schematic illustration of the reversible folding process of a linear polymer into a single-chain nanoparticle, using green light as a fuel. As long as the light source is switched on, fuel is consumed and the architecture is in a folded non-equilibrium state. If the light source is turned oﬀ, the architecture unfolds again and reaches the equilibrium point. (b) Chemical structures of the statistical RAFT-copolymers P1 and P2 of functional naphthalene monomers and methyl methacrylate, used in the folding/unfolding process with BisTAD crosslinkers. equilibrium SCNPs unfold back into linear polymer chains, enabling for the rst time to gate reversible folding of synthetic polymer chains with light. Introduction Such a reversible folding was thus far only achieved through the addition of chemical triggers,28,29 change of solvent,30,31 pH32 and high thermal33,34 or mechanical energy,35,36 which limit areas of applications since it prevents covalently bonded SCNPs to be reversibly folded under mild conditions. Moreover, light-gated reversible folding remained unprecedented in the past, as photoreversible cycloadditions have shown to forfeit their reversibility in the conned envi- ronment of the polymer chain.37–39 reversibly control the transition of a viscoelastic liquid into a cross-linked light-stabilized dynamic material (LSDM).41 To apply the photo-Diels–Alder reaction for the non-equilibrium folding of single polymer chains, two copolymers (P1 and P2, Scheme 1b) of methyl methacrylate (MMA) and naphthalene- containing comonomer were synthesized via reversible addi- tion–fragmentation chain-transfer polymerization (RAFT) (Table 1). Based on previous small molecule studies, a 2-carboxyl naph- thalene derivative was used to introduce the aromatic side chains, as its electronic structure enables a rapid formation and close to quantitative cycloreversion of the corresponding TAD-adduct.41 To avoid undesired reactivity that might potentially occur between the residual dithiobenzoate RAFT group and the TAD moieties, the polymer end group was removed prior to the photochemical experiments (see ESI 4.2.3 and 4.2.4†).42 The resulting naphthalene-containing polymers were subsequently crosslinked using a bivalent 1,6-hexamethylene bistriazolinedione (BisTAD). It Table 1 Synthesized polymers P1 and P2 with their respective Mn, Đ, percentage of naphthalene units and crosslinking points Polymer Mn a/g mol1 Đa % of naphthalene unitsb Crosslinking pointsc P1 8200 1.1 14 9 P2 11 200 1.2 12 11 a Obtained from THF-SEC data calibrated on PMMA standards. b The monomer ratios were calculated from 1H NMR spectra in THF-d8 (Fig. S19 and S21). c Crosslinking points calculated from 1H NMR spectra (ESI chapter 2). Results and discussion The herein explored underlying visible light-fueled reaction is based on the reversible photoinduced cycloaddition of a tri- azolinedione (TAD) with naphthalene, which was rst reported in 1984 (ref. 40) yet only recently applied in materials science to Table 1 Synthesized polymers P1 and P2 with their respective Mn, Đ, percentage of naphthalene units and crosslinking points Polymer Mn a/g mol1 Đa % of naphthalene unitsb Crosslinking pointsc P1 8200 1.1 14 9 P2 11 200 1.2 12 11 a Obtained from THF-SEC data calibrated on PMMA standards. b The monomer ratios were calculated from 1H NMR spectra in THF-d8 (Fig. S19 and S21). c Crosslinking points calculated from 1H NMR spectra (ESI chapter 2). a Obtained from THF-SEC data calibrated on PMMA standards. b The monomer ratios were calculated from 1H NM S21). c Crosslinking points calculated from 1H NMR spectra (ESI chapter 2). ed by the Royal Society of Chemistry Chem. Sci., 2021, 12, 1302–1310 \| 1 © 2021 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2021, 12, 1302–1310 \| 1303 Edge Article View Article Online Edge Article View Article Online Chemical Science should be noted that an external crosslinker-mediated folding strategy was chosen over a hybrid TAD- and naphthalene- containing polymer, since TAD units on the polymer may degrade over time or undergo a competing light-induced poly- merization.43,44 To investigate the SCNP folding, a mixture of P2 in dry, deoxygenized acetonitrile (c ¼ 0.05 mg mL1) and BisTAD (3 eq.) was irradiated with a green LED (10 W, lmax ¼ 525 nm, Ee ¼ 60 mW cm2). The course of the reaction during the irradiation process was monitored by UV/vis spectroscopy (Fig. S1†). The recorded spectra display the expected decrease in absorption during irradiation, resulting from the change in aromaticity of the naphthalene unit (Scheme 1b) as well as the consumption of the chromophoric azobond of the BisTAD moiety.41 However, since the absorption of both species overlaps in the UV region, the end point of the cycloaddition is not immediately evident. Indeed, whereas the absorption of the BisTAD moiety is prominent between l ¼ 450–550 nm, its decrease throughout the period of irradiation only conrms TAD consumption, either arising from the desired cycloaddition or the occurrence of unwanted side reactions. Open Access Article. Published on 17 November 2020. Downloaded on 6/8/2021 3:18:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To achieve a maximum contraction of SCNP2, the equiva- lents of crosslinker were found to be a key parameter. Indeed, when less than 3 eq. of BisTAD are used for the folding of P2, the obtained single-chain nanoparticle did not reach its maximum folded state (Fig. S10,† for 1 and 2 eq. BisTAD). It should be noted that the use of such super-stoichiometric amounts is a well-known requirement to achieve external crosslinker- mediated SCNP folding.47 When the amount of BisTAD excee- ded 3 eq., however, intermolecular crosslinking of the polymer strands became apparent by the notable shoulder in the SEC traces at shorter elution times (Fig. S10,† for 6 and 9 eq. Bis- TAD). The observed intermolecular crosslinking can be ratio- nalized by a higher propensity of TAD–TAD homo- polymerization with increased TAD concentration and a larger number of monovalently bound crosslinker moieties that can undergo intermolecular polymer reactions. Next to the crosslinker equivalents, it is well-known that the solvent choice can also have a pronounced inuence on the SCNP formation.48 To investigate the solvent eﬀect, identical folding procedures were also carried out in dichloromethane, ethyl acetate and hexane. In all cases, no change in elution time was observed from SEC analysis (Fig. S11†), although upon folding P2 in dichloromethane, a second polymer distribution was noted at shorter elution times, thus indi- cating an expansion rather than the expected compaction. Thus, unlike acetonitrile, dichloromethane was identied as a non-preferred solvent for SCNP formation, which is likely attributed to its ability to promote TAD-homo-polymerization upon irradiation.44 The solvent choice thus has to meet a ne balance between polarity to dissolve both the polymer To conrm that the decrease in absorption also results in the desired macromolecular response of the system, size exclusion chromatography (SEC) traces were recorded as soon as possible aer 20 min of irradiation in order to minimize the system's dark time instability (Fig. 1a). Compared to the parent polymer P1 (black solid line), the resulting SCNP1 (dark green solid line) has a signicantly increased elution time, which results from a decrease in the hydrodynamic volume upon intramolecular crosslinking-induced compaction of the polymer chain.42,45,46 Along with the crosslinker-mediated folding, the apparent molecular weight of P1 decreased from Mn ¼ 8200 g mol1 to Mn ¼ 7100 g mol1. To verify the similar decrease in apparent molecular weight for P2 (i.e. Results and discussion Nonetheless, both BisTAD and naphthalene units show a strong absorption between l ¼ 240–400 nm (UV/vis spectra of both individual compounds are shown in Fig. S2†). The charac- teristic absorption pattern of the naphthalene moiety (lmax ¼ 280 nm) was hence monitored during irradiation, which reached a plateau aer 10 min. Whereas an overlap in absorption of both chromophores prevents an exact quantication of the reaction progress, a decrease of the characteristic naphthalene bands indicates that naphthalene crosslinking points are consumed for the intramolecular reaction (Fig. S1 and S2†).42 10 000 g mol1, Fig. S7 and S8†), resulting from an absolute decrease in hydrodynamic diameter, diﬀusion ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) measurements were carried out. The hydrodynamic diameter (DH) of the parent polymer P2 (DH ¼ 5.50 nm) indeed decreased by 11% upon single chain folding into SCNP2 (DH ¼ 4.92 nm). Furthermore, oﬄine 1H NMR of SCNP2 veried that the observed contraction can be attributed to the transformation of the naphthalene side chain moieties into their corresponding TAD/naphthalene cycloadducts, with approximately 45% of naphthalene consumption reached 2 h aer irradiation (d ¼ 8.75–8.50 ppm, Fig. S23–S26†). Open Access Article. Published on 17 November 2020. Downloaded on 6/8/2021 3:18:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Mn ¼ 11 200 g mol1 to Mn ¼ Fig. 1 (a) Normalized UV-SEC-trace at 250 nm of the crosslinker mediated folding of P1 (black line) with BisTAD using green light (lmax ¼ 525 nm). The resulting collapsed polymer chain is displayed in dark green (SCNP1). (b) Normalized UV-SEC-traces at 250 nm of the stepwise unfolding process of SCNP1 over time in the dark. (c) Evolution of MP during the stepwise unfolding process over time of SCNP1. Fig. 1 (a) Normalized UV-SEC-trace at 250 nm of the crosslinker mediated folding of P1 (black line) with BisTAD using green light (lmax ¼ 525 nm). The resulting collapsed polymer chain is displayed in dark green (SCNP1). (b) Normalized UV-SEC-traces at 250 nm of the stepwise unfolding process of SCNP1 over time in the dark. (c) Evolution of MP during the stepwise unfolding process over time of SCNP1. © 2021 The Author(s). Published by the Royal Society of Chemistry 1304 \| Chem. Sci., 2021, 12, 1302–1310 Chemical Science View Article Online Chemical Science View Article Online Chemical Science View Article Online Edge Article Edge Article and TAD crosslinker and suppressing competing TAD photopolymerization. process via UV/vis spectroscopy displayed an increased absorption in the naphthalene area at 280 nm over time (Fig. S3†). The starting intensity was, however, not fully recov- ered in our experiments, although this can be attributed to a slow degradation of the excess of BisTAD crosslinker over such a long time scale.49,50 Such irreversible TAD degradation was indeed observed when a plain solution of the BisTAD was irra- diated in the absence of P1 or P2 at the concentrations used for the folding process (0.025 mg mL1, irradiation for nearly 4 h, monitored via UV/vis absorption, see Fig. S4†). To investigate the dissipative unfolding of SCNP1, the reac- tion solution was deprived of its fuel for 32 h at ambient temperature by placing it in the dark and the kinetics of the unfolding process were examined. In the absence of light, the macromolecular architecture is expected to unfold since the spontaneous thermal cycloreversion of the TAD/naphthalene adduct would not be compensated for by the light-induced cycloaddition reaction (as was assessed for the cycloaddition/ cycloreversion of naphthalene derivative N1 and 4-n-butyl- TAD, Fig. S31†). To reach its initial conformation, the SCNP must thus proceed through several consecutive unfolding steps – each representing one covalent bond cleavage. Open Access Article. Published on 17 November 2020. Downloaded on 6/8/2021 3:18:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Following the single chain polymer compaction upon irra- diation, the macromolecule is thus shown to spontaneously unfold when lein the dark. The close to complete unfolding of the SCNPs can be rationalized by the dynamic nature of the TAD/naphthalene bond within the closed chemical system. The TAD/naphthalene cycloaddition reaches an equilibrium state in the dark, which strongly favors the starting reagents in the case of unconned small molecules of naphthalene and TAD.41 Because of the high local concentrations within the conned environment of the polymer coil and a less pronounced loss of entropic contribution upon addition compared to non-polymer bound molecules, the equilibrium is apparently shied towards the cycloadduct, resulting in a slightly folded SCNP124 h-Dark with regard to its parent polymer P1. A notable small degree of TAD/naphthalene cycloaddition occurring in the dark was also observed, as evidenced by the slight increase of elution time of P1 when stored with BisTAD overnight without being subjected to irradiation (Fig. S9†). To further assess the unfolding of the non-equilibrium SCNPs, DOSY-NMR was employed, revealing a hydrodynamic diameter for SCNP224 h-Dark of DH ¼ 5.36 nm. Thus, the polymer chain unfolds to 97% of the initial parent polymer diameter within 24 h. As observed from the 1H NMR of SCNP2 recorded aer 24 h of standing in the dark, 96% of free naphthalene is present (d ¼ 8.75–8.50 ppm, Fig. S23†), whereas 3% of cyclo- adduct is still present, which might account for the residual contraction of the polymer. Further analysis of the unfolding Aer having established that the single polymer chain can be pushed from its equilibrium into a higher energetic folded conformation using green light as the fuel, it was next explored how the folded conformation can be sustained. Firstly, the ability to modulate the energetic landscape to make the folded state thermodynamically favored was investigated (Fig. 2a). If the double bond generated by the cycloaddition is removed Fig. 2 Schematic display of hypothetic energy landscapes (top) and normalized UV-SEC traces (bottom) of the (a) kinetically trapped non- equilibrium macromolecular architecture, (b) chemically stabilized folded macromolecular architecture, and (c) light-fueled stabilization of the non-equilibrium macromolecular architecture. Fig. 2 Schematic display of hypothetic energy landscapes (top) and normalized UV-SEC traces (bottom) of the (a) kinetically trapped non- equilibrium macromolecular architecture, (b) chemically stabilized folded macromolecular architecture, and (c) light-fueled stabilization of the non-equilibrium macromolecular architecture. Chem. Open Access Article. Published on 17 November 2020. Downloaded on 6/8/2021 3:18:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The unfolding process was followed via SEC measurements aer diﬀerent time intervals in the absence of light (Fig. 1b). In comparison to the collapsed SCNP1 (dark green solid line), the SEC traces of the resulting SCNP1 aer 2, 4, 8 and 24 h of darkness (dashed line, see Fig. 1b) show a signicant decrease in elution time. The apparent MP increases with increasing time in the absence of light, thereby indicating the unfolding of the macromolecular architecture over time. Specically, the process of unfolding is rapid in the beginning, yet slows down over time, with the MP increasing by 4.2% in the rst 2 h compared to only an addi- tional 3.5% between 8 and 24 h (Fig. 1c). Aer 24 h, a plateau MP value is reached, at 96% recovery of the starting polymer. Open Access Article. Published on 17 November 2020. Downloaded on 6/8/2021 3:18:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Sci., 2021, 12, 1302–1310 \| 1305 © 2021 The Author(s). Published by the Royal Society of Chemistry Edge Article View Article Online Edge Article View Article Online Chemical Science These observations demonstrate the kinetic trapping of the folded architecture and indicate that low temperatures provide a suitably high thermal barrier to halt the cycloreversion and thus SCNP unfolding. under reductive conditions, the cycloreversion can no longer take place and the folded architecture should become more thermodynamically favored.41,51 To remove the double bond and hence chemically stabilize the SCNP, P2 was subjected to a dii- mide-mediated reduction aer being folded (see ESI section 4.2.6†). Thus, the SCNP can be trapped at the maximum folded state, resulting in a longer elution time than the SCNP2 ob- tained directly aer irradiation, which already unfolds during the SEC run. Finally, the fuel driven equilibrium state was investigated. To stabilize SCNP1, formed upon 30 min green light irradiation of in the presence of BisTAD, the reaction mixture containing SCNP1 was continuously subjected to light and SEC traces were recorded aer 2 h and 16 h of extended irradiation, respectively. The obtained traces (Fig. 2c, green dashed lines) display the same elution times and demonstrate that the macromolecular architecture can be kept in the non-equilibrium state as long as fuel is provided. In contrast, leaving SCNP1 – obtained aer 30 min of irradiation – standing in the dark for 24 h results in an increase of the hydrodynamic volume back to the equilibrium of the freely dissolved polymer (Fig. 2c, red solid line). Open Access Article. Published on 17 November 2020. Downloaded on 6/8/2021 3:18:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To maintain the folded architecture without chemical modication, low temperatures were investigated to kinetically trap the non-equilibrium state by lowering the available energy for the system below the activation barrier required to allow for the cycloreversion to become feasible (Fig. 2b). When the collapsed SCNP1 is stored well below ambient temperature, the unfolding rate is indeed drastically reduced. Specically, storage for 24 h at 21 C (dark blue dashed line) results in an identical SEC elugram compared to SCNP1 immediately measured aer irradiation. In contrast, aer 24 h storage at 6 C, a slight shitowards higher Mn is visible, which indicates the slow unfolding of the architecture (light blue dashed line). Open Access Article. Published on 17 November 2020. Downloaded on 6/8/2021 3:18:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. One advantage of dynamic non-equilibrium systems is the inherent reversibility of the system. It is thus possible to undergo several folding and unfolding steps in a closed system with the same starting polymer. To investigate repetitive poly- mer compaction/unraveling, P1 was irradiated with BisTAD (3 eq.) for 20 min to obtain SCNP1 (green dashed line, compaction of 20%). The unfolded P10, obtained aer keeping SCNP1 24 h in the dark, displays a clear decrease in elution time. Following the rst cycle, the solution was again irradiated for 20 min, resulting in a collapsed SCNP10 (red dashed line, compaction of 9%) that subsequently unfolded aer 24 h in the dark to P100. Finally, P100 was again irradiated for 20 min to obtain SCNP100 (blue dashed line, compaction of 7%), whereas P100 was retrieved aer leaving SCNP100 unfold for 24 h in the dark (Fig. 4). Throughout the three consecutive folding/unfolding cycles, a signicant change in the volume of the macromolec- ular architecture occurs during each cycle. To elucidate the The conned multivalent environment of the SCNP structure thus appears to be key for the eﬃcient light-stabilization. Indeed, for the non-conned small molecule, the TAD and naphthalene moiety are expected to diﬀuse apart more readily aer cycloreversion. Upon subsequent photoexcitation, the Fig. 4 Reversible folding and unfolding of the macromolecular architecture. In a closed system, P1 (black line) was folded to SCNP1 (green dashed line, 20% contraction) upon 20 min of irradiation (525 nm). After 24 h SCNP1 unfolds to P10 (green solid line), which was folded again, resulting in SCNP10 (red dashed line, 9% folding). This unfolds back to P100 (solid red line), which can be folded once more into SCNP100 (blue dashed line, 7% folding). In the last step, SCNP100 unfolds again to form P100 (blue solid line). Fig. 4 Reversible folding and unfolding of the macromolecular architecture. In a closed system, P1 (black line) was folded to SCNP1 (green dashed line, 20% contraction) upon 20 min of irradiation (525 nm). After 24 h SCNP1 unfolds to P10 (green solid line), which was folded again, resulting in SCNP10 (red dashed line, 9% folding). This unfolds back to P100 (solid red line), which can be folded once more into SCNP100 (blue dashed line, 7% folding). In the last step, SCNP100 unfolds again to form P100 (blue solid line). © 2021 The Author(s). Published by the Royal Society of Chemistry Chem. Open Access Article. Published on 17 November 2020. Downloaded on 6/8/2021 3:18:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The continued light irradiation thus provides the necessary driving force to maintain the photostationary state, yielding the thermally unstable cycloadduct. An important consideration, however, is that even in the presence of green light, the cyclo- adduct is expected to constantly cleave and reform over short Fig. 3 Analysis of long-term light-fueled stabilization of TAD/naphthalene cycloadducts. (a) Small molecule reaction equation with naphthalene N1. The dashed line displays a break since diﬀerent regio-isomers are formed. (b) 1H NMR analysis (in CD3CN) of the light-stabilization on smal molecule scale with continuous irradiation with NMR spectra displayed before irradiation, after the ﬁrst irradiation (0 h) and at 24, 48 and 72 h of irradiation. The dashed marked areas display the naphthalene signal 1 (starting material), bridge protons 2a and 2b (cycloadducts) and aliphatic TAD-signals 3. (c) Plot of percentage of cycloadduct present in the solution at diﬀerent times. (d) Normalized UV-SEC traces (at 250 nm) measurements after 8, 24, 48 and 72 h of light-fueled stabilization of SCNP2. (e) Evolution of MP during the light stabilization of SCNP2. Fig. 3 Analysis of long-term light-fueled stabilization of TAD/naphthalene cycloadducts. (a) Small molecule reaction equation with naphthalene N1. The dashed line displays a break since diﬀerent regio-isomers are formed. (b) 1H NMR analysis (in CD3CN) of the light-stabilization on small molecule scale with continuous irradiation with NMR spectra displayed before irradiation, after the ﬁrst irradiation (0 h) and at 24, 48 and 72 h of irradiation. The dashed marked areas display the naphthalene signal 1 (starting material), bridge protons 2a and 2b (cycloadducts) and aliphatic TAD-signals 3. (c) Plot of percentage of cycloadduct present in the solution at diﬀerent times. (d) Normalized UV-SEC traces (at 250 nm) measurements after 8, 24, 48 and 72 h of light-fueled stabilization of SCNP2. (e) Evolution of MP during the light stabilization of SCNP2. © 2021 The Author(s). Published by the Royal Society of Chemistry 1306 \| Chem. Sci., 2021, 12, 1302–1310 Chemical Science View Article Online Chemical Science View Article Online Edge Article time intervals during the fuel-driven process, although with the equilibrium strongly favoring the cycloadduct side (as veried with an exchange experiment during irradiation, see Fig. S32†). Since prolonged irradiation of freely existing TAD moieties can also promote undesired transformations such as photo- degradation or photopolymerization, it is essential to further probe the limits of the light-fueled reaction over much longer time frames. Therefore, a naphthalene derivative N1 was synthesized, which mimics the used naphthalene monomer and hence allows for small molecule investigations concerning the TAD-based photocycloaddition (Fig. 3a). The irradiation of N1 in the presence of BisTAD (0.4 eq.) was conducted at a rela- tively high concentration (3.6 mg mL1) in deuterated deoxy- genated acetonitrile in order to monitor the cycloadduct conversion via 1H NMR (Fig. 3a, note that diﬀerent regioisomers are formed, see ESI 4.2.7†). Aer 40 min of irradiation, complete BisTAD consumption can be observed from the 1H NMR spec- trum (d ¼ 3.6 ppm), indicating quantitative formation of the cycloadduct (Fig. 3b, 0 h aer irradiation). This marked the starting point of the prolonged irradiation experiment, where the percentage of intact cycloadduct decreased signicantly with percentages as low as 28% reached aer 72 h (compared to 100% cycloadduct at the start of prolonged irradiation, see Fig. 3c). In contrast to the model study, the light-stabilization of the single polymer chain was much more reliable over the course of 72 h of irradiation. Indeed, as long as fuel could be consumed, the non-equilibrium architecture was maintained, enabling its stabilization for up to three days (Fig. 3d and e). time intervals during the fuel-driven process, although with the equilibrium strongly favoring the cycloadduct side (as veried with an exchange experiment during irradiation, see Fig. S32†). Since prolonged irradiation of freely existing TAD moieties can also promote undesired transformations such as photo- degradation or photopolymerization, it is essential to further probe the limits of the light-fueled reaction over much longer time frames. Therefore, a naphthalene derivative N1 was synthesized, which mimics the used naphthalene monomer and hence allows for small molecule investigations concerning the TAD-based photocycloaddition (Fig. 3a). The irradiation of N1 in the presence of BisTAD (0.4 eq.) was conducted at a rela- tively high concentration (3.6 mg mL1) in deuterated deoxy- genated acetonitrile in order to monitor the cycloadduct conversion via 1H NMR (Fig. 3a, note that diﬀerent regioisomers are formed, see ESI 4.2.7†). Edge Article Aer 40 min of irradiation, complete BisTAD consumption can be observed from the 1H NMR spec- trum (d ¼ 3.6 ppm), indicating quantitative formation of the cycloadduct (Fig. 3b, 0 h aer irradiation). This marked the starting point of the prolonged irradiation experiment, where the percentage of intact cycloadduct decreased signicantly with percentages as low as 28% reached aer 72 h (compared to 100% cycloadduct at the start of prolonged irradiation, see Fig. 3c). In contrast to the model study, the light-stabilization of the single polymer chain was much more reliable over the course of 72 h of irradiation. Indeed, as long as fuel could be consumed, the non-equilibrium architecture was maintained, enabling its stabilization for up to three days (Fig. 3d and e). TAD/naphthalene reaction therefore will have to compete to a larger extent with de-excitation processes or TAD-based side reactions such as polymerization and photodegradation, resulting in a loss fraction of cycloadduct. On the other hand, in the conned environment of the SCNP, released TAD moieties are more likely to remain in close proximity to either their previous naphthalene binding partner or a number of other available pending naphthalene moieties, which is believed to facilitate the (re)formation of the desired TAD/naphthalene cycloadduct in the presence of light. Unlike purely photo- chemically gated systems based on photoreversible cycloaddi- tions – where the macromolecular connement favours the folding reaction to the point where unfolding becomes impos- sible37–39,52 – it herein appears that the combination of photo- chemical folding and thermal unfolding is ideal to execute these reactions selectively. Open Access Article. Published on 17 November 2020. Downloaded on 6/8/2021 3:18:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Sci., 2021, 12, 1302–1310 \| 1307 Edge Article View Article Online Edge Article View Article Online Chemical Science Chemical Science formation was also observed with H-NMR, were the conversion was followed via the appearance and disappearance of the bridge-protons of the Diels–Alder product (Fig. 5c, bridge protons 2a and 2b, 6.2–5.8 ppm). Aer 24 h in the dark, close to 84% of naphthalene N1 was restored at the end of each cycle. Following each irradiation step, the amount of unreacted naphthalene increased (Fig. 5d), and therefore the amount of formed cycloadduct decreases, although the observed photo- bleaching aer each irradiation step suggests the complete consumption of BisTAD (Fig. S29†). This is a rst indication that the hysteresis results from TAD depletion, rather than from irreversible degradation of the naphthalene or cycloadduct. Remarkably, even aer four days following the last reversion step, still 8% of cycloadduct was present in the reaction mixture (Fig. S30†). In fact, this obtained equilibrium point observed on a small molecule scale explains why the folded polymer chains did not entirely reform the starting polymers P1 and P2 aer the fuel was removed (cf. Fig. 1b, 3a, b and 4). reason for the hysteresis, it was investigated whether the naphthalene crosslinking points are no longer accessible because of irreversible side reactions, or whether the BisTAD crosslinker availability depletes as a result of its limited stability when being stored in solution for several days at ambient temperature. In addition, also photodegradation of the cross- linker could cause the noted hysteresis. Thus, the repetitive TAD/naphthalene cycloaddition/ reversion was investigated by means of small molecule studies on a closed system of N1 (3.6 mg mL1) in acetonitrile (0.7 mL) containing a substoichiometric amount of BisTAD (0.4 eq.). The resulting mixture was deoxygenated and then irradiated for 40 min. For three consecutive cycles, UV/vis (Fig. 5a and b) as well as 1H NMR (Fig. 5c and d) were measured before and aer irradiation, as well as aer 24 h upon standing in the dark. In the resulting UV/vis spectra (Fig. 5a) the characteristic TAD absorption band at 520–530 nm increases again aer disap- pearance following every irradiation. The recovered signal results from the liberation of free TAD moieties aer every irradiation. Nevertheless, a hysteresis is visible, hence resulting in a decrease of maximum intensity aer every cycle (Fig. 5a and b highlighted for 320 nm and 520 nm). Reversible cycloadduct Open Access Article. Published on 17 November 2020. Downloaded on 6/8/2021 3:18:30 PM. References 1 M. Kathan and S. Hecht, Chem. Soc. Rev., 2017, 46, 5536– 5550. 1 M. Kathan and S. Hecht, Chem. Soc. Rev., 2017, 46, 5536– 5550. Chemical Science This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To further investigate the source of hysteresis in the revers- ible process of each small molecule reversion, mass spectrom- etry was used to analyze the SCNP and small molecule reaction solutions. Thereby, 11 plausible TAD-based side-products were Fig. 5 Reversible folding studies on small molecule scale using naphthalene N1. (a) UV/vis-spectra before and after three consecutive irradiation cycles (reversion in the dark). (b) Evolution of the UV/vis absorbance as a function of time at 526.5 nm (lmax TAD absorption) over the three irradiation and reversion cycles. (c) 1H NMR analysis recorded during the irradiation cycles in CD3CN. Measurements were carried out before irradiation, after irradiation and after being kept 24 h in the dark, respectively. The dashed lines highlight the evolution of representative reso- nances, including naphthalene proton 1 (starting material) and the bridge protons 2a and 2b (cycloadduct). (d) Plot of the percentage of naphthalene N1 present in the sample directly after irradiation and after 24 h of cycloreversion in the dark. Fig. 5 Reversible folding studies on small molecule scale using naphthalene N1. (a) UV/vis-spectra before and after three consecutive irradiation cycles (reversion in the dark). (b) Evolution of the UV/vis absorbance as a function of time at 526.5 nm (lmax TAD absorption) over the three irradiation and reversion cycles. (c) 1H NMR analysis recorded during the irradiation cycles in CD3CN. Measurements were carried out before irradiation, after irradiation and after being kept 24 h in the dark, respectively. The dashed lines highlight the evolution of representative reso- nances, including naphthalene proton 1 (starting material) and the bridge protons 2a and 2b (cycloadduct). (d) Plot of the percentage of naphthalene N1 present in the sample directly after irradiation and after 24 h of cycloreversion in the dark. © 2021 The Author(s). Published by the Royal Society of Chemistry 1308 \| Chem. Sci., 2021, 12, 1302–1310 Chemical Science View Article Online Chemical Science View Article Online Chemical Science View Article Online The authors declare no conict of interests. The authors declare no conict of interests. The diminished reversibility of the folding process can readily be overcome through the here exploited polymer design based on an external crosslinker mediated SCNP folding strategy. Indeed, additional TAD can readily be introduced into the system at any time to compensate for TAD depletion and hence re-instate the complete folding of the naphthalene- containing polymers. Thus, when additional BisTAD was added in a separate system aer every unfolding (Fig. S12†), no signicant change in elution time of the SCNP aer the second folding cycle was observed (compaction of 10%). In contrast, when additional BisTAD was added before the third folding step into SCNP100 – which showed the most pronounced hysteresis in the case of P100 to SCNP100 – a notable increase in elution time was noted as compared to the SEC elugram obtained without the addition of new BisTAD (contraction of 11%, i.e. 4% more than without new TAD aliquots). Reversibility of the developed SCNP system can thus still be obtained, albeit not only upon consumption of light as a fuel, but also BisTAD over the course of time. Acknowledgements A. G., H. F. and C. B.-K. acknowledge continued support from the Queensland University of Technology (QUT) and through the Centre for Materials Science. H. F. acknowledges support by the Australian Research Council (ARC) in the form of a DECRA Fellowship and C. B.-K. in the form of a Laureate Fellowship enabling his photochemical research program. F. E. D. P. and H. A. H. thank the Research Foundation-Flanders (FWO) and Bijzonder Onderzoeksfonds (BOF)-UGent for nancial support. Some of the data reported in this paper were obtained at the Central Analytical Research Facility (CARF) operated by the Institute for Future Environments (QUT). Access to CARF is supported by generous funding from the Science and Engi- neering Faculty (QUT). © 2021 The Author(s). Published by the Royal Society of Chemistry Edge Article detected (Fig. S13 and Table S1†).53,54 When the UV/vis experi- ment was repeated at signicantly lower concentrations for a better comparison with the concentration regime of SCNP folding, an even more pronounced change in the reversibility can be observed (concentrations 4 times higher than on SCNP scale, Fig. S5 and S6†). Whereas aer irradiation the aborption intensity of the TAD moiety decreases similarly to the previous results (l ¼ 526.5 nm, Fig. S6†), the following reversion cycle recovers less than 50% of the initial intensity. The yield of regenerated TAD aer subsequent reversion cycles decreases even further. These ndings are in agreement with the result obtained for the SCNP folding where also very little recovery was visible in the UV/vis (Fig. S3†). In other words, these results indicate that the observed folding/unfolding hysteresis results from degradation of released BisTAD, while the polymer bound naphthalene moieties stay intact. detected (Fig. S13 and Table S1†).53,54 When the UV/vis experi- ment was repeated at signicantly lower concentrations for a better comparison with the concentration regime of SCNP folding, an even more pronounced change in the reversibility can be observed (concentrations 4 times higher than on SCNP scale, Fig. S5 and S6†). Whereas aer irradiation the aborption intensity of the TAD moiety decreases similarly to the previous results (l ¼ 526.5 nm, Fig. S6†), the following reversion cycle recovers less than 50% of the initial intensity. The yield of regenerated TAD aer subsequent reversion cycles decreases even further. These ndings are in agreement with the result obtained for the SCNP folding where also very little recovery was visible in the UV/vis (Fig. S3†). In other words, these results indicate that the observed folding/unfolding hysteresis results from degradation of released BisTAD, while the polymer bound naphthalene moieties stay intact. structure was also brought to a new thermodynamic minimum by a chemical modication that halts the cycloreversion. In a closed system, it was possible to repeat the folding/ unfolding process three times, albeit with a small hysteresis that was attributed to a depleted BisTAD availability. Upon addition of new BisTAD to the closed system, however, the hysteresis was overcome. 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https://openalex.org/W2508047096	http://www.scielo.br/pdf/rlae/v24/0104-1169-rlae-24-02801.pdf	English	null	Strengths of primary healthcare regarding care provided for chronic kidney disease	Revista latino-americana de enfermagem	2,016	cc-by	5,848	Strengths of primary healthcare regarding care provided for chronic kidney disease1 Descriptors: Process Assessment (Health Care); Outcome Assessment (Health Care); Delivery of Health Care; Chronic Kidney Disease. aper extracted from Master’s Thesis “Evaluation of the attention to chronic kidney disease in the hiperdia minas center Juiz de Fora”, resented to Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil. This research was supported by Fundação de Amparo à Pesquisa o Estado de Minas Gerais (FAPEMIG). 2 MSc, RN, Hospital Escola da Universidade Federal de Pelotas, Pelotas, RS, Brazil. 3 PhD, Full Professor, Faculdade de Medicina, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil. 4 PhD, Adjunct Professor, Faculdade de Medicina, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil. 5 PhD, Physician, Secretaria Municipal de Saúde, Juiz de Fora, MG, Brazil. 6 Doctoral student, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil. 7 MSc, Physician. Strengths of primary healthcare regarding care provided for chronic kidney disease1 Elaine Amaral de Paula2 Mônica Barros Costa3 Fernando Antonio Basile Colugnati4 Rita Maria Rodrigues Bastos5 Chislene Pereira Vanelli6 Christiane Chaves Augusto Leite7 Márcio Santos Caminhas8 Rogério Baumgratz de Paula3 Objective: to assess the structure and results obtained by the “Chronic Renal Patients Care Program” in a Brazilian city. Method: epidemiological, cross-sectional study conducted in 14 PHC units and a secondary center from 2010 to 2013. The Donabedian Model was the methodological framework used. A total of 14 physicians, 13 supervisors, and 11 community health agents from primary healthcare were interviewed for the assessment of structure and process and 1,534 medical files from primary healthcare and 282 from secondary care were consulted to assess outcomes. Results: most units lacked sufficient offices for physicians and nurses to provide consultations, had incomplete staffing, and most professionals had not received proper qualification to provide care for chronic renal disease. Physicians from PHC units classified as capable more frequently referred patients to the secondary care service in the early stages of chronic renal disease (stage 3B) when compared to physicians of units considered not capable (58% vs. 36%) (p=0.049). Capable PHC units also more frequently presented stabilized glomerular filtration rates (51%) when compared to partially capable units (36%) and not capable units (44%) (p=0.046). Conclusion: patients cared for by primary healthcare units that scored higher in structure and process criteria presented better clinical outcomes. Objective: to identify the coping strategies of family members of patients with mental disorders and relate them to family member sociodemographic variables and to the patient’s clinical variables. Method: this was a descriptive study conducted at a psychiatric hospital in the interior of the state of São Paulo, with 40 family members of hospitalized patients over the age of 18, and who followed the patient before and during hospitalization. We used tools to characterize the subjects and the Folkman and Lazarus Inventory of Coping Strategies. Results: the coping strategies most often used by family members were social support and problem solving. Mothers and fathers used more functional strategies (self-control p=0.037, positive reappraisal p=0.037, and social support p=0,021). We found no significant differences between the strategies and other variables examined. Conclusion: despite the suffering resulting from the illness of a dear one, family members make more use of functional strategies, allowing them to cope with adversities in a more well-adjusted way. 2 MSc, RN, Hospital Escola da Universidade Federal de Pelotas, Pelotas, RS, Brazil. 3 PhD, Full Professor, Faculdade de Medicina, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil. 4 PhD, Adjunct Professor, Faculdade de Medicina, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil. 5 PhD, Physician, Secretaria Municipal de Saúde, Juiz de Fora, MG, Brazil. 6 Doctoral student, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil. 7 MS Ph i i Rev. Latino-Am. Enfermagem 2016;24:e2801 DOI: 10.1590/1518-8345.1234.2801 www.eerp.usp.br/rlae Original article Strengths of primary healthcare regarding care provided for chronic kidney disease1 Rev. Latino-Am. Enfermagem 2016;24:e2801 DOI: 10.1590/1518-8345.1234.2801 www.eerp.usp.br/rlae Original article Strengths of primary healthcare regarding care provided for chronic kidney disease1 Rev. Latino-Am. Enfermagem 2016;24:e2801 DOI: 10.1590/1518-8345.1234.2801 www.eerp.usp.br/rlae Original article Original article Rev. Latino-Am. Enfermagem 2016;24:e2801 DOI: 10.1590/1518-8345.1234.2801 www.eerp.usp.br/rlae Strengths of primary healthcare regarding care provided for chronic kidney disease1 Method This was a cross-sectional epidemiological study conducted in 14 PHC units and a secondary healthcare center enrolled in the Hiperdia Minas Program in Juiz de Fora, MG, Brazil between 2010 and 2013. Specifically in the case of CKD, severe problems still persist due to a low level of problem-solving capacity in primary healthcare (PHC)(3-4). Studies show that this disease is highly prevalent and mainly affects elderly individuals, hypertensive and diabetic patients, and is often under-diagnosed, untreated, or addressed only later on(5-6). The Donabedian Model(10) was used to construct the structure, process and results indicators. The variables related to structure include the qualification of human resources and the availability of physical and material resources in the units. The process variables refer to screening actions of risk factors for CKD, criteria used to diagnose this disease and to refer patients to the Hiperdia Minas Program. Results were assessed based on clinical indicators, such as: average of the last two blood pressure measurements; fasting glucose; and serum creatinine recorded in the medical files of individuals monitored by the hypertension and diabetes mellitus groups from the units under study. Data concerning the world population reveals that the prevalence of CKD is between 8% and 16%(6). To date, there is no definitive information concerning the epidemiology of CKD in Brazil. Data obtained by a clinical laboratory in Juiz de Fora, MG, Brazil from 24,248 adult individuals when the disease was diagnosed using glomerular filtration rate (GFR) taking two creatinine blood test with a minimum interval of three months, revealed that 2.3% of the individuals presented CKD stages 3B, 4 or 5. If this percentage is extrapolated to the Brazilian adult population, an estimate of thee million individuals with CKD in advanced stage is obtained(7). The high prevalence of this disease associated with the complexity of its treatment results in the consumption of 10% of the annual budget of the Brazilian Ministry of Health, in addition to loss of productivity and of quality of life(8). The technical and normative guidelines established for the Hiperdia Minas Program were considered in this study. According to the State Health Department’s Resolution No. How to cite this article Paula EA, Costa MB, Colugnati FAB, Bastos RMR, Vanelli CP, Leite CCA, et al. Strengths of primary healthcare regarding care provided for chronic kidney disease. Rev. Latino-Am. Enfermagem. 2016;24:e2801. [Access ___ __ ____]; Available in: ____________________. DOI: http://dx.doi.org/10.1590/1518- 8345.1234.2801. day month year URL Paula EA, Costa MB, Colugnati FAB, Bastos RMR, Vanelli CP, Leite CCA, et al. Strengths of primary healthcare regarding care provided for chronic kidney disease. Rev. Latino-Am. Enfermagem. 2016;24:e2801. [Access ___ __ ____]; Available in: ____________________. DOI: http://dx.doi.org/10.1590/1518- 8345.1234.2801. day month year URL Rev. Latino-Am. Enfermagem 2016;24:e2801 2 Introduction Health Services Networks. In Minas Gerais, this proposal was consolidated with the creation of the HIPERDIA Minas Program, characterized by the supply of care shared between PHC units and Hiperdia Centers(9). The Brazilian health system is experiencing a situation characterized by high rates of morbidity and mortality caused by chronic conditions, among which the following stand out: type 2 diabetes mellitus, hypertension, chronic kidney disease (CKD), and cardiovascular diseases(1). In this model, the PHC is the entry way to the health system and should monitor the entire path the patient goes through the network by using specific tools such as: clinical records, guidelines, a referral and counter- referral system, and effective communication strategies between PHC teams and specialists. Concomitantly, infectious diseases persist, along with the strong growth of external causes, which combine to form a complex epidemiological situation defined as “triple burden of disease”(2). Understanding the complexity of care provided to patients with CKD and the role of PHC in the coordination of the Integrated Health Services Networks, this study’s objective was to assess the structure, process, and results obtained by the Chronic Renal Patients Care Program in a Brazilian city. Changes in the population’s epidemiological profile are not properly heeded by the organization of the healthcare system so that inconsistency between the population’s needs and the current healthcare model is observed(2). Hence, coping with chronic conditions is a challenge faced by managers and professionals in all the fields of the healthcare system, as well as by patients. Method 2,606/2010, patients with hypertension and/or diabetes mellitus presenting the following conditions must be referred to the chronic kidney disease ambulatory at the Hiperdia Minas Center in Juiz de Fora: - CKD stage 3B, 4 or 5; estimated annual loss of glomerular filtration ≥5 mL/min/year (initial GFR – final GFR/number of months of observation x 12); - proteinuria>1.0 g/day or proteinuria<1.0 g/day accompanied by hematuria; - abrupt increase of serum creatinine (≥30%); 25% decrease in estimated GFR Given the need to design new proposals to guide prevention strategies and the management of chronic diseases, the Ministry of Health has developed policies to reorganize the health services focusing on Integrated www.eerp.usp.br/rlae 3 3 Paula EA, Costa MB, Colugnati FAB, Bastos RMR, Vanelli CP, Leite CCA, et al. Paula EA, Costa MB, Colugnati FAB, Bastos RMR, Vanelli CP, Leite CCA, et al. the PHC units selected for the study in the period in which the program was implemented (from September 2010 to August 2013) were also selected. when initiating some medication that blocks the renin- angiotensin- aldosterone axis. These guidelines were provided to the PHC units at the time the program was implemented in the city. According to the progression of the GFR over the follow-up period, patients were classified as: - non-progressing (GFR did not decrease); - slowly progressing (GFR decreased up to 5 mL/min/year); - rapidly progressing (GFR decreased ≥5 mL/min/year). Blood pressure was considered to be under control when systolic blood pressure was ≤ 140 mmHg and/ or diastolic blood pressure was ≤ 90 mmHg among hypertensive individuals. Those with diabetes should have their systolic blood pressure ≤ 130 mmHg and/or diastolic blood pressure ≤ 80 mmHg(13). The PHC units were selected according to region criteria and the frequency with which patients were referred to the secondary health care service. Hence, two units of each of the seven administrative regions in the city (north, south, east, west, northeast, southeast and center) were selected: the one with the highest number of referrals and another with the lowest number of referrals to the Hiperdia Minas Center in Juiz de Fora. The professionals were randomly selected for the interviews. The PHC units selected for this study represented 23% of all units in the city, covering approximately 63,000 users of the healthcare system. The variables selected were pre-encoded and stored in SPSS® (Statistical Package for the Social Sciences) version 15.0. Method Data were interpreted using descriptive analysis, such as means and prevalence. The relationships among variables were assessed with Chi- square test. A level of significance of 5% and confidence interval of 95% were adopted for all statistics. The study was approved by the Institutional Review Board at the university hospital of Juiz de Fora (report No. 133.399). The participants signed free and informed consent forms. The instrument used(11) was originally designed for a normative assessment of the care provided to individuals with diabetes mellitus. This instrument was adapted for this study to allow the care provided to patients with CKD to be assessed. Indicators and actions were selected according to the “Guidelines for Care Provided to Chronic Kidney Disease”(12). To analyze the capability of PHC units, scores were assigned to each criterion of the questionnaire and these scores were totaled within the structure or process’ subcomponents. This way, we have two columns: one presenting the sum of the structure’s subcomponents and another presenting the sum of the process’s subcomponents for each interview. The structure dimension received a weight of 4 and the process dimension received a weight of 6. The scores obtained by the three interviewees in the subcomponents of both structure and process were totaled to reach a PHC unit’s total score, which in turn represents a unit’s capability. This score was then divided into terciles of classification and, based on the total scores, each PHC unit was classified as “capable”, “partially capable”, or “not capable”, that is, these classifications concern the PHC units’ capacity to implement preventive actions and monitor CKD. Results Among the 14 PHC units selected, 11 were Family Health Strategy (FHS) units, two were traditional PHC units, and one was mixed (FHS and traditional PHC). A total of 14 physicians, 13 supervisors, and 11 community health agents were interviewed. The average time the professionals worked in the PHC unit was 6.0±6.12 years, 8.3±6.31 and 8.6±3.55 years, for physicians, supervisors and community health agents, respectively; 57% of the physicians, 39% of the supervisors and 23% of the community health agents had worked less than five years in the unit. In regard to the physical structure, nine (61%) units did not have a sufficient number of offices for physicians and nurses to simultaneously give consultations, while ten (69%) units did not have sufficient amounts of basic medication to meet the demand they experienced. A total of 1,534 medical files from PHC that corresponded to the micro-area of the physician interviewed were selected to be analyzed. These files contained the clinical information of the patients cared for by the hypertension and/or diabetes mellitus groups, beginning in 2010. Another 282 files of patients with CKD who were referred to the Hiperdia Center by Staffing for only four (31%) of the 14 PHC units was complete. In regard to qualification, eight physicians (54%) and 11 nurses (77%) had specialization in Family Health and seven (67%) community health agents had attended the introductory course in Family 4 Rev. Latino-Am. Enfermagem 2016;24:e2801 Rev. Latino-Am. Enfermagem 2016;24:e2801 were found in 398 (26%) files, while only one record was found in another 261 (17%) files. Health. Additionally, only eight (54%) physicians, six (46%) nurses and one community health agent had received specific qualification to provide care and assist patients with CKD. CKD was diagnosed in 250 (16%) individuals based on the GFR. Table presents the description of the sample’s sociodemographic and clinical characteristics. None of the 14 PHC units had guidelines specific for CKD. The interviews with the physicians revealed that six (43%) of them knew the criteria used to refer patients to the Hiperdia Minas Center and nine physicians (64%) used serum creatinine as the main criterion to refer patients to a secondary care service. Additionally, only five (36%) interviewees reported including urinary protein excretion as an assessment criterion. Results These figures are even lower when the referral criteria, that is, checking for glomerular filtration rate and microalbuminuria screening (which are used to establish how severe the disease is), are considered. Hence, less than one third of the physicians reported using the GFR and only one mentioned that microalbuminuria was an assessment criteria. Two participants reported using GFR provided by the clinical analysis laboratory. Table 1 – Socidemographic and clinical characterization of chronic renal patients cared for in PHC units in the city of Juiz de Fora, MG, Brazil, 2010 to 2013 Variables Total (N=250) Average age, years (Standard deviation) 67 (10.9) Men, n(%) 204 (82) Comorbidities Hypertension, n(%) 175 (70) Diabetes mellitus, n(%) 75 (30) Chronic kidney disease staging Stage 3 A 143 (57) Stage 3B 88 (35) Stage 4 15 (6) Stage 5 4 (2) In regard to the coordination of the flow of patients within the network, the interview with the professionals from the PHC units revealed there is no committee to regulate referrals to the specialist in the city. Therefore, considering the PHC units’ structure and process aspects, their capacity to prevent risk factors, and early detection and control of CKD, was classified as followed: five (36%) units were classified as capable, five (36%) as partially capable, and four (28%) as not capable. The analysis of clinical indicators showed that 61% of the patients cared for by the capable PHC units had their average blood pressure under control, while only 39% of those cared for by PHC units were classified either as partially or not capable had their average blood pressure under control (p=0.92). When analyzing the 1,534 medical files of patients monitored by the hypertension and diabetes mellitus groups from the PHC units, we observed errors in the recording of routine laboratory exams. In four years of follow-up, no data regarding fasting glucose were recorded in 18% (276) of the medical files; 163 (44%) files of diabetic patients presented no records of glycated hemoglobin; and no records of serum creatinine, which refers to screening for CKD, In regard to the GFR progression, 51% of the patients monitored in the capable PHC units had stabilized their GFR, while 56% of those monitored in PHC units that were not capable presented slow or rapid decline of the GFR (p=0.046) (Figure 1). www.eerp.usp.br/rlae 5 Paula EA, Costa MB, Colugnati FAB, Bastos RMR, Vanelli CP, Leite CCA, et al. Results Figure 1 – Progression of the GFR of patients monitored by PHC unit, according to their classification. Juiz de Fora, Minas Gerais, Brazil, 2010 to 2013 (N=250) Figure 1 – Progression of the GFR of patients monitored by PHC unit, according to their classification. Juiz de Fora, Minas Gerais, Brazil, 2010 to 2013 (N=250) the “not capable” PHC units less frequently referred patients to the ambulatory unit compared to “capable” and “partially capable” units (p<0.001) (Figure 2). The analysis of the conditions under which patients were referred to the Hiperdia Minas Center’s chronic kidney disease ambulatory unit in Juiz revealed that Figure 2 – Frequency in which patients were referred to the Hiperdia Minas Center in Juiz de Fora, according to the PHC unit’s classification. Juiz de Fora, Minas Gerais, Brazil, 2010 to 2013 (N=282) Figure 2 – Frequency in which patients were referred to the Hiperdia Minas Center in Juiz de Fora, according to the PHC unit’s classification. Juiz de Fora, Minas Gerais, Brazil, 2010 to 2013 (N=282) www.eerp.usp.br/rlae 6 Rev. Latino-Am. Enfermagem 2016;24:e2801 that being at stage 3B is a criterion to refer a patient to a secondary care service, the analysis of the patients’ stages at the time of referral according to the PHC units’ classifications revealed that 58% of patients originating from units considered capable were at stage 3B. In turn, those units classified as not capable had referred most of their patients (65%) when they were already at stage 4 or 5 of CKD (p=0.049) (Figure 3). that being at stage 3B is a criterion to refer a patient to a secondary care service, the analysis of the patients’ stages at the time of referral according to the PHC units’ classifications revealed that 58% of patients originating from units considered capable were at stage 3B. In turn, those units classified as not capable had referred most of their patients (65%) when they were already at stage 4 or 5 of CKD (p=0.049) (Figure 3). The PHC units classified as “capable” more frequently presented knowledge regarding the criteria used to refer patients to secondary services (80%), compared to units considered partially capable (40%) or not capable (50%). When assessing the stages of the CKD at the time of referral, 61 (22%) patients were at stage 3A, 99 (35%) were at stage 3B, 101 patients (36%) were at stage 4, and 21 (7%) were at stage 5. Results Considering Figure 3 – Stage of the chronic kidney disease among patients referred to the Hiperdia Minas Center in Juiz de Fora, according to the PHC unit classification. Juiz de Fora, MG, Brazil, 2010 to 2013 (N = 221). Figure 3 – Stage of the chronic kidney disease among patients referred to the Hiperdia Minas Center in Juiz de Fora, according to the PHC unit classification. Juiz de Fora, MG, Brazil, 2010 to 2013 (N = 221). Discussion patients having a permanent vascular access to initiate dialysis(20). Late referral was also observed in England, where approximately one quarter of the patients were referred to a specialist only one month before the need to initiate renal replacement therapy. Late referral was associated with a lower prevalence of preventive interventions, worse clinical status at the beginning of renal replacement therapy, longer hospitalizations, and lower survival rates(21). On the other hand, early referral of patients with CKD to the secondary care level is associated with a better prognosis. A recent study addressing 3,273 chronic patients at stages 3 to 5 verified that those referred to a nephrologist at advanced stages of the disease were at a greater risk of death before dialysis was initiated(22). In addition to early referral of patients to a specialist, a multidisciplinary approach to renal disease can lead to more satisfying outcomes. A recent study reports that the average annual decline in the glomerular filtration rate was twice as high among patients monitored by a nephrologist only compared to when the treatment was conducted by a multidisciplinary team(23). Similar results were obtained in the Hiperdia Minas Center in Juiz de Fora from 934 patients received cared over a period of two years. The speed of annual GFR loss in this sample was reduced or stabilized in two thirds of the patients, suggesting the multidisciplinary team was efficient in managing CKD(24). These results become more of a problem if we consider that both Brazilian and international guidelines recommend annual measurement of creatinine in hypertensive and diabetic patients and the use of GFR to screen for CKD(12-13,16). Failure to comply with these guidelines when monitoring CKD is not limited to the state of Minas Gerais. Other authors list severe deficiencies in the management of renal disease within the PHC sphere. A study conducted in the south of Brazil reports that only 8% of diabetic patients and 5% of hypertensive patients had their GFR assessed by physicians in the PHC service(17). In the state of São Paulo, records of microalbuminuria were found in only 1.4% of the medical files(18). Discussion and nurses to concomitantly provide consultations and insufficient medication to meet the demands of patients. This study’s results reveal the weaknesses and strengths presented by the healthcare network available to chronic renal patients in Juiz de Fora, MG, Brazil. If, on the one hand, assessment of the capacity of PHC units to provide care to individuals with CKD showed a lack of resources and systematization of preventive and monitoring actions to manage kidney disease, on the other hand, strengths such as improved results were observed in the care provided to individuals with chronic conditions in PHC units that have more well-established structure and processes. We also observed that most professionals were not specifically qualified to provide care to patients with CKD. In regard to this aspect, the obstacle most frequently mentioned by the units’ managers was the low number of workers with the appropriate profile and technical qualification to enable the team’s expansion process. Other factors, such as lack of social recognition, difficulties attending continuing education programs, poor working conditions, and difficulties faced in the management of the staff, also contribute to high turnover and consequent fragmentation of the network(14-15). A deficient physical structure and a lack of material resources in the units were also observed. Most units had an insufficient number of offices for physicians In addition to the structural and human resources deficiencies, weaknesses regarding the process were www.eerp.usp.br/rlae 7 Paula EA, Costa MB, Colugnati FAB, Bastos RMR, Vanelli CP, Leite CCA, et al. Paula EA, Costa MB, Colugnati FAB, Bastos RMR, Vanelli CP, Leite CCA, et al. also observed. Thus, a lack of guidelines concerning CKD and a lack of knowledge concerning criteria used to refer patients to secondary care services observed among the units under study possibly resulted in delayed diagnoses and referrals. As seen in the interviews with the PHC physicians, serum creatinine was the main parameter used to assess renal function, at the expense of the GFR. Additionally, most physicians failed to mention microalbuminuria as a criterion to be considered in the referral of patients to specialized care. Similarly, analysis of the medical files of hypertensive and diabetic patients cared for in the PHC units revealed that only 57% of the files contained two creatinine measurements during the period under study and, even more concerning, 43% of the files contained only one creatinine measure or no reference to this information, at all. www.eerp.usp.br/rlae Conclusion 8. Sancho LG, Dain S. Análise de custo efetividade em relação às terapias renais substitutivas: como pensar estudos em relação a essas intervenções no Brasil? Cad Saúde Pública. 2008;24(6):1279-90. doi: http:// dx.doi.org/10.1590/S0102-311X2008000600009  9. Secretaria do Estado De Minas Gerais (SES). Institui o Programa Hiperdia Minas e dá outras providências. Resolução No 2606, de 7 de dezembro de 2010. 2010 [Acesso 9 dez 2014]. Disponível em: http://www.saude. mg.gov.br/sobre/institucional/resolucoes. This study showed that the PHC units with higher scores concerning structure and process presented better clinical outcomes regarding the care provided to CKD, characterized by earlier referrals and lower declines in patient GFR rates. These findings suggest that the healthcare network is more efficient in providing care to chronic renal patients when PHC services develop appropriate clinical management processes, characterized by preventive, management and treatment actions directed to CKD. 10. Donabedian A. Striving for Quality in Health Care. An Inquity into Policy and Practice. Ann Arbor, Michigan: Health Administration Press; 1991. ISBN: ISBN-10: 0910701695 Acknowledgment 11. Pereira PMH. Avaliação da atenção básica para o diabetes mellitus na Estratégia Saúde da Família [Internet]. Recife. Dissertação [Saúde Pública]. Centro de Pesquisas Aggeu Magalhães da Fundação Oswaldo Cruz; 2007. [Acesso 6 nov 2014]. Disponível em: http:// www.cpqam.fiocruz.br/bibpdf/2007pereira.pmh.pdf. Gratitude to IMEPEN (Minas Gerais Foundation Institute of Study and Research in Nephrology) and the State Health Department of Minas Gerais for supporting the study. Discussion Favorable outcomes related to the progression of renal function among patients monitored in PHC units classified as “capable”, allied with greater knowledge of early referral criteria, indicate the need to improve the structure and to systematize the work processes of those units classified as partially or not capable in order to harmonize the actions directed to patients with CKD among the various levels of healthcare services. Another study conducted in the United States reports that 64% of PHC physicians report a lack of knowledge concerning criteria used to refer patients to specialized care services and 16% were not able to estimate a patient’s glomerular filtration rate. The same study shows that 53% of the nephrologists interviewed believed that PHC physicians were late in referring patients to specialized assessment(3). Even though a single city was addressed in this study, which can be considered one of this study’s limitation, the results can support the sensitization of managers concerning the need to invest in the qualification of professionals and in management tools, coupled with a care plan that is shared between PHC and a multidisciplinary team, factors that can positively impact the care provided to individuals with CKD(25). Late referral of patients with CKD implies increased risk of mortality, worse metabolic status in dialysis, complications related to the use of temporary vascular access, longer hospitalizations, more difficult access to preemptive kidney transplantation, and a consequent increase in health services costs(5,19). It is believed that the care provided to individuals with CKD in most Brazilian cities is even worse, considering there is a lack of programs connecting the services and harmonizing the care provided for this health condition. A retrospective study conducted in a dialysis center revealed that 22% of the patients were referred to secondary care services when in stage 5 of CKD (GFR< 15mL/min/1.73 m²), which resulted in a lower number of Rev. Latino-Am. Enfermagem 2016;24:e2801 8 References 12. Secretaria do Estado de Minas Gerais. Atenção à saúde do adulto: Conteúdo técnico da Linha. Guia de hipertensão arterial sistêmica, diabetes mellitus e doença renal crônica. 3ª edição. Belo Horizonte; 2013. [Acesso 16 dez 2014]. Disponível em: www.fhemig. mg.gov.br 12. Secretaria do Estado de Minas Gerais. Atenção à saúde do adulto: Conteúdo técnico da Linha. Guia de hipertensão arterial sistêmica, diabetes mellitus e doença renal crônica. 3ª edição. Belo Horizonte; 2013. [Acesso 16 dez 2014]. Disponível em: www.fhemig. mg.gov.br 1. Alwan A, Maclean DR, Riley LM, d’Espaignet ET, Mathers CD, Stevens GA, et al. Monitoring and surveillance of chronic non-communicable diseases: progress and capacity in high-burden countries. Lancet. 2010;376(9755):1861-8. doi:10.1016/S0140- 6736(10)61853-3 1. Alwan A, Maclean DR, Riley LM, d’Espaignet ET, Mathers CD, Stevens GA, et al. Monitoring and surveillance of chronic non-communicable diseases: progress and capacity in high-burden countries. Lancet. 2010;376(9755):1861-8. doi:10.1016/S0140- 6736(10)61853-3 13. VI Diretrizes Brasileiras de Hipertensão Arterial (DBH). Avaliação Clínica e Laboratorial e Estratificação de Risco. J Bras Nefrol. 2010;32(Suppl I)(1):14-8. doi: http://dx.doi.org/10.1590/S0066-782X2010001700001 14. André AM, Ciampone TMH, Santelle O. Tendências de gerenciamento de unidades de saúde e de pessoas. Rev Saúde Pública. 2013;47(1):158-63. doi: http:// dx.doi.org/10.1590/S0034-89102013000100020 15. Matumoto S, Fortuna CM, Kawata LS, Mishima SM, Pereira MJB. Nurses’ clinical practice in primary care: a process under construction. Rev. 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Late referral of patients with end-stage renal disease: an in-depth review and suggestions for further actions. NDT Plus. 2010;3:17– 27. doi: doi: 10.1093/ndtplus/sfp050 6. Jha V, Garcia-Garcia G, Iseki k, Li k, Naicker S, Plather B, et al. Chronic Kidney disease: global dimension and perspectives. Lancet. 2013;382(9888):260-72. doi: 10.1016/S0140-6736(13)60687-X. 7. Bastos RMR, Bastos MG, Ribeiro LC, Bastos RV, Teixeira MTB. Prevalência da doença renal crônica nos estágios 3, 4 e 5. Corresponding Author: Elaine Amaral de Paula Universidade Federal de Juiz de Fora. Faculdade de Medicina Rua Carangola, nº 630 Bairro: Democrata CEP: 36035-220, Juiz de Fora, MG, Brasil E-mail: elaineamp@hotmail.com Received: Sept. 10th 2015 Accepted: Apr. 12th 2016 Copyright © 2016 Revista Latino-Americana de Enfermagem This is an Open Access article distributed under the terms of the Creative Commons (CC BY). 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https://openalex.org/W2592284210	https://europepmc.org/articles/pmc5338776?pdf=render	English	null	A camera-phone based study reveals erratic eating pattern and disrupted daily eating-fasting cycle among adults in India	PloS one	2,017	cc-by	8,652	Editor: Karen L. Gamble, University of Alabama at Birmingham, UNITED STATES Copyright: © 2017 Gupta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. A camera-phone based study reveals erratic eating pattern and disrupted daily eating- fasting cycle among adults in India Neelu Jain Gupta1,2, Vinod Kumar1, Satchidananda Panda3,4 Neelu Jain Gupta1,2, Vinod Kumar1, Satchidananda Panda3,4 1 Department of Zoology, University of Delhi, Delhi, India, 2 Department of Zoology, MMH College, Ghaziabad, Uttar Pradesh, India, 3 Salk Institute of Biological Sciences, La Jolla, California, United States of America, 4 UC San Diego Center for Circadian Biology, San Diego, California, United States of America 1 Department of Zoology, University of Delhi, Delhi, India, 2 Department of Zoology, MMH College, Ghaziabad, Uttar Pradesh, India, 3 Salk Institute of Biological Sciences, La Jolla, California, United States of America, 4 UC San Diego Center for Circadian Biology, San Diego, California, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * drneelujgupta@hotmail.com (NJG); satchin@salk.edu (SP) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Data Availability Statement: All relevant data are within the paper. Funding: This work was partially supported by grants from American Federation of Aging Research #M14322, Leona M. and Harry B. Helmsley Charitable Trust’s grant #2012-PG- MED002, the Glenn Center for Aging, Joe W. and Dorothy Dorsett Brown Foundation to SP and UGC research award (F-30-50/2011) to NJG, and a joint grant from the Indo-US Science and Technology Forum (IUSSTF) IUSSTF/JC- Biological Timing/JC- 15- 2013/2014-15 to VK (http://www.iusstf.org/). Abstract The daily rhythm of feeding-fasting and meal-timing are emerging as important determinants of health. Circadian rhythm research in animal models and retrospective analyses of human nutrition data have shown that reduced length of overnight fasting or increased late night eating increases risk for metabolic diseases including obesity and diabetes. However, the daily rhythm in eating pattern in humans is rarely measured. Traditional methods to collect nutrition information through food diary and food log pay little attention to the timing of eating which may also change from day to day. We adopted a novel cell-phone based approach to longitudinally record all events of food and beverage intake in adults. In a feasibility study daily food-eating patterns of 93 healthy individuals were recorded for 21 days using camera phones. Analysis of the daily eating patterns of these individuals indicates deviation from conventional assumption that people eat three meals-a-day within a 12 h interval. We found that eating events are widespread throughout the day, with <30% of calories consumed before noon and >30% consumed in evening and late night hours. There was little difference in eating pattern between weekdays and weekends. In this cohort more than 50% of people spread their caloric intake events over 15 h or longer. One decile of the cohort who were spouses of shift-workers or had flexible work schedule spread their caloric intake over 20 h. Although the nutrition quality and diversity of food consumed is different between South- East Asian and Western countries, such overall disruption of daily eating-fasting rhythm is similar. Therefore, in view of hypothesis that disrupted daily eating pattern may contribute to the global increase in metabolic diseases and modification of daily eating pattern is a poten- tial modifiable behavior to contain these diseases, monitoring eating pattern is an important aspect of lifestyle. RESEARCH ARTICLE OPEN ACCESS Citation: Gupta NJ, Kumar V, Panda S (2017) A camera-phone based study reveals erratic eating pattern and disrupted daily eating-fasting cycle among adults in India. PLoS ONE 12(3): e0172852. doi:10.1371/journal.pone.0172852 Editor: Karen L. Gamble, University of Alabama at Birmingham, UNITED STATES Received: August 16, 2016 Accepted: February 11, 2017 Published: March 6, 2017 Copyright: © 2017 Gupta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Karen L. Gamble, University of Alabama at Birmingham, UNITED STATES Received: August 16, 2016 Accepted: February 11, 2017 Published: March 6, 2017 Disrupted circadian eating pattern in India circadian rhythms have evolved to temporally coordinate activity-sleep and feeding-fasting to the appropriate time of the day-night cycle. This instructive pressure has resulted in daily oscil- lations in activities of thousands of genes in a tissue specific manner to offer metabolic fitness to the organism [1]. Industrialization and access to electrical lighting at night has changed human lifestyle from predominantly agrarian—with plenty of access to daylight and food during the day—to post-industrial lifestyle marked by predominantly indoor living and extended period of access to electrical lighting. This in turn has allowed humans to stay awake for longer periods of time under artificial light and have prolonged access to food. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. There is a complex interaction between the quality of nutrition and circadian rhythms. In the most widely used animal model of obesity and chronic metabolic diseases, rodents are fed a high fat diet ad libitum (ad lib). This Diet-Induced Obesity (DIO) model also predisposes to diabetes, high cholesterol, fatty liver disease, and increased cardiovascular disease risks [2, 3]. These diseases are among the top ten causes of morbidity and mortality among adults in mod- ern societies [4]. Interestingly, ad lib access to high fat diet also changes the daily eating pattern of rodents and they switch from a primarily nocturnal eating pattern to erratic eating habits throughout the day and night [5]. This erratic eating pattern under ad lib high fat feeding changes the global diurnal gene expression pattern in metabolic organs [6]. Conversely, time- restricted feeding (TRF) in which isogenic mice are given the isocaloric energy dense diet within an 8–12 h period are prevented from obesity, diabetes and associated metabolic diseases in both male and female rodents [7–10]. Even among diurnal animals, TRF prevents body weight gain and age- or diet- induced deterioration of heart function [11]. These animal stud- ies in which detailed genomic and metabolic characterizations have elucidated the underlying molecular changes, raise a possibility for health improvement in human beings through tem- poral re-organization of daily food consumption. Sleep, activity, and the associated eating pattern are overt outputs of circadian rhythms. Introduction Animals and humans evolved on our rotating planet with predictable daily rhythms in activity- rest and associated rhythms in period of feeding and fasting. Accordingly, tissue-autonomous 1 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 Methods This study was approved by the human ethics committee of MMH College, Ghaziabad, Uttar Pradesh. Participants were recruited through paper leaflets/flyers and notice board advertise- ments in public places. Inclusion criteria were as follows. Healthy adults 18 years or older, no- shift-work, no history of major sickness within the past 6 months, non-smoker, no diagnosed diabetes were included in this study. Female adults who were not pregnant or whose youngest child was older than 1 year, not enrolled in a weight-loss or weight-management program, not taking any medication that is meant for or has a known effect on appetite, no psychiatric disor- der or on anti-depressant medicine, no out-of-town travel planned during the study period, no surgery in recent past were included in this study. Subjects were screened for inclusion and exclusion criteria by telephone and in-person interview. Based on profession (to screen out shift-workers) reported, subjects largely belonged to lower to medium income groups. At the first visit, goals and methods of this study were described to the participants and those willing to participate in the program provided written consent. Each participant was given a functional camera phone and was asked to record all of his/her food, beverage and water intake using the camera function of the phone. In order to avoid potentially expensive data usage during transferring digital pictures, the subjects were asked to save the food pictures in their phone cameras. Subjects’ height and weight were measured using a calibrated scale and tape measure at the beginning and during their visit after the end of the 3 week monitor- ing period. At the end of the 3 week period, a follow-up questionnaire was filled by each partic- ipant. Participants were nominally compensated for their time and effort in this study. In order to monitor eating pattern in this study, data were collected from a Tuesday/ Wednesday midnight of the first week to Tuesday/Wednesday midnight three weeks later. Subjects received instructions to record every item consumed (food, drink, water), regardless of serving size, using the camera phone. Participants received a phone call or text message at random times during the day, reminding them to log their food data. The leftovers from items that were not completely consumed were to be recorded again and noted. Participants that fell sick and required regular medications or diet modifications were removed from the study. Cir- cadian and sleep disruption has long been associated with increased predisposition to obesity, diabetes and metabolic diseases [12–14]. Shift-work with erratic lifestyle also predisposes an individual to these diseases [15–17]. In humans, an aberrant eating pattern, such as late night caloric intake, increases the risk of developing coronary heart disease by as much as 55%, after controlling for diet and lifestyle [18]. These observations in humans are now replicated in controlled animal studies [19], lending further support to the notion that chronic circadian disruption contributes to risks for chronic diseases. Conversely, there is growing number of observations that constraining all caloric intake to <12 h can reduce breast cancer risk and improve prognosis [20, 21]. Similarly, in weight-loss studies, early ingestion of major meals during the day has been shown to enhance weight-loss [22]. These observations have prompted a renewed interest on daily pattern of food intake [23]. India is among the top 5 countries that account for more than 50% of the world’s diabetic patients [24]. Over the past 5 decades, it has rapidly moved from a largely agrarian society to an advanced economy with accelerated increase in electrification and increase in non-farm employment [25]. These changes in socio-economic parameters contribute significantly towards changes in circadian lifestyles and associated eating pattern. Contrary to conventional wisdom that humans in modern societies primarily eat three meals within a 12 h period, we hypothesize more than 50% of adults spread their caloric intake over >12 h. Specifically, an erratic eating pattern with food intake spread over a long period in the 24 hours within a day might exacerbate circadian disruption. If this behavior is sustained over years, it may increase the risk for metabolic diseases. Although 24-h food recollection, food diary and food frequency questionnaire are typically used in nutrition studies, they are not intended to capture daily eat- ing pattern data and its day to day variation. Therefore, methods to collect evidence-based and time-stamped ingestion data and to analyze them for daily pattern of eating and fasting are PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 2 / 15 Disrupted circadian eating pattern in India being developed [26]. Camera phones are increasingly being used in nutritional studies [27, 28]. In this study, we tested the feasibility of monitoring daily eating pattern among healthy adults with no diagnosed disease using camera phones. PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 Methods After 21 days, the pictures from their camera were downloaded and analyzed for time stamp and food pictures. All participants were re-contacted to verify one or more of three spe- cific questions about the data; any unrecognizable food picture, whether they observed any religious fasting on days with <3 records, and abnormal eating pattern resembling that of a shift-worker. As many individuals in India follow religious practices to observe partial-, com- plete- fast, or modified diet on religious days or festivities, days with less than 3 total events (including non-caloric content items) were flagged and verified with the participants for any observed fasting day or whether they forgot to log data. If they had <3 events and the reason was failure to record data, those days were removed from analyses. Individuals with erratic eat- ing pattern (most of the food items consumed very late at night, or a switch from day to night time eating on 3 or more days of the week) were asked if they or someone in their family does shiftwork or flexible work that would explain such eating pattern. Each participant was assigned a random alpha-numeric code and the food picture names were tagged with the par- ticipant ID as a prefix. Each picture was renamed using windows ImageRenaming application to include anonymous name and time-stamp in the format: SubjectID_YYYY-MM-DD_HH- MM-SS. Each picture was sorted into water or non-water items. The name and portion size of PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 3 / 15 Disrupted circadian eating pattern in India food items were independently assessed by three researchers. A fourth researcher verified any discrepancy between annotations or randomly checked the images and the annotations. The non-water items were further annotated by the research team by looking up Food And Nutri- ent Database For Dietary Studies (FNDDS) website of USDA National Nutrient Database for Standard Reference [27]., CalorieKing and MyFitnessPal websites. The FNDDS has caloric val- ues for several food items commonly consumed in India (fruits, raw vegetables, rice, bread, coffee, tea, milk etc.). Some of the foods consumed in India may have different names, but they have similar composition to several FNDDS listed items (e.g. channa in India is garbanzo beans, Daal is lentil soup, Bhel is rice crispie etc.). When a food item or its equivalent was not found in FNDDS, it was looked up in CalorieKing or MyFitnessPal websites (e.g. Methods Dosa, samosa, vada). All data were imported to GraphPad Prism for subsequent analyses and statistical tests. Time of caloric events for each participant was analyzed in GraphPad Prism to derive 95 per- centile eating interval or “eating duration”. Time of caloric events for each individual was also plotted as a scatter plot to visualize the spread of caloric events within 24 hours. Beginning of the eating duration was considered as breakfast or time of first caloric intake and the end of the duration was considered the last caloric intake. Time of eating at all events from all partici- pants was pooled and frequency distribution of eating events in one hour bin over 24 h of the day was derived. For each individual, difference between two consecutive events was calculated and if it was less than15min, it was considered as one meal. Frequency distribution of all inter- meal intervals was calculated to find the 25-, 50-, and 75 percentile intervals. Resting energy expenditure for each individual was calculated using modified Harris Bene- dict equation [29]. Reported daily caloric intake for each participant was calculated from their food pictures. PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 Results In India, the vast diversity of non-standard food items, language diversity, and insufficient awareness of portion size, makes it difficult to adopt standard method of nutrition studies in which the participants are asked to describe food and portion size. Therefore, we adopted a smartphone-based method to collect images of food that transfers the burden of food annota- tion from users to the researchers (Fig 1A and 1B). We used the picture taking function of smartphones to collect food consumption data from a cohort of healthy males and females in National Capital Territory of India (Delhi-NCR)—a cosmopolitan city of India. Out of 102 participants who consented to the study and started the data collection, eight dropped out due to study-unrelated sickness, leaving 94 participants who completed the study (Table 1). There was no significant reduction in the body weight of participants during the 3 weeks self-logging of nutrition data (Fig 1C and 1D), suggesting that self-reporting did not adversely affect food intake leading to significant weight loss. The use of camera phone along with daily reminders to record ingestion events was effec- tive as we could collect 1940 days of eating data out of (94 participants21 days) 1974 days of intended data collection. Out of 17622 total images collected from 94 participants over a 3 week period, 7473 pictures contained only water, 5990 contained only one food/beverage and the rest contained multiple food and/or beverages. Unlike in the Western countries, low calo- rie soda is not marketed nor consumed to a great extent in India. The most common beverages consumed in India are tea or coffee with milk and sugar. So, all non-water consumptions were considered caloric containing beverages. The time-stamps of non-water items, numbering 17238, were used to illustrate the eating pattern of all participants. This translates to ~8.5 inges- tion events/day, which is not unusual as people typically drink tea/coffee/beverages and snacks 4 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 Disrupted circadian eating pattern in India Fig 1. A simple camera phone based method to collect human nutrition information. (A) Schematic of the study to collect eating pattern data for 3 weeks. (B) Work flow for data collection, download and annotation. Reminders were sent to the user’s preferred phones at random time of the day. PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 A parsi- monious interpretation of such temporally widespread pattern of caloric intake is that the indi- viduals ate more frequently and/or had large day to day variation in their ingestion events. Upon arranging the eating pattern of all participants according to increasing duration of the day throughout which they spread their caloric intake, the top decile were found to spread their caloric intake throughout the 24 h (Fig 2D). Three subjects (marked as in Fig 2D) logged <5% of all their non-water ingestion events between 10 am-6 pm and in follow-up interview with these participants, they confirmed an erratic lifestyle of being stay-at-home spouses of shift-workers, even though they themselves were not employed as shift workers. As has been shown earlier [26], we considered all non-water events recorded within 15 min of each other as part of one meal. At group level, 25% of all meals were within 1 h 27 min of another meal and the median inter-meal interval was 3 h 15 min. Only 25% of the meals occurred after > 7 h 44 min of fasting (Fig 3A). The post-prandial physiological response marked with an elevation in blood glucose and insulin action for absorption and nutrient utili- zation in anabolic metabolism can last for >90min. So, when ingestion events occur within 90 min of a previous meal, physiology likely sustains at an anabolic state between meals. Average and 95% conﬁdence interval values (in parentheses) are shown. Paired t-test p values for body weight before and after observation period were >0.05 (not signiﬁcant). Resting energy expenditure was calculated for each individual using the modiﬁed Harris Benedict equation [29]. doi:10.1371/journal.pone.0172852.t001 PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 6 / 15 The non-water events were binned into 1 h interval over 24 h and were found to populate a large segment of the 24 h day (Fig 2B) leaving only 6 h between 11 pm and 5 am when the number of events/h were <1% of total events (Fig 2B). As has been found in a comparable study in the US [26], we considered 4 am as the onset of “metabolic day”, so that consumption from 4 am to 3:59:59 am was considered a 24 h day. Results Although the user could potentially review collected data, it did not affect their body weight at baseline and after 3 weeks of data collection as seen in individual body weights (C) and (D) in median body weight (±max/min range, paired t-test p>0.05). d i 10 1371/j l 0172852 001 Fig 1. A simple camera phone based method to collect human nutrition information. (A) Schematic of the study to collect eating pattern data for 3 weeks. (B) Work flow for data collection, download and annotation. Reminders were sent to the user’s preferred phones at random time of the day. Although the user could potentially review collected data, it did not affect their body weight at baseline and after 3 weeks of data collection as seen in individual body weights (C) and (D) in median body weight (±max/min range, paired t-test p>0.05). doi:10.1371/journal.pone.0172852.g001 doi:10.1371/journal.pone.0172852.g001 several times a day. Time-stamps of all non-water ingestion events over 21 days were plotted in a scatter plot for each individual along the vertical axis (Fig 2A). If subjects ate three meals a day and were consistent with their meal times, one would expect to observe the time-stamps cluster into three clusters. However, if the subjects ate more than 3 calorie-containing meals a day or if the timing of three meals was random on different days, the time-stamps would scat- ter throughout the day. In contrast to the self-reported 3 meals/day structure of meals from most of the participants, a breakfast-lunch-dinner temporal pattern was largely absent (Fig 2A, 2C and 2D). 5 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 Disrupted circadian eating pattern in India Table 1. Composition of the cohort, their anthropometric measurements before and after the observation period, and estimated energy intake dur- ing observation period. Male Female All n 35 59 94 Age 36.97 31.51 33.54 (19–58) (19–52) (19–58) Height 169.81 157.45 162.05 (149.86–192) (124.36–176) (124.36–192) Initial BMI (before baseline) 23.9 22.19 22.83 (16.19–32.02) (14.84–34.78) (14.84–34.78) Final BMI (after baseline) 24.07 22.16 22.87 (15.55–31.9) (15.23–34.42) (15.23–34.42) Change in BMI (after baseline) -0.1724 0.02615 -0.0478 Paired t-test P-value p = .036; “t = 2.175 df = 34” ns ns Calculated REE (Kcal) 1623 1321 1422 (1378–1467) (1294–1347) (1540–1706) Average daily caloric intake reported (Kcal) 1347 1320 1329 (1230–1464) (1247–1393) (1268–1390) Percentage of REE intake reported 83.86 100.2 94.71 (76.19–91.52) (94.52–106.0) (89.91–99.51) Average and 95% conﬁdence interval values (in parentheses) are shown. Paired t-test p values for body weight before and after observation period were >0.05 (not signiﬁcant). Resting energy expenditure was calculated for each individual using the modiﬁed Harris Benedict equation [29]. eir anthropometric measurements before and after the observation period, and estimated energy intake dur- Average and 95% conﬁdence interval values (in parentheses) are shown. Paired t-test p values for body weight before and after observation period were >0.05 (not signiﬁcant). Resting energy expenditure was calculated for each individual using the modiﬁed Harris Benedict equation [29]. The non-water events were binned into 1 h interval over 24 h and were found to populate a large segment of the 24 h day (Fig 2B) leaving only 6 h between 11 pm and 5 am when the number of events/h were <1% of total events (Fig 2B). As has been found in a comparable study in the US [26], we considered 4 am as the onset of “metabolic day”, so that consumption from 4 am to 3:59:59 am was considered a 24 h day. Although at individual participant level three clusters of food intake was largely absent, when all ingestion events from the entire cohort was analyzed together, there were three peaks of events at 8 am, 1 pm, and 8 pm (Fig 2B). Each of these three peaks accounted for 8% of all events and the fraction of all events in every hour was >4% in a contiguous period of 16 h spanning 7 am-10 pm (Fig 2B). During this 16 h interval ingestion events also accounted for >5%/h except at 3 pm and 4 pm. Disrupted circadian eating pattern in India Fig 2. Daily eating pattern is highly erratic at individual level. (A) Scatter plot of all non-water events collected from 94 healthy subjects, where each vertical array is color coded for time-stamped ingestion events from each subject over 21 days. The subjects are arranged from left to right with increasing portion of 24 h day where they ingested food or beverages. (B) Frequency distribution of all non-water ingestion events as percentage of total number of events binned in hourly interval shows very low intake at cohort level during midnight-5am and three peaks approximately corresponding to the conventional breakfast, lunch and dinner. However, at individual level, the day to day variations in eating pattern blurs these pattern both among (C) subjects who eat during a shorter time interval or those (D) who spread their ingestion events throughout a 24 h period. Subjects in C and D are subsets of data shown in (A). Each column in Fig 2a,c,d represents data from individual subject. doi:10 1371/journal pone 0172852 g002 Fig 2. Daily eating pattern is highly erratic at individual level. (A) Scatter plot of all non-water events collected from 94 healthy subjects, where each vertical array is color coded for time-stamped ingestion events from each subject over 21 days. The subjects are arranged from left to right with increasing portion of 24 h day where they ingested food or beverages. (B) Frequency distribution of all non-water ingestion events as percentage of total number of events binned in hourly interval shows very low intake at cohort level during midnight-5am and three peaks approximately corresponding to the conventional breakfast, lunch and dinner. However, at individual level, the day to day variations in eating pattern blurs these pattern both among (C) subjects who eat during a shorter time interval or those (D) who spread their ingestion events throughout a 24 h period. Subjects in C and D are subsets of data shown in (A). Each column in Fig 2a,c,d represents data from individual subject. doi:10.1371/journal.pone.0172852.g002 doi:10.1371/journal.pone.0172852.g002 As every food image was annotated for estimated caloric content, we tested the temporal pattern of caloric intake by this cohort. The fraction of total calories consumed in every hourly bin starting after 4 am showed three clear peaks that followed the temporal pattern of a num- ber of reported events (Figs 2B and 3B). PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 Although at individual participant level three clusters of food intake was largely absent, when all ingestion events from the entire cohort was analyzed together, there were three peaks of events at 8 am, 1 pm, and 8 pm (Fig 2B). Each of these three peaks accounted for 8% of all events and the fraction of all events in every hour was >4% in a contiguous period of 16 h spanning 7 am-10 pm (Fig 2B). During this 16 h interval ingestion events also accounted for >5%/h except at 3 pm and 4 pm. A parsi- monious interpretation of such temporally widespread pattern of caloric intake is that the indi- viduals ate more frequently and/or had large day to day variation in their ingestion events. Upon arranging the eating pattern of all participants according to increasing duration of the day throughout which they spread their caloric intake, the top decile were found to spread their caloric intake throughout the 24 h (Fig 2D). Three subjects (marked as in Fig 2D) logged <5% of all their non-water ingestion events between 10 am-6 pm and in follow-up interview with these participants, they confirmed an erratic lifestyle of being stay-at-home spouses of shift-workers, even though they themselves were not employed as shift workers. As has been shown earlier [26], we considered all non-water events recorded within 15 min of each other as part of one meal. At group level, 25% of all meals were within 1 h 27 min of another meal and the median inter-meal interval was 3 h 15 min. Only 25% of the meals occurred after > 7 h 44 min of fasting (Fig 3A). The post-prandial physiological response marked with an elevation in blood glucose and insulin action for absorption and nutrient utili- zation in anabolic metabolism can last for >90min. So, when ingestion events occur within 90 min of a previous meal, physiology likely sustains at an anabolic state between meals. 6 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 Given that we considered 4 am as the start of a meta- bolic day, in the first 8 h or by noon, the cohort consumed only 28.8% or less than 1/3rd of the daily caloric intake. By 6 pm, they consumed 60.1% and in the 4 h of evening between 7 pm-11 pm, they consumed more calories (32.2%) than in the first 8 h of the day (Fig 3B). In summary, there was a clear trend towards larger portion of daily caloric intake being consumed at night. Having observed the surprisingly large variance in the first and last caloric intake and the absence of a clear 3 meals/day eating pattern at individual level (Fig 2A, 2C and 2D), a better PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 7 / 15 Disrupted circadian eating pattern in India Fig 3. Temporal aspects of daily eating pattern. (A) Frequency distribution of time interval between two consecutive non-water ingestion events. Events with <15min intermeal intervals were considered as one me and were not included in this analysis. Extended intermeal intervals of >16 h constituted a small fraction of data points and were likely due to cultural fasting or the subjects forgetting to log their food. (B) Percentage o all caloric intake in hourly bins show three prominent peaks corresponding to conventional times for breakfa lunch and dinner. However, subjects consumed more food at dinner than at breakfast or lunch. (C) The day 0172852 M h 6 2017 8 / Fig 3. Temporal aspects of daily eating pattern. (A) Frequency distribution of time interval between two Fig 3. Temporal aspects of daily eating pattern. (A) Frequency distribution of time interval between two consecutive non-water ingestion events. Events with <15min intermeal intervals were considered as one meal and were not included in this analysis. Extended intermeal intervals of >16 h constituted a small fraction of data points and were likely due to cultural fasting or the subjects forgetting to log their food. (B) Percentage of all caloric intake in hourly bins show three prominent peaks corresponding to conventional times for breakfast, lunch and dinner. However, subjects consumed more food at dinner than at breakfast or lunch. (C) The day to Fig 3. Temporal aspects of daily eating pattern. (A) Frequency distribution of time interval between two consecutive non-water ingestion events. Events with <15min intermeal intervals were considered as one meal and were not included in this analysis. Extended intermeal intervals of >16 h constituted a small fraction of data points and were likely due to cultural fasting or the subjects forgetting to log their food. (B) Percentage of all caloric intake in hourly bins show three prominent peaks corresponding to conventional times for breakfast, lunch and dinner. However, subjects consumed more food at dinner than at breakfast or lunch. (C) The day to PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 8 / 15 Disrupted circadian eating pattern in India day variation in breakfast or dinner time for the cohort (median+25%ile and min-max range) showed no significant difference between days. However, there was larger variation for dinner than for breakfast time. doi:10.1371/journal.pone.0172852.g003 day variation in breakfast or dinner time for the cohort (median+25%ile and min-max range) showed no significant difference between days. However, there was larger variation for dinner than for breakfast time. doi:10.1371/journal.pone.0172852.g003 description of eating pattern would be the daily interval when a person is likely to eat. So we defined the eating duration as the time interval (4 am onwards) that contained 95% (2.5%ile- 97.5%ile) of all intake events during the monitoring period (Fig 4A). The median eating dura- tion was 15.53 h with 25-75th percentile interval being 15.05–17.42 h. The top decile with erratic eating pattern during the monitoring period (Fig 2D) had an eating duration of >20h (Fig 4B). Beginning of the eating interval approximates the likely first caloric intake and the end of the duration corresponds to the last caloric intake of the day. Overall the median break- fast and time of last caloric intake were 6:58 am (6:10 am-7:27 am; 25-75th percentile interval), and 10:45 pm (10:18 pm– 11:58 pm; 25-75th percentile interval). Unlike what was observed in a comparable US study [26], there was no statistically significant difference in the median breakfast or dinner time between weekdays and weekends. The time of first caloric intake neg- atively correlated with the last caloric intake (r2 = 0.2335, p<0.0001) (Fig 5A). Eating duration positively correlated with the time of last caloric intake (r2 = 0.8443) (Fig 5B), while it inversely correlated with the time of breakfast (r2 = 0.6233) (Fig 5C) or with BMI (r2 = 0.017) (Fig 5D). The weak correlation (r2 = 0.025, P = 0.1258) between the eating duration and BMI could be due to the smaller sample size, heterogeneity of participants in terms of gender and age, and the fact that the eating pattern recorded in the monitoring period is a short-term snapshot of a person’s diet-related behaviors. In summary, in contrast to the conventional thinking that humans eat 3 meals a day within approximately 12 h interval, the study found both intra-indi- vidual and inter-individual variations in daily eating pattern. PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 Discussion We report a feasibility study to use camera phones as a method to collect evidence-backed nutrition metadata containing what, when, and how much a person eats in a developing country. Such studies have been done in controlled settings with users meticulously placing a size/color reference next to the meal, taking a picture from a recommended angle and dis- tance [28]. However, in free living conditions, such methods may not be feasible for longitu- dinal studies where daily eating pattern and day to day or weekday-weekend variability is a parameter to be assessed. While smartphones have been ubiquitous with fast internet con- nectivity and relatively cheap data plans, in many developing countries like India, the use of fully functional smartphones as nutrition data collection devices has yet to be established. Therefore, we tested whether relatively cheap camera phone can be used as an alternate device to collect nutrition metadata. We found that in India the participants were as meticu- lous in recording data as participants in the US. The number of data points collected per individual in India is equivalent to what was observed in a recent study using smartphones in the US [26]. Nevertheless, it is difficult to estimate the false negative (when an individual ingests and forgets to record) reporting and we cannot rule out the possibility that some fraction of actual intake was not reported. In a recent study using smartphones, a random push notification and user’s response to the notification was used to estimate false negative reporting to be ~10% [26]. It is reasonable to assume that the actual energy intake for moder- ately active individuals would be higher than the resting energy expenditure (REE). The reported daily caloric intake was only 83.86% of REE for men and 100.2% of REE for women (Table 1), which indicates some caloric intake was not reported. The data collection method appears to have no immediate impact on reducing energy intake that would result in weight loss. Unlike in the US study, where the participants could not review PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 9 / 15 Disrupted circadian eating pattern in India th i f d d t d i th d t ll ti i d th ti i t i thi t d ld il Fig 4. Eating duration for majority of individuals is longer than 12h. PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 Discussion (A) Eating duration of 94 participants arranged with increasing time of last meal. (B) Frequency distribution of eating duration in 30min bins and cumulative percentage shows ~60% of the study cohort eat for 15 h or longer. doi:10.1371/journal.pone.0172852.g004 Fig 4. Eating duration for majority of individuals is longer than 12h. (A) Eating duration of 94 participants arranged with increasing time of last meal. (B) Frequency distribution of eating duration in 30min bins and cumulative percentage shows ~60% of the study cohort eat for 15 h or longer. doi:10.1371/journal.pone.0172852.g004 their food data during the data collection period, the participants in this study could easily review their own nutrition data stored in the local phone. But they were advised not to review the food pictures stored in their phones. We found that there was no acute effect of this poten- tial user nutrition data review on reducing food intake, as there was no significant decline in body weight in this cohort (Fig 1C and 1D). Therefore, this method has potential for adoption in developing and under-developed countries as a data collection method. This is of significant importance as more than half of world’s metabolic disease patients live in these countries and nearly 78 million diabetic patients live in India [24]. Furthermore, frequent malnutrition in their food data during the data collection period, the participants in this study could easily review their own nutrition data stored in the local phone. But they were advised not to review the food pictures stored in their phones. We found that there was no acute effect of this poten- tial user nutrition data review on reducing food intake, as there was no significant decline in body weight in this cohort (Fig 1C and 1D). Therefore, this method has potential for adoption in developing and under-developed countries as a data collection method. This is of significant importance as more than half of world’s metabolic disease patients live in these countries and nearly 78 million diabetic patients live in India [24]. Furthermore, frequent malnutrition in 10 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 Disrupted circadian eating pattern in India Fig 5. Inter-relation of various daily eating parameters. The timing of first meal inversely correlates with (A) the timing of last meal and with (B) duration. (C) The time of last meal strongly correlates with the eating duration. However, the eating duration does not show a simple correlation with BMI. Fig 5. Inter-relation of various daily eating parameters. The timing of first meal inversely correlates with (A) the timing of last meal and with (B) duration. (C) The time of last meal strongly correlates with the eating duration. However, the eating duration does not show a simple correlation with BMI. doi:10.1371/journal.pone.0172852.g005 some of these countries is also a risk for stunted growth and increased disease susceptibility. As both nutrition and eating pattern are increasingly recognized as modifiable factors for alleviat- ing these disease risks, methods to collect nutrition data in these countries is urgently needed. Furthermore, people in India and in many developing countries eat a diverse range of home-cooked food with very little similarity to the standardized recipes or ready to eat pre- packaged food items consumed in the Western countries. Therefore, it is often difficult to find nutrition values for food consumed in developing countries. By adopting an image-based data collection system, we could overcome the barrier to food data collection which typically requires matching food data to a standard library and assessing portion size. We acknowledge the lack of accurate nutrition values for some items may introduce inaccuracy, but it is also a powerful survey tool to collect the diversity of food items consumed, so that effort can be focused on characterizing the most frequently eaten food. We found many similarities and differences in the eating pattern of comparable sized non- shiftwork cohorts (of equivalent age and gender composition) in the US and in India. In both countries we found people don’t restrict their daily nutrition intake to breakfast, lunch and dinner. Rather, in addition to these three meals, there is frequent caloric intake throughout the 11 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 Disrupted circadian eating pattern in India 24 h day. In the US cohort, parallel measurement of sleep by actigraphy devices showed that reduced sleep correlated with frequent eating throughout the wakeful hours. Although we did not objectively measure sleep, we also found erratic eating pattern covered a large portion of the 24 h day. In the US cohort >50% adults spread their caloric intake over 15 h or longer, and in the present study a similar trend was found. Surprisingly, although one of our exclusion criteria was shift-work, we found nearly 10% of the cohorts had an extremely erratic lifestyle similar to that of shift-work; their eating time was spread well over 20 h during the 3 weeks observation period. Upon close examination we found they included spouses of shift-workers and people employed with flexible hours. This is of specific health interest as these two sub- groups are generally not included in national surveys on shift-work. While shift-work is known to be a risk factor for several non-infectious chronic diseases including cancer, the extent of circadian disruptions among flexible workers and of family members of shift or flexi- ble workers and their disease risks is rarely addressed. As the developing countries like India have a sizeable portion of shift-workers and flexible hour workers, erratic lifestyle in these unaccounted subgroups can be a hidden population risk for NCDs. We also found a nocturnal shift in eating pattern both in the US and in India. While among the US cohort, <25% calories were consumed by noon and people consumed more than 1/3rd of their daily caloric intake after 6 pm, in our study people consumed ~28% of their calories by noon and ~40% food after 6 pm. Specifically, between 7–11 pm, the cohort ate more than 30% of daily caloric intake (Fig 3B). Importantly, the diversity of food consumed during day or night was distinct; high glycemic foods such as cooked rice, ice cream, Indian sweets were con- sumed at night (Fig 6). This nocturnal shift of caloric intake combined with the recent finding Fig 6. Daily consumption pattern of different food shows a unique pattern. Percentage of pictures containing a given food item in hourly bins are shown. Data for the presumptive daytime from 6am to 6pm is shown in cyan blue. doi:10.1371/journal.pone.0172852.g006 Fig 6. Daily consumption pattern of different food shows a unique pattern. Percentage of pictures containing a given food item in hourly bins are shown. Data for the presumptive daytime from 6am to 6pm is shown in cyan blue. doi:10.1371/journal.pone.0172852.g006 PLOS ONE \| DOI:10.1371/journal.pone.0172852 March 6, 2017 12 / 15 Disrupted circadian eating pattern in India that melatonin can suppress post-prandial insulin release [30], raises the hypothesis that the nocturnal food intake might contribute to rising diabetes trend in both countries [24]. There was also a remarkable difference in weekday and weekend eating pattern in two populations. While in the US, the cultural normal is at least 2 days of weekend off days, in India 6 working days is more of a norm in non-farm employment. Accordingly, in the Indian cohort we did not see any significant shift in breakfast time during the weekend as was reported in the US cohort. They showed a slight delay in time of dinner consumption on Sunday, which was not statistically significant (Fig 3C). Despite the observed prevalence of erratic eating pattern in both countries, there was no simple correlation between eating duration and BMI in either study. Although increased BMI is considered a risk for metabolic diseases, it does not always correlate with diabetes [31] or cardiovascular diseases [32]. Even rodents that are fed a high fructose or high sucrose diet, may eat erratically and develop type2 diabetes without gaining excessive body weight. Glucose intolerance and metabolic diseases in these normal weight mice is prevented by a short daily eating duration [8]. Similarly, a review of time-restricted feeding in both rodents and humans has shown metabolic health benefits may arise from TRF in controlled studies without signifi- cant reduction in body weight [33]. Therefore, future studies with larger cohorts and broader evaluation of disease states is necessary to assess the contribution of eating pattern along with diet type to the risk for metabolic diseases. 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Cancer epidemiology, biomarkers & prevention: a publication of the American Association for Cancer Research, cosponsored by the Ameri- can Society of Preventive Oncology. 2015; 24(5):783–9. Epub 2015/04/22. References Worldwide trends in diabetes since 1980: a pooled analysis of 751 population- based studies with 4.4 million participants. Lancet. 2016; 387(10027):1513–30. Epub 2016/04/12. doi: 10.1016/S0140-6736(16)00618-8 PMID: 27061677 25. Shetty PS. Nutrition transition in India. Public Health Nutr. 2002; 5(1A):175–82. Epub 2002/05/25. doi: 10.1079/PHN2001291 PMID: 12027282 26. Gill S, Panda S. A Smartphone App Reveals Erratic Diurnal Eating Patterns in Humans that Can Be Modulated for Health Benefits. Cell metabolism. 2015; 22(5):789–98. Epub 2015/09/29. doi: 10.1016/j. cmet.2015.09.005 PMID: 26411343 27. Six BL, Schap TE, Kerr DA, Boushey CJ. Evaluation of the Food and Nutrient Database for Dietary Studies for use with a mobile telephone food record. 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https://openalex.org/W4383863831	https://zenodo.org/records/8133174/files/67-70.pdf	Latin	null	THE WIDE USAGE OF VISCOUS SEISMIC PROTECTION IN IMPROVING THE RESISTANCE OF BUILDINGS FOR EARTHQUAKE	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,814	THE WIDE USAGE OF VISCOUS SEISMIC PROTECTION IN IMPROVING THE RESISTANCE OF BUILDINGS FOR EARTHQUAKE PhD student, Kamalov Bobur, Prof. Mirolimov Mirrakhim Tashkent University of Architecture and Civil Engineering, bobur514@gmail.com Annotatsiya: Ushbu maqolada hozirgi kundagi dolzarb muammolardan biri bo’lgan seysmik faol hududlardagi binolarning zilzilabardoshliligini oshirishda qo’llaniladigan zilzilaviy himoya vositalari, ularning turlari va qo’llanilishi haqida so’z boradi. Bundan tashqari, ko’p qavatli binolarda qavatlararo zilzilaviy himoya vositalaridan foydalanish orqali ularning zilzilabardoshliligini takomillashtirish haqida so’z yuritilgan. Kalit so’zlar: zilzilaviy himoya vositalari, seysmik demfer, viscus demfer, metal chevron tirgak, dioganal tirgak, ustun tizimi, vaqtinchalik ta’sir, poydevor himoya tizimi. Аннотатция: В данной статье рассматриваются средства сейсмозащиты, их виды и области применения, которые используются для повышения сейсмостойкости зданий в сейсмоактивных районах, что является одной из актуальных проблем. Кроме того, говорят о повышении сейсмостойкости многоэтажных зданий за счет применения межэтажных устройств сейсмозащиты. Ключевые слова: устройства защиты от землетрясений, сейсмический демпфер, демпфер вязкости, металлический шевронный раскос, диагональный раскос, колонная система, временный эффект, система защиты фундамента. Abstract: In this article deals with earthquake protection tools, their types and applications, which are used to increase the earthquake resistance of buildings in seismically active areas, which is one of the current problems. In addition, there is talk about improving the earthquake resistance of multi- story buildings by using inter-floor seismic protection devices. y g y g p Key words: earthquake protection means, seismic damper, fluid viscous damper, steel chevron brace, diagonal bracing, outrigger system, transient shock, base-isolation system. Nowadays, in the construction practice of the developed countries of the world, the issues of ensuring the seismic stability of multi-story buildings by using devices that extinguish the energy of earthquake force occupy a leading place. Some progress has been made in this regard, and one of the important tasks is the development of structural solutions and anti-earthquake measures to ensure stability and earthquake resistance in the construction and reconstruction of multi-story buildings, as well as the improvement of theoretical calculation methods. At the same time, the disastrous consequences of a series of earthquakes in different countries, in which many destructions of buildings built in different periods reveal a number of problems that require urgent solutions. Taking into account the scale of damages and victims, in seismically active areas, including Uzbekistan, it is of particular importance to ensure the seismic safety of buildings and structures, as well as their anti-seismic strengthening, the use of earthquake protection means and economically appropriate spending of material and labor resources. INNOVATIVE TECHNOLOGIES IN CONSTRUCTION 2023/1, PART 1 IVE TECHNOLOGIES IN CONSTRUCTION 2023/1, PART PART 1 INNOVATIVE TECHNOLOGIES IN CONSTRUCTION 2023/1, INNOVATIVE TECHNOLOGIES IN CONSTRUCTION 2023/1, NNOVATIVE TECHNOLOGIES IN CONSTRUCTION 2023/1, and vibration disturbances. Conventional approach would dictate that the structure must passively attenuate or dissipate the effects of transient inputs through a combination of strength, flexibility, deformability and energy absorption. The level of damping in a conventional structure is very low, and hence the amount of energy dissipated during transient disturbances is also very low. During strong motions, such as earthquakes, conventional structures usually deform well beyond their elastic limits, and remain intact only due to their ability to inelastically deform. Therefore, most of the energy dissipated is absorbed by the structure itself through localized damage. p y g g The concept of added-on dampers within a structure assumes that some of the energy input to the structure from a transient will be absorbed, not by the structure itself, but rather by supplemental damping elements. An idealized supplemental damper would be of a form such that the force being produced by the damper is of such a magnitude and occurs at such a time that the damper forces do not increase overall stress in the structure. Properly implemented, an ideal damper should be able to simultaneously reduce both stress and deflection in the structure. y Many methods exist to implement distributed damping in a structure, the typical underlying concept is to connect the moving masses (floor levels) with dampers so that as they move or deflect relative to one-another in a shearing-type motion, the dampers capture this motion and resist in both tension and compression directions with an opposing force. This concept works well in typical moment frame, shear wall or braced frame office or residential type buildings, and can be applied to short, medium and tall structures. It is noted that all of these systems are passive, meaning that no external power is needed to make the dampers function. The dampers simply react at any time they are deflected. THE WIDE USAGE OF VISCOUS SEISMIC PROTECTION IN IMPROVING THE RESISTANCE OF BUILDINGS FOR EARTHQUAKE In world’s experience, great importance is attached to the creation of devices designed to increase the seismic strength of existing and newly constructed buildings, to reduce the impact of seismic forces on buildings and possible damage caused by them. In this regard, including the use of seismic force damping devices in buildings, improving the methods of calculating the impact of earthquakes on buildings, and installing inter-floor seismic protection devices (dampers) in existing buildings, calculating the effect of seismic forces, calculating with the help of a computer program, earthquake energy that reduces the energy of earthquake force appearing in buildings. It is one of the important tasks to carry out targeted scientific research in directions such as the development of a design method using protective means (damper). Damping is one of many different methods that have been proposed for allowing a structure to achieve optimal performance when it is subjected to seismic, windstorm or other types of transient shock 64 INTERNATIONAL SCIENTIFIC CONFERENCE rg/index.php/goldenbrain/issue/view/80 INTERNATIONAL SCIENTIFIC CONFERENCE https://researchedu.org/index.php/goldenbrain/issue/view/80 INTERNATIONAL SCIENTIFI Figure 1. Dampers in Chevron Braced Frames. Chevron frames are depicted in Figure 1 In this configuration, the dampers are placed horizontally, and connected to a frame (chevron) that is intended to be near rigid with the floor it is connected to. The advantage with this direct damping orientation is that the horizontal flexibility of the structure injects this full movement directly into the horizontal orientation of the damper. However, a small amount of motion can be lost due to the constraints of the attainable stiffness of an economical chevron frame. 65 65 https://researchedu.org/index.php/goldenbrain/issue/view/80 INTERNATIONAL SCIENTIFIC CONFERENCE https://researchedu.org/index.php/goldenbrain/issue/view/80 INTERNATIONAL SCIENTIFIC CONFERENCE INNOVATIVE TECHNOLOGIES IN CONSTRUCTION 2023/1, PART 1 INNOVATIVE TECHNOLOGIES IN CONSTRUCTION 2023/1, PART 1 Figure 2. Dampers in Diagonal Braced Frames. Figure 2. Dampers in Diagonal Braced Frames. Dampers in Diagonal bracing schemes are depicted in Figure 2 In this orientation, the horizontal movement of the structure only allows an angular component of the full deflection to go into the damper, but thence takes this motion directly to the next floor level, straight through a strong tension/compression member. Often this diagonal bracing scheme is considered the most basic, or simplistic method to apply distributed damping in a structure. Additionally, an outrigger solution to apply damping to taller, more slender building systems can be used where it is determined that the gross motion of the structure does not fall into the traditional shearing-type movement pattern, but exhibits more of an overall tension/compression on the opposing outer columns of the building. Often outrigger damping can be accomplished by creating a rigid level near the top of a building that moves with the core and connecting dampers between the rigid level and the outer columns of the building. This useful system is shown in Figures 3 and 4. Figure 3. Dampers in Outrigger Systems (a). Figure 4. Dampers in Outrigger Systems (b). Dampers can also be distributed in base isolation systems, as depicted in Figure 5, where the damper is used to augment the vertically supporting isolators, and most often provide viscous (velocity dependent) damping to significantly improve the performance and usefulness of the isolators. Figure 3. Dampers in Outrigger Systems (a). Figure 4. Dampers in Outrigger Systems (b). Figure 4. Dampers in Outrigger Systems (b). g p gg y ( ) Dampers can also be distributed in base isolation systems, as depicted in Figure 5, where the damper is used to augment the vertically supporting isolators, and most often provide viscous (velocity dependent) damping to significantly improve the performance and usefulness of the isolators. g p gg y Dampers can also be distributed in base isolation systems, as depicted in Figure 5, where the damper is used to augment the vertically supporting isolators, and most often provide viscous (velocity dependent) damping to significantly improve the performance and usefulness of the isolators. 66 66 INNOVATIVE TECHNOLOGIES IN CONSTRUCTION 2023/1, PART 1 INNOVATIVE TECHNOLOGIES IN CONSTRUCTION 2023/1, PART 1 Figure 5. Dampers in Base-Isolation Systems. Currently, most of the scientific research carried out in our country is focused only on the study of the connection between the foundation of the building and the ground, and they are mainly devoted to the improvement of the model of the ground soil, issues of interaction with the foundation under the influence of external forces, issues of the building-foundation-ground system and their solutions. But in the world experience, we can see that the practice of using inter-floor seismic protection devices is also widely used in multi-story buildings. But taking into account the fact that the researches conducted in our country are very few, the need to study this type of earthquake protection means in the field of defense has been recognized by the scientists of the field. 66 https://researchedu.org/index.php/goldenbrain/issue/view/80 INTERNATIONAL SCIENTIFIC CONFERENCE https://researchedu.org/index.php/goldenbrain/issue/view/80 INTERNATIONAL SCIENTIFIC CONFERENCE REFERENCES: 1. Chandnani D., Joshi R., Trivedi K. Characteristics & applications of different types of dampers as seismic energy dissipater //Int J Comput Sci Netw. – 2016. – Т. 5. – №. 2. – С. 369-372. 1. Chandnani D., Joshi R., Trivedi K. Characteristics & applications of different types of dampers as seismic energy dissipater //Int J Comput Sci Netw. – 2016. – Т. 5. – №. 2. – С. 369-372. p gy p p 2. AISC (American Institute of Steel Construction). (2016a) Specification for Structural Steel Buildings Standard ANSI/AISC 360-16. AISC, Chicago, IL p gy p p 2. AISC (American Institute of Steel Construction). (2016a) Specification for Structural Steel Buildings Standard ANSI/AISC 360-16. AISC, Chicago, IL g g 3. Kamalov, Bobur. "SIZE-PLANNING AND URBAN PLANNING OF BUILDINGS IN SEISMICALLY ACTIVE REGIONS REQUIREMENTS FOR SOLUTIONS." Results of National Scientific Research International Journal 1.6 (2022): 556-562.\ 3. Kamalov, Bobur. "SIZE-PLANNING AND URBAN PLANNING OF BUILDINGS IN SEISMICALLY ACTIVE REGIONS REQUIREMENTS FOR SOLUTIONS." Results of National Scientific Research International Journal 1.6 (2022): 556-562.\ f 4. Principles of Regulation of Thermal Protection of Enclosing Structures and Their Impact on the Energy Efficiency Of Buildings MM Miralimov - Design Engineering, 2021 4. Principles of Regulation of Thermal Protection of Enclosing Structures and Their Impact on the Energy Efficiency Of Buildings MM Miralimov - Design Engineering, 2021 5. Modern methods of increasing energy efficiency of buildings in the Republic of Uzbekistan at the design stage S Sadriddin, MM Mirmakhmutovich, MS Makhmudovich…- International Journal of Scientific and Technology…, 2019 67 https://researchedu.org/index.php/goldenbrain/issue/view/80 INTERNATIONAL SCIENTIFIC CONFERENCE
https://openalex.org/W2985448638	https://repositorio.iscte-iul.pt/bitstream/10071/20059/1/2019_Discriminative%20characteristics%20of%20marginalized%20NPS%20users.pdf	English	null	Discriminative Characteristics of Marginalised Novel Psychoactive Users: a Transnational Study	International journal of mental health and addiction	2,019	cc-by	10,180	International Journal of Mental Health and Addiction https://doi.org/10.1007/s11469-019-00128-8 International Journal of Mental Health and Addiction https://doi.org/10.1007/s11469-019-00128-8 ORIGINAL ARTICLE * Zsolt Demetrovics demetrovics.zsolt@ppk.elte.hu Katalin Felvinczi1 & Annemieke Benschop2 & Róbert Urbán1 & Marie Claire Van Hout3 & Katarzyna Dąbrowska4 & Evelyn Hearne5 & Susana Henriques6 & Zsuzsa Kaló1 & Gerrit Kamphausen7 & Joana Paula Silva6 & Łukasz Wieczorek4 & Bernd Werse7 & Michal Bujalski4 & Zsolt Demetrovics1 & Dirk Korf2 Katalin Felvinczi1 & Annemieke Benschop2 & Róbert Urbán1 & Marie Claire Van Hout3 & Katarzyna Dąbrowska4 & Evelyn Hearne5 & Susana Henriques6 & Zsuzsa Kaló1 & Gerrit Kamphausen7 & Joana Paula Silva6 & Łukasz Wieczorek4 & Bernd Werse7 & Michal Bujalski4 & Zsolt Demetrovics1 & Dirk Korf2 Katalin Felvinczi1 & Annemieke Benschop2 & Róbert Urbán1 & Marie Claire Van Hout3 & Katarzyna Dąbrowska4 & Evelyn Hearne5 & Susana Henriques6 & Zsuzsa Kaló1 & Gerrit Kamphausen7 & Joana Paula Silva6 & Łukasz Wieczorek4 & Bernd Werse7 & Michal Bujalski4 & Zsolt Demetrovics1 & Dirk Korf2 # The Author(s) 2019 Extended author information available on the last page of the article * Zsolt Demetrovics demetrovics.zsolt@ppk.elte.hu Extended author information available on the last page of the article * Zsolt Demetrovics Abstract New psychoactive substances (NPS) continue to be considered as a major public health concern in many European countries. The study was implemented within the framework of a transnational project of six European countries (Germany, Hungary, Ireland, Netherlands, Poland, Portugal). Our aim here is to report on the distinct and differentiating characteristics of marginalised NPS users. Three subgroups of a total of 3023 adult NPS users (socially marginalised, night life, online community) were examined regarding their socio- demographic characteristics, substance use, and external motives towards NPS use. Poland and Hungary reported higher rates of NPS use in comparison to traditional controlled drugs. The external/contextual motives did not play a central role in the background of NPS use, the least important motives were alleged legality and non-detectability of these substances. Marginalised (defined as those accessing low threshold harm reduction services) users’ substance use patterns are different from the other two groups in terms of showing more intense and riskier drug use. The most important variables which contributed to be categorised as a marginalised NPS user were lower level education, being older, having an unfavourable labour market position and using drugs intravenously. Contextual motives did not play a decisive role in being categorised as a marginalised user when drug use pattern was controlled. These identified discriminative features of marginalised drug users should inform policy makers to develop and implement tailor-made interventions targeting this user group to successfully tackle the elevated public health concerns associated with NPS use. Keywords NPS use . User groups . Contextual motivation . Public health concern . Drug policy Extended author information available on the last page of the article International Journal of Mental Health and Addiction New psychoactive substances (NPS) are a major public health and policy concern in several parts of the world. NPS are defined as “synthetic or naturally occurring sub- stances that are not controlled under international law, and often produced with the intention of mimicking the effects of controlled drugs” (EMCDDA 2014, p.27). In general, NPS represent a multitude of synthetic and natural compounds marketed as alternatives to conventional illicit drugs (i.e. cannabis, heroin, cocaine, speed, ecstasy), the most popular being cathinone derivatives (i.e. mephedrone, 4-MEC, MDPV), other amphetamine-type substances, and synthetic cannabinoids, with the phenomenon ob- served since 2007 (Caudevilla 2016; EMCDDA 2016). Abstract The legislative procedures related to NPS are very much varied in the different EU member states, covering, and initiating a wide range of measures to tackle the problem. Neicun et al. (2019) concluded that in most countries the general psychoactive substance-related legislation is in place to establish a temporary or permanent ban on new psychoactive substances; the actual legal consequences related to personal use (possession, cultivation, purchasing) of these substances is depending on the general rigour or strictness of the penal code of the given country. The number of compounds labelled as NPS and observed through the Early Warning System (EWS) of the EMCDDA has grown from 13 (in 2004) to 650 (in 2016). General population-based prevalence estimates are rare and inconsistent with regard to trend monitoring of NPS use (Korf et al. 2019). According to the Eurobarometer (telephone surveys on drugs among young people aged 15–24 years in Europe), lifetime prevalence of NPS use increased from 5 to 8% between 2011 and 2014 (Eurobarometer 2011 and 2014). The most recent ESPAD study in 2015 reported 4% life time and 3% last year prevalence of NPS use among the 15–16-year-old European school population (ESPAD group 2016), with large differences among the different countries. In England and Wales, the 2010/2011 British Crime Survey identified a 0.6% lifetime ketamine use and 1.4% lifetime mephedrone use (Smith and Flatley 2011). The 2015 National Survey on Addiction Problems in Hungary found a 1.9%, 1.3% and 0.6% lifetime prevalence rate for synthetic cannabinoids, designer stimulants and mephedrone, respectively (Paksi et al. 2016). A national prevalence estimates among high-risk drug users in Belgium showed that the average reported use was 26%, in case of synthetic cannabinoid receptor agonists (SCRA) 19%, whilst for mephedrone 12.5% (European Monitoring Centre for Drugs and Drug Addiction 2017a). In the Czech Republic, one-third of the high-risk users had experience (LTP) with NPS (European Monitoring Centre for Drugs and Drug Addiction 2017b). Surveys targeting special (vulnerable) populations of NPS use are rare and struggle with methodological challenges; the available data suggest that these groups are more prone to NPS use, and show higher prevalence rates across all NPS types (MacLeod et al. 2016). Studies exploring the knowledge related to NPS in different countries and age groups revealed that the relevant knowledge is unevenly distributed among young respondents, with young people from rural areas showing less knowledge. Abstract These studies also suggested that there is a strong association between NPS use and the risk of binge drinking (Martinotti et al., 2015). The NPS situation has become even more challenging due to the emerging synthetic opioid crisis, which seems to be less alerting in most European countries than in the USA (Schifano et al. 2019); however, opiate use and related death rates are also increasing in the EU countries. The synthetic opioids— mostly fentanyl—play a significant role in those seeking specialised treatment in Europe (EMCDDA 2018). International Journal of Mental Health and Addiction Little knowledge is available about the specific motivations for NPS use and more in-depth research is needed to better understand these reasons (Moore et al. 2013; Korf et al. 2019). There are studies suggesting that the main motivations towards NPS use are curiosity, the enjoyable effects, or enhancement of social situations, similar to other conventional/classical drugs (Corazza et al. 2014; Measham et al. 2010; Werse and Morgenstern 2012; Winstock et al. 2015). Studies which have assessed the motivational background of NPS use have applied the four-factor motivation scale of Cooper (1994) which is used to measure alcohol consumption motivations, and was later further developed to measure cannabis (Simons et al. 1998) and amphetamine use related motivations (Thurn et al. 2017). These motivation scales are based on the Cox and Klinger’s Motivational Model (Cox and Klinger 1988) and concentrate on internal motivations such as coping, curiosity, conformity and enhancement. The internal mo- tivation of different user groups and the psychometric properties of a new motivation scale appropriate for cross national study of NPS motivation were also analysed based on the dataset of this research. Apart from identifying the psychometric properties and usability of the scale in a transnational setting and in a special population, it was found that marginalised users showed a higher endorsement in coping motives. The reported results also suggest that marginalisation can be considered as a cross-cultural factor behind instrumental substance use (Benschop et al. 2019 submitted). Studies assessing contextual and/or external (pragmatic) motivations towards NPS use, revealed that temporary legal status, easy availability, low price, high perceived or expected quality/purity of these substances might play an important role in predicting use of NPS (e.g., Werse and Morgenstern 2012; Barratt et al. 2013; Soussan et al. 2018; Sutherland et al., 2017). The aim of the present article is (i) to present some short term (last month, last year) frequencies of substance use; (ii) to identify the role of external/contextual motivation factors in NPS use of different user groups; and most importantly (iii) to identify a set of variables (discriminating features) which might contribute to being categorised as marginalised NPS user. Sample In most cases the socially marginalised users were recruited from the capital of the participat- ing country. The German marginalised sample consisted of, almost exclusively, persons from the Munich scene, which is probably the only urban hard drugs users’ setting with substantial (and mostly stimulant) NPS use in Germany. 2. Users in nightlife are recreational drug users who frequent clubs, raves and/or festi- vals. They were mainly recruited face-to-face on-site at clubs, raves and festivals. Users in night life mostly self-completed either a pen-and-paper or online question- naire to which they were referred by a flyer containing a link and an individual code; 3. Users in online communities are users who are very active on the internet, and actively participate in drug forums. They were recruited by posting messages on drug-related social media and internet forums (i.e. eve-rave.ch/forum; www.legal-high-inhaltsstoffe.de; www.daath.hu; www.drugs-forum.com; www.legalhighsforum.com). Users in online communities were only given access to the online questionnaire. Sample The work was undertaken as part of a large transnational interdisciplinary research project (“New Psychoactive Substances: transnational project on different user groups, user characteristics, extent and patterns of use, market dynamics, and best practices in prevention” [NPS-t]) spanning six European countries (Germany, Hungary, Ireland, Netherlands, Poland and Portugal). The data collection was carried out on a conve- nience sample of three different user groups supposed to have diverging consumption habits, motivations and socioeconomic background in both countries. Despite the high number of respondents (N = 3023), none of our findings can be generalised for the countries involved or for Europe as such. The survey was conducted between April and November 2016. The different user groups reached have an uneven representation in the sample; marginalised users proved to be hard to reach, mostly in Germany, the Netherlands and Portugal. International Journal of Mental Health and Addiction Three inclusion criteria were set as (1) recent NPS use (at least once in the past 12 months); (2) being resident of one of the participating countries; and (3) an age of 18 years or older. To reach the most heterogeneous sample of NPS users as possible, three subgroups were aimed to examine (for more details see Van Hout et al. 2018; Korf et al. 2019): 1. Socially marginalised users were defined by their (known and supposed) drug using habits on one hand and by their availability (low threshold, harm reduction services) and also by the venue of substance use, on the other. Marginalised users were reported as using opioids, (crack) cocaine and or (meth) amphetamine—often and/ or frequently intravenously or through smoking. They were recruited and interviewed face-to-face by trained fieldworkers or care professionals in the street, or through care and treatment facilities (e.g., drug services, shelters) and through snowball sampling; In most cases the socially marginalised users were recruited from the capital of the participat- ing country. The German marginalised sample consisted of, almost exclusively, persons from the Munich scene, which is probably the only urban hard drugs users’ setting with substantial (and mostly stimulant) NPS use in Germany. In most cases the socially marginalised users were recruited from the capital of the participat- ing country. The German marginalised sample consisted of, almost exclusively, persons from the Munich scene, which is probably the only urban hard drugs users’ setting with substantial (and mostly stimulant) NPS use in Germany. 1 The current paper is not presenting findings on all motivation related items; it is focusing exclusively on the newly added 6 items. The motivation scale without the newly added items (its psychometric properties and cross- cultural usability) was analysed in another article (Benschop et al. 2019, submitted). Measures The questionnaire contained items on demographics, routes of administration of sub- stances, motives and frequencies of NPS and other drug use, social and health problems related to it, modes of obtaining NPS, and possible perceived ways of tackling NPS problems. Seven categories of NPS were initially included in the study based on their epidemiological and clinical relevance: i.e., ‘herbal blends (e.g., ‘Spice’)’; ‘synthetic cannabinoids (obtained pure)’; ‘branded stimulants (e.g., “bath salts”)’; ‘stimulants/ empathogens/nootropics (obtained pure, e.g., mephedrone, MDPV, a-PVP)’; ‘psyche- delics (e.g., NBOMe-x, 2C-x’); ‘dissociatives (e.g., methoxetamine)’; and ‘other’. However, survey responses indicated that participants were generally unable to either properly categorise the NPS they had used or differentiate between certain categories (i.e. between herbal blends and synthetic cannabinoids obtained pure and similarly between branded stimulants and stimulants obtained pure) (Van Hout et al. 2018; Korf et al. 2019). Therefore, these uncertain categories were merged into the following categories: synthetic cannabinoids (including herbal blends names/ingredients); NPS International Journal of Mental Health and Addiction stimulants (including bath salts and ingredients). In case of NPS dissociatives and NPS psychedelics no merging of the categories was necessary. These NPS substance cate- gories were used during the process of statistical analysis and throughout the presen- tation of the results. The questionnaire also contained motivations related items1 to investigate what were the most important reasons to use NPS. These questions had to be answered on a 5-point Likert scale (1 = very unimportant; 5 = very important). The items of the questionnaire were based on the Marijuana Motivation Scale (MMM, Simons et al. 1998) and its further developed amended and psychometrically tested version (Benschop et al. 2015). Six items reflecting external/contextual motives unique for NPS use and not covered by the revised MMM were added: poor quality of other (traditional) drugs, price, alleged legality, expecting different or new experiences (regarding drug effects), non-detectability and low availability of other (traditional) drugs. Statistical Analysis During the statistical analysis, SPSS 25.0 programme was used. Descriptive statistics are presented on substance use frequencies and on external/contextual motives. Prin- cipal component analysis was carried out on the external motivation items to assess if these items construct a coherent scale. Variance analysis was carried out to assess the differences regarding external/contextual motives in different user groups and/or coun- tries, and finally, binary logistic regression models were developed to assess the possible factors contributing to be categorised as a marginalised user. 2 We deliberately chose last month frequency of use to present the differences among user groups and countries. The inclusion criteria for all the respondents were NPS use at least once in the preceding 12 months; consequently, the measured last year frequency of use was considerably high in all user groups. According to the recently introduced new “High Risk Drug Use Indicator” by the EMCDDA (2013) this “…..indicator area focuses on “High-risk drug use”, “recurrent drug use that is causing actual harms (negative consequences) to the person (including dependence, but also other health, psychological or social problems) or is placing the person at a high probability/risk of suffering such harms. High risk drug use is measured as the use of psychoactive substances by high risk pattern (e.g. intensively) and/or by high risk routes of administration….” EMCDDA, 2013, p.3.). As the main objective of this paper is to describe the discriminating features of marginalised users we wanted to capture the most visible elements of high-risk drug use, among them short-term (last month) use (as recurrent and intensified drug use) and the most harmful modes of administering substances (injecting). Descriptive Statistics—Sample Characteristics The number of respondents reached in the different countries and user groups varied significantly across the participating countries (see Table 1). The highest number of marginalised users was recruited in Hungary, whilst the highest number of online users was reached in the Netherlands. The most balanced sample (in number of respondents belonging to the different user groups) was achieved in Poland. As there was no way to carry out a probabilistic and random sample selection in these special populations none of the samples were representative of the participating countries. The sample was very much varied as far as the socio-demographic characteristics of the respondents are concerned. Most of them were male, and lived in larger cities, the most noticeable differences can be seen in the level of education (the lowest level education was observed among marginalised users and the highest in the on-line community) and in the employment conditions; most marginalised users were unemployed whilst night life and online community representatives were either students or full-time workers (for further details see Korf et al. 2019). International Journal of Mental Health and Addiction Table 1 Composition of the sample according to countries and user groups (number and percentage of respondents) Marginalised Nightlife Online Total Germany 23 (32.47%) 98 (14.78%) 542 (81.75%) 663 Hungary 101 (37%) 15 (5.51%) 156 (57.35%) 272 Ireland 48 (77.42%) 3 (4.84%) 11(17.74%) 62 The Netherlands 1(0.08%) 189 (17.04%) 1000 (90.17%) 1190 Poland 86 (17.43%) 172 (28.86%) 338 (56.71%) 596 Portugal 7 (2.92%) 170 (70.83%) 63 (26.25%) 240 TOTAL 266 (8.80%) 647 (21.40%) 2.110 (69.80%) 3.023 Short Term (Last Month2) Frequency of Substance Use in Different User Groups According to last month frequency of substance use, all user groups, regardless of their country of residence, are also using controlled substances; apart from Hungary and Poland, these frequencies are the highest in all user groups. Due to the fact that the different user groups were reached in an uneven proportion in the different countries, the following results would describe the short-term frequencies of substance use in different user groups. To be able to show the problematic nature of substance use, frequencies of intravenous drug use were also included into the analysis. Users in Online Community In the online NPS user group, after the most prevalent controlled substances, NPS stimulants rank second and NPS psychedelics the third place (43% and 17%, respective- ly). Synthetic cannabinoids are the fourth most popular NPS substance used in this group during the month preceding the data collection. Last month frequency of intravenous drug use is low, 1.20% in this user group (Table 2). Short-term frequencies of substance use show that NPS users do not form an independent group of substance users, traditional controlled substances still play a significant role in the poly-substance use patterns of NPS users. The last month frequencies of substance use, especially when we compare them with data stemming from general adult population surveys of the last 3 years, show that recent NPS users in the current sample are very intensive substance users. The frequencies of substance use estimates suggest that the most popular new psychoactive substances (synthetic cannabinoids, NPS stimulants) are 2–3 times more fre- quently used monthly among recent NPS consumers (the frequencies are between 33.80 and 45.50%) than any controlled substances in the general population of the same countries. For the most popular substances (cannabis and MDMA) the highest prevalence was 15.70% and 9.20% respectively (EMCDDA 2018). However, not just NPS are used heavily among recent NPS users, but the last month frequencies of substance use for all controlled substances is four to five times higher among recent NPS users than in the general population, as it is presented in the European Drug Report (EMCDDA 2018). Table 2 Last month frequencies of substance use (%) according to user groups User groups Marginalised N = 266 Night life N = 647 Internet N = 2110 χ2 (p) Substance categories Synthetic cannabinoids, N(%) 90 (33.8)a 78 (12.1)b 243 (11.5)b 101.8* NPS stimulants N(%) 121 (45.50)a 162 (25.00)b 914 (43.30)a 73.4* NPS psychedelics N(%) 15 (5.60)a 130 (20.10)b 366 (17.30)b 29.0*** NPS dissociatives N(%) 16 (6.00)a 21 (3.20)a 118 (5.60)a 6.1* All controlled substances N(%) 174 (66.40)a 533 (84.50)b 1712 (81.60)a 41.5* Intravenous drug use N(%) 133 (50.00)a 9 (1.40)b 26 (1.20)b 1084.1* Synthetic cannabinoids included synthetic cannabinoids pure, herbal brands; NPS stimulants included branded stimulants (e.g. “bath salts”), empathogens, nootropics, (obtained pure, e.g.. mephedrone, MDPV, a-PVP); NPS Psychedelics included e.g. NBOMe-x. 2C-x′; NPS dissociatives included, e.g. methoxetamine; controlled substances included cannabis, amphetamine, xtc, cocaine, crack-cocaine, heroin, magic mushroom, LSD, metamphetamine, ketamin, GHB. Marginalised Users Last month frequency of controlled substance use is lower among marginalised users than in the other two user groups (66.4% vs. above 80%). The most popular substances among new psychoactive substances are the NPS stimulants and the synthetic cannabinoids. The highest frequency of intravenous drug use was observed among this user group, half of them applied this route of administration in the month preceding the data collection. NPS psychedelic use is very rare among marginalised users, less than 6% of them used this type of substance in the last month. International Journal of Mental Health and Addiction Users in Night Life Controlled substances are the most prevalent substances in the night life user group (84.5%) as well, the second most prevalent substances in this user group are NPS stimulants and NPS psychedelics (25% and 20.1%, respectively). Synthetic cannabi- noids are also popular in this user group as every 9th respondents used this substance during the month before the data collection was carried out. Almost no intravenous drug use can be observed in this user group (1.40%). Perceived Context of NPS Use among Different User Groups The focus of our current analysis regarding motives was to find out if the external/pragmatic items added to the motivation scale can deepen our understanding regarding the motivation towards NPS use. The mean scores of the sample show that these external/contextual motives were rated 3.1 or less on the five-grade Likert scale. Items related to the legal status and to the perceived non- detectability of NPS were given ratings lower than 2. There are considerable differences among user groups; in general, marginalised users rate these items higher (consider them more important) than the other user groups with the only exception of “Expecting different or new experiences (regarding drug effects)”. This item is rated highest among night life users (see Table 3). Even though the analysis found relatively low ratings of external/contextual motives towards NPS use, during the next step, for methodological clarity, we studied if the newly included items of the motivation scale construct a coherent factor to measure external/contextual motivations towards NPS use. The principal component analysis on these items extracted only one component with the following items: ‘alleged legality’, ‘price’, ‘low availability of other (traditional) drugs’, ‘non-detectability’ and ‘poor quality of other (traditional) drugs’ whilst ‘expecting different or new experiences (regarding drug effects)’ did not become part of this factor. The identified factor was able to explain more than 44% of the variances. The component loadings ranged between 0.60 and 0.73. This factor was given the contextual motivation factor label. The Cronbach Alpha for these items was 0.682, which is on the threshold of acceptability. As the main focus of the present analysis is to identify the discriminative features of marginalised users compared to other user groups, we analysed the variances of the external/ contextual motivation factor scores in marginalised and non-marginalised users across the different countries. To be able to do so, we applied a re-coding during which we kept the marginalised group as it was (N = 265), and the night life and online community groups were merged into one variable labelled as non-marginalised (N = 1568). The Netherlands was left out from this analysis as only one marginalised respondent was interviewed in this country. As the figure below (Fig. Users in Online Community p < 0.05; *p < 0.001. Different subscript letters indicate a significant (p ≤ 0.05) differences in proportions Table 2 Last month frequencies of substance use (%) according to user groups Synthetic cannabinoids included synthetic cannabinoids pure, herbal brands; NPS stimulants included branded stimulants (e.g. “bath salts”), empathogens, nootropics, (obtained pure, e.g.. mephedrone, MDPV, a-PVP); NPS Psychedelics included e.g. NBOMe-x. 2C-x′; NPS dissociatives included, e.g. methoxetamine; controlled substances included cannabis, amphetamine, xtc, cocaine, crack-cocaine, heroin, magic mushroom, LSD, metamphetamine, ketamin, GHB. p < 0.05; **p < 0.001. Different subscript letters indicate a significant (p ≤ 0.05) differences in proportions Synthetic cannabinoids included synthetic cannabinoids pure, herbal brands; NPS stimulants included branded stimulants (e.g. “bath salts”), empathogens, nootropics, (obtained pure, e.g.. mephedrone, MDPV, a-PVP); NPS Psychedelics included e.g. NBOMe-x. 2C-x′; NPS dissociatives included, e.g. methoxetamine; controlled substances included cannabis, amphetamine, xtc, cocaine, crack-cocaine, heroin, magic mushroom, LSD, metamphetamine, ketamin, GHB. p < 0.05; **p < 0.001. Different subscript letters indicate a significant (p ≤ 0.05) differences in proportions International Journal of Mental Health and Addiction Perceived Context of NPS Use among Different User Groups 1) shows, contextual/external motivation factor scores are higher among marginalised users in all countries included into the analysis, whilst in two countries Table 3 The variance of contextual motives among user groups Motives User groups Statistics Marginalised N = 266 Night life N = 647 Internet N = 2110 F Alleged legality, mean (SD) 1.79 (1.26)a 1.36 (0.84)b 1.57 (1.16)c 14.93* Poor quality of other traditional drugs, Mean (SD) 2.45 (1.58)a 1.48 (0.94)b 1.78 (1.26)c 53.34* Price, mean (SD) 2.46 (1.64)a 1.53 (1.08)b 1.81 (1.24)c 47.60* Expecting new experiences, mean (SD) 2.55 (1.51)a 3.08 (1.54)b 2.79 (1.53)a 13.2* Non-detectability, mean (SD) 1.94 (1.42)a 1.31 (0.94)b 1.50 (1.12)c 28.70* Lack of traditional drugs, mean (SD) 2.35 (1.52)a 1.44 (0.92)b 1.77 (1.31)c 47.05* Contextual motive scale, mean (SD) 2.23 (0.96)a 1.49 (0.70)b 1.91 (0.91)c 75.720*** ANOVA p < 0.001 The Games-Howell test was used as post hoc test. Different subscript letters indicate a significant difference (p ≤ 0.05) in the parameters Contextual motive scale results are presented without the Netherlands, the N for the user groups is marginalised, 265; night life, 458; Internet 1110 Table 3 The variance of contextual motives among user groups Table 3 The variance of contextual motives among user groups International Journal of Mental Health and Addiction 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 Non marginalised Marginalised Germany Hungary Ireland Portugal Poland Means Fig. 1 The variance of contextual motivation according to user groups and countries (N = 1801). Note: F value for countries, 5.811; for user groups, 16.343*; for countryuser group interaction, 1.612, * p < 0.001 Fig. 1 The variance of contextual motivation according to user groups and countries (N = 1801). Note: F value for countries, 5.811; for user groups, 16.343*; for countryuser group interaction, 1.612, *** p < 0.001 (Hungary and Poland), the differences in the means between marginalised and non- marginalised user groups are much smaller than in the others. These comparisons show that contextual motivations presumed to be NPS specific play a different role depending on the country of residence of the respondents. Discriminating Features of Marginalised NPS Use—Binary Logistic Regression In the next phase, we studied the factors that might describe the discriminating features of marginalised NPS users. For this purpose, two binary logistic regression models were created; in each case, the dependent variable was if the respondent was categorised as marginalised vs. non-marginalised user. The Netherlands was left out from this analysis as the total number of marginalised users reached during the interview was only one person. The total number of respondents included into this analysis was 1833. In the first model, we included only socio- demographic characteristics (education, current status of employment, gender, age, size of the settlement and living conditions). The low level of accomplished education, living on benefits, being unemployed, being older, living with friends, using homeless-type accommodation arrangements and living in bigger settlements significantly increased the probability of being a marginalised user (see Table 4). Country of residence seems to be associated with significant and high odds ratios for being categorised as a marginalised user, though these findings might just be the consequence of sample attainment. As the number of marginalised users reached during the data collection was very much varied we carried out a country based binary logistic regression analysis as well during which the same set of variables were used to find out the country specific characteristics of the marginalised user group. Level of education played a role in being categorised as a marginalised user in the case of Hungary and Germany. In Hungary, less education elevated the risk of being part of the marginalised user group whilst in Germany any accomplished education increased this risk (very probably due to the high number of participants with higher education attainment in the sample). The employment status proved to be an active ingredient of being categorised as a marginalised user in Poland. Living on benefits or being unemployed elevated this risk 32–9.2 times higher than being a student. Age also had a different role depending on the country of International Journal of Mental Health and Addiction Table 4 Binary logistic regression analysis of socio-demographic factors for being categorised as a marginalised user Variables OR (95% C.I.) p Country of residence Hungary 46.71 (16.80–129.84) < 0.001 Ireland 72.00 (16.78–308.92) < 0.001 Portugal 4.56 (1.38–14.99) 0.012 Poland 19.09 (7.56–48.21) < 0.001 Germany Ref. Discriminating Features of Marginalised NPS Use—Binary Logistic Regression Level of education None 26.26 (1.83–377.20) 0.016 Primary school 9.72 (1.17–80.88) 0.035 Secondary school 1.91 (0.24–15.32) 0.541 College 0.49 (0.06–4.09) 0.508 MA or higher Ref. Current status of employment Full-time worker 0.84 (0.25–2.75) 0.768 Part-time worker 1.70 (0.59–4.90) 0.326 Self-employed 1.16 (0.32–4.17) 0.825 Benefits 12.47 (3.88–40.08) < 0.001 Retired 2.61 (0.19–35.28) 0.471 Home duties 8.14 (0.51–129.14) 0.137 Unemployed 4.05 (1.53–10.73) 0.004 Other 0.69 (0.02–25.91) 0.841 Student Ref. Age categories 18–24 Ref. 25–34 3.32 (1.64–6.72) < 0.001 35–44 7.96 (3.19–19.88) < 0.001 45– 16.56 (4.41–62.14) < 0.001 Living conditions Own home Ref. With parents/family 1.80 (0.72–4.62) 0.201 With friends/in friends’ home 13.15 (3.80–45.53) < 0.001 Residential care 64.34 (16.01–258.58) < 0.001 Rent apartment or room 1.48 (0.59–3.70) 0.403 Homeless accommodation/hostel 81.60 (18.94–351.66) < 0.001 Other 31.36 (6.29–156.43) < 0.001 Size of the settlement Below 50,000 Ref. Between 50 and 100,000 4.30 (1.46–12.64) 0.008 More than 100,000 8.43 (3.49–20.35) < 0.001 Ref., Reference category Binary logistic regression analysis (FSTEP), 8 iterations All variables remained in the equation, though just a few of them contributed significantly to the categorisation All variables remained in the equation, though just a few of them contributed significantly to the categorisat residence of the respondent. In case of Hungary and Poland, being older than 24 highly elevates the risk of being part of the marginalised group. Living with friends just in Germany, residential care in Germany and Poland and homeless accommodation in Germany, Hungary and Poland contributed to the marginalised categorisation. Size of the settlement where the respondent was living did not add to this categorisation. In the case of Portugal and Ireland, none of the variables revealed any patterns (see detailed result in Appendix Table 6). Another binary logistic regression model was developed on substance use–related behav- iour (Table 5). We included NPS and controlled substance use frequencies (life time, last year, last month), venue of substance use, problems (physical, social and mental health) experienced after using NPS, parallel usage of different substances, and contextual motivation factor. The model shows that characteristics of problem drug use (EMCDDA, 2009) are clearly associated with being a marginalised respondent. Those who used heroin or methamphetamine during International Journal of Mental Health and Addiction Table 5 Binary logistic regression analysis of substance use related factors for being categorised as a marginalised user Name of the variable OR (95% C.I. ) p. Ref., reference category Binary logistic regression analysis (FSTEP), 7 iterations Synthetic cannabinoids included synthetic cannabinoids pure. Herbal brands; NPS stimulants included branded stimulants (e.g.: “bath salts”), empathogens, nootropics (obtained pure, e.g.: mephedrone. MDPV, a-PVP), NPS psychedelics included, e.g. NBOMe-x. 2C-x′, NPS dissociatives included, e.g. methoxetamine; controlled substances included cannabis, amphetamine, xtc, cocaine, crack-cocaine, heroin, magic mushroom, LSD, metamphetamine, ketamin, GHB Variables not remained in the equation during the FSTEP procedure: Substance use related variables: synthetic cannabinoids last year consumption; stimulants/empathogens/ nootropics pure last year and last month consumption; dissociative last year and last month consumption, psychedelics last month consumption, cannabis las year consumption, cocaine last year, last month consumption, heroine last year, last month consumption, crack last year consumption, LSD last month consumption, metamphetamine last month consumption, NPS use together with another NPS Venue of the substance use related items: work place, special places created for drug users Contextual motive scale their life time or in the last year have a 2.1 to 3.1 times bigger chance to be categorised as marginalised than those who did not. In case of those who had ever or during the last year injected any kind of substances, the odds ratios are considerably elevated. Frequencies of current substance use reveal that using synthetic cannabinoids or crack also highly increases the odds ratios for being categorised as marginalised user, whilst using cannabis or amphet- amine during the last month decreased the chance for belonging to this category. If someone is using substances in residential care, the chance for being categorised as a marginalised user is 5.4 times higher, whilst using NPS and alcohol or controlled substances together decreases the likelihood of being categorised as a marginalised user. their life time or in the last year have a 2.1 to 3.1 times bigger chance to be categorised as marginalised than those who did not. In case of those who had ever or during the last year injected any kind of substances, the odds ratios are considerably elevated. Frequencies of current substance use reveal that using synthetic cannabinoids or crack also highly increases the odds ratios for being categorised as marginalised user, whilst using cannabis or amphet- amine during the last month decreased the chance for belonging to this category. Discriminating Features of Marginalised NPS Use—Binary Logistic Regression Last year/life time substance use Last year NPS psychedelic use 0.25 (0.13–0.47) < 0.001 Last year ecstasy use 0.60 (0.34–1.03) 0.066 Life time heroin use 2.06 (1.10—3.85) 0.023 Last year methamphetamine use 3.09 (1.70–5.59) < 0.001 Last year LSD use 0.41 (0.19–0.85) 0.017 Life time intravenous drug use 8.32 (4.28–16.20) < 0.001 Last month substance use Last month synthetic cannabinoids use (incl. all herbal blends) 2.28 (1.38–3.77) 0.001 Last month cannabis use 0.37 (0.23–0.59) < 0.001 Last month amphetamine use 0.40 (0.21–0.77) 0.006 Last month crack use 6.93 (2.18–22.05) 0.001 Last month intravenous drug use 5.22 (2.52–10.81) < 0.001 Problems and risk behaviour Perceived social problems 0.48 (0.30–0.76) 0.002 NPS consumption together with controlled substances 0.54 (0.34–0.85) 0.008 NPS consumption together with alcohol 0.49 (0.31–0.77) 0.002 Venue of consumption At own home 0.24 (0.15–0.39) < 0.001 At a friend’s house 0.57 (0.35–0.91) 0.018 Night life 0.57 (0. 34–0.95) 0.029 In residential care 5.42 (1.62–18.09) 0.006 Discussion The study has provided unique insight into NPS and controlled drug using patterns of different user groups and identified a series of demographic and external contextual factors potentially contributing to being categorised as marginalised user in six European countries. Based on frequencies, and mostly on short-term frequencies of substance use (last month) we can say that among recent NPS users, much higher frequencies of substance use can be observed than in adult population surveys, suggesting that we studied a very specific population. The special nature of the population we studied was illustrated in the results chapter, based on comparing our own data and the ones presented in the 2018 European Drug Report (EMCDDA 2018). Based on this comparison, it can be assumed that those three user groups which were investigated in the context of a large European transnational project are much more intense, and in some cases, much more problematic users than those representing the adult population in the relevant countries. The impression of intense substance use in these groups might also be the consequence of poly drug using tendencies of NPS users; NPS use very frequently happens in the context of polysubstance use (European Monitoring Centre for Drugs and Drug Addiction 2017b). The problematic nature of substance use is supported by the frequent intravenous substance use especially among marginalised users. Another warning sign is that the respondents were not necessarily able to say what kind of substance they were using, and they were not able to reflect on the active ingredients of the substances (see further details in Van Hout et al. 2018; Korf et al. 2019). Epidemiological research so far has not yet revealed the discriminating features of different NPS user groups as large-scale representative studies cannot reflect on the specificities of special groups. There are plenty of studies which describe the socioeconomic properties of substance users (including legal and illegal substances); these studies consistently claim that disadvantageous socioeconomic situation significantly contributes to elevated risk of heavy/ problematic substance use (see e.g. Janicijevic et al. 2017). In the light of the few adult population surveys implemented in the last few years, it can be assumed that NPS play a substantial role in the poly-consumption patterns of drug users (i.e. Paksi et al. 2016, 2017). According to our analysis and in accordance with other studies (i.e. Van Hout et al. 2018), the substance-related preferences of different user groups vary. Ref., reference category If someone is using substances in residential care, the chance for being categorised as a marginalised user is 5.4 times higher, whilst using NPS and alcohol or controlled substances together decreases the likelihood of being categorised as a marginalised user. As we found considerable differences in the sociodemographic variables according to countries of residence, we analysed the country-specific contributing factors of substance International Journal of Mental Health and Addiction use (the same set of variables as described above) in being categorised as a marginalised user. (see Appendix, Table 7). The analysis revealed that last year and last month use of heroin and intravenous drug use (in Germany and Poland), amphetamine, methamphetamine use (in Poland) and synthetic cannabinoid and crack use (in Hungary), highly elevated the risk of being categorised as a marginalised user. The parallel use of different new substances (NPS) contributed to being categorised as a marginalised user in Germany (OR = 6.53), whilst the venue of substance use found to be influential in the case of Poland where usage in residential care substantially increased the odds ratios of this type of categorisation. Contextual motives did not contribute to being categorised as a marginalised user among German respondents. In the case of Portugal and Ireland none of the variables revealed any patterns, probably due to the low sample size reached in these countries. Discussion In all user groups (marginalised users, night life and online), the most frequently used substances are the controlled ones, with NPS being just part of poly-substance using repertoires. Based on our data and in accordance with other research (Potter and Chattwin 2018), it can be said that NPS use does not constitute an independent category of drug use; NPS users are still using controlled substances, though International Journal of Mental Health and Addiction marginalised users are using significantly less of them than the other user groups. In the case of marginalised users, regardless of their country of residence, the rates for current use show that NPS play a relatively bigger role in their drug using preferences than traditional controlled substances, which is the reverse in the case of other user groups (night life and internet community). The most popular NPS type substances among marginalised users are synthetic cannabinoids, and NPS stimulants. In some countries, it is worth noting, as qualitative research also revealed (Kaló et al. 2017), that some individuals in disadvantageous social-economic situations start drug-using careers or pathways with NPS. The early onset of psychoactive substance use and especially the preference towards NPS, contributes substantially to the development of particularly destructive forms of drug use in socially marginalised groups. Our analysis also revealed that the newly added items to the motivation scale are organised in one factor named contextual factor. The analysis also revealed that the perceived (reported) importance of these motives do not play a decisive role in drug using preferences of recent NPS users. We can conclude that, if at all, particularly low availability and poor quality of controlled substances as well as the relatively low price of NPS may play a more important role in the motivation towards these substances in case of marginalised users compared with other recent NPS user groups. The respondents from all user groups rated the contextual motivation items relatively low, the least important motives are the alleged legality and non- detectability of these substances. The relative unimportance of these items might be the consequence of the legal status of different new psychoactive substances in the different countries. Another possible explanation of this finding might also be that all recent NPS users are (intensively) using controlled substances; consequently, the temporary legal status of any given NPS is not a significant issue to articulate their choices for consumption. Discussion This finding might be highly relevant in the drug policy/legislation debates in connection to the current regulatory efforts. Some of the external/contextual motivation items received higher ratings from marginalised respondents with significant differences depending on country of residence. Data describing the drug use patterns of the three diverse NPS user groups and the regression model, including a wide range of socio-demographic variables (age, level of education, labour market position, recent use of NPS and controlled substances, route of administration), strongly suggest that marginalised users are the most at-risk population with lower education and with the most risky drug using patterns. As our selection criteria regarding marginalised users was very much concentrating on the venue of substance use (streets, public places) and/or being clients of low threshold/harm reduction services and their networks, it is very much reassuring that our analysis revealed that the marginalised users reached have indeed a well identifiable socio-demographic profile, describing their elevated vulnerability. These findings not just mirror the sample selection criteria, but it draws a more refined picture regarding the socio-demographic factors and the discriminating substance use habits of marginalised users; outlining the profile of high-risk problematic drug use. The binary logistic regression model, which included all respondents from the participating countries (apart from the Netherlands) is conclusive regarding the main sociodemographic characteristics and substance use patterns of the marginalised user group. More detailed country-based analysis suggests that variables supposed to be contributing to the marginalised—non-marginalised categorisation of the respondents in different countries are very much varied, at some points, the findings are even contradictory. Therefore, it is suggested that further epidemiological studies should be carried out to obtain reliable data from different countries on the relative weight of NPS use in different user groups and on other factors contributing to enhance our user group specific understanding of NPS use and, in International Journal of Mental Health and Addiction general, problematic drug use behaviour. Further research is especially needed as our data cannot be considered as representative for the participating countries or for the chosen user groups. Contextual motives, as the logistic regression model shows, do not contribute to be categorised as a marginalised user when other variables were controlled. Discussion The intense drug use of marginalised NPS users should call the attention of policy makers to strengthen tailor-made harm reduction services and targeted prevention activities in these groups, to properly respond to the public health and legal concerns in some European countries where NPS use is especially prevalent among low social status marginalised users. Conclusions This study contributes to the better understanding of NPS use in special populations, especially in the marginalised user group, by describing the discriminating features of substance use, its setting and the sociodemographic properties of respondents in this group. This statement might remain to be true even though the data collection was carried out on a convenience sample (with different but standardised recruitment strategies in each user group) and the number of respondents in the different user groups was very much uneven, and we have no sufficient reliable information on the prevalence of NPS use in the general population. Our data can inform decision-makers about the necessity of enhancing complex interventions targeting high risk marginalised users and also the scientific community that large-scale epidemiological surveys in special populations should be carried out to further develop our understanding regarding NPS use in certain populations. Funding Information This study was supported by the European Union (New Psychoactive Substances: transnational project on different user groups, user characteristics, extent and patterns of use, market dynamics, and best practices in prevention [HOME/2014/JDRU/AG/DRUG/7077]), the Hungarian National Research, Development and Innovation Office (Grant number: KKP126835), and the Hungarian Ministry of Human Capacities (ELTE Institutional Excellence Program, 783-3/2018/FEKUTSRAT). The study was also supported for the realisation of this international co-financed science project in 2016-2017 by the Polish Ministry of Science and Higher Education. The funding institutions had no role in the study design or the collection, analysis and interpretation of the data, writing the manuscript, or the decision to submit the paper for publication. Compliance with Ethical Standards Conflict of Interest The authors (Katalin Felvinczi, Annemieke Benschop, Róbert Urbán, Marie Claire Van Hout, Katarzyna Dąbrowska, Evelyn Hearne, Susana Henriques, Zsuzsa Kaló, Gerrit Kamphausen, Joana Paula Silva, Łukasz Wieczorek, Bernd Werse, Michal Bujalski, Zsolt Demetrovics, Dirk Korf) declare that they do not have any conflicts of interest that could constitute a real, potential or apparent issue with respect to their involvement in the publication. The authors also declare that they do not have any financial or other relations (e.g. directorship, consultancy or speaker fee) with companies, trade associations, unions or groups (including civic associations and public interest groups) that may affect the results or conclusions in the study. Sources of funding are acknowledged. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, dis- tribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. International Journal of Mental Health and Addiction Table 6 Binary logistic regression analysis of socio-demographic factors for being categorised as a marginalised user according to country of residence Country name of the variable GER HU IR PL PT OR [95% CI] Level of education None 0.087 [0.003–2.814] x x x x Primary school 0.046* [0.003–0.675] 193.911* [5.079–7402.937] x x x Secondary school 0.015* [0.001–0.266] 34.166* [1.051–1110.678] x x x College 0.028* [0.001–0.527] 1.430 [0.05–40.92] x x x MA/MSc or higher Ref. Ref. Ref. Ref. Ref. Current status of employment Full-time worker x x x 0.531 [0.055–5.148] x Part-time worker x x x 1.984 [0.246–15.396] x Self-employed x x x 1.402 [0.097–20.204] x Benefits x x x 32.098* [3.784–272.12] x Retired x x Home duties x x Unemployed x x x 9.221* [1.813–46.902] x Other x x 1 x x Student Ref. Ref. Ref. Ref. Ref. Age 18–24 Ref. Ref. Ref. Ref. Ref. 25–34 x 5.691* [1.021–31.703] x 2.223 [0.608–8.129] x 35–44 x 54.236* [5.316–553.321] x 34.739** [4.982–242.241]x x 45+ x 24.787 [0.892–689.086] x 33.168* [2.16–509.312] x Gender Female Ref. Ref. Ref. Ref. Ref. Compliance with Ethical Standards Male 9.479* [1.872–48.009] Living conditions With parents/family 0.584 [0.031–11.13] 0.679 [0.096–4.82] x 1.592 [0.281–9.037] x With friends/in friends’ home 19.933* [1.247–318.649] 5.758 [0.342–96.836] x 19.891* [1.515–261.155] x Residential care 106.556* [4.904–2315.114] 48.227 [0.607–3832.513] x 142.338 [13.485–1502.466] x Rent apartment or room 2.690 [0.278–31.567] 0.487 [0.054–4.383] x 1.287 [0.222–7.48] x tional Journal of Mental Health and Addiction International Journal of Mental Health and Addiction Table 6 (continued) Country name of the variable GER HU IR PL PT Homeless accommodation/hostel 169.212 [11.502–2489.414] 93.947* [4.723–1868.553] x x x other 47.130* [1.734–1281.187] 17.095* [1.046 × 279.35] x 99.617* [4.503–2203.68] x Own home Ref. Ref. Ref. Ref. Ref. Size of the settlement Below 50.000 Ref. Ref. Ref. Ref. Ref. Between 50 × 100.000 x x x 7.273* [1.43–36.988] x More than 100.000 x x x 2.848 [0.616–13.174] x p < 0.05; p < 0.001; x the variable is not in the equation or the OR not within the confidence interval International Journal of Mental Health and Addicti Table 6 (continued) Country name of the variable GER HU IR PL PT Homeless accommodation/hostel 169.212* [11.502–2489.414] 93.947* [4.723–1868.553] x x x other 47.130* [1.734–1281.187] 17.095* [1.046 × 279.35] x 99.617* [4.503–2203.68] x Own home Ref. Ref. Ref. Ref. Ref. Size of the settlement Below 50.000 Ref. Ref. Ref. Ref. Ref. Between 50 × 100.000 x x x 7.273* [1.43–36.988] x More than 100.000 x x x 2.848 [0.616–13.174] x p < 0.05; p < 0.001; x the variable is not in the equation or the OR not within the confidence interval Table 6 (continued) Country name of the variable GER HU IR PL PT Homeless accommodation/hostel 169.212* [11.502–2489.414] 93.947* [4.723–1868.553] x x x other 47.130* [1.734–1281.187] 17.095* [1.046 × 279.35] x 99.617* [4.503–2203.68] x Own home Ref. Ref. Ref. Ref. Ref. Size of the settlement Below 50.000 Ref. Ref. Ref. Ref. Ref. Compliance with Ethical Standards Between 50 × 100.000 x x x 7.273* [1.43–36.988] x More than 100.000 x x x 2.848 [0.616–13.174] x p < 0.05; p < 0.001; x the variable is not in the equation or the OR not within the confidence interval International Journal of Mental Health and Addiction Table 7 Binary logistic regression analysis of substance use related factors for being categorised as a marginalised user according to country of residence Country name of the variable GER HU IR PL PT OR [95% CI] Frequency of life time/last year substance use Cannabis x x x 0.179 [0.048–0.661] x NPS psychedelics x 0.055* [0.008–0.4] x x x Ecstasy x 0.247* [0.079–0.766] x 0.245* [0.078–0.772] x Amphetamine x x x 14.902** [4.504–49.301] x Cocaine x x 0.079* [0.016–0.403] x Heroin 45.241* [4.874–419.894] x x x x Methamphetamine x x x 4.721* [1.641–13.587] x Intravenous drug use x 87.058 [24.871–304.73] x 28.784 [8.943–92.64] x Synthetic cannabinoids (incl. All herbal blends) x x x 6.533* [2.268–18.819] x Frequency of last month substance use Synthetic cannabinoids (incl. All herbal blends) x 4.938* [1.661–14.684] x x x NPS psychedelics x x x 0.017* [0.001–0.278] x cannabis x x x 0.176* [0.062–0.501] x ecstasy xx x x x amphetamine 0.218* [0.054–0.886] x x 0.09* [0.019–0.433] x cocaine x x x x x crack x 66.594* [2.125–2086.848] x x x NPS dissociatives x x x 0.106 [0.007–1.56] x intravenous drug use 18.733 [5.008–70.073] x x 80.204 [12.109–531.231] x PROBLEMS AND RISK BEHAVIOUR mental and physical side effects related to drug use x x x 0.393 [0.164–0.941] 0.064* NPS use together with alcohol x 0.2* [0.068–0.588] x x x NPS use together with other NPS 6.532* [1.38–30.923] x x x x NPS use together with controlled substances x 0.144* [0.045–0.458] x x x Venue of consumption At own home 0.168* [0.04–0.703] 0.202* [0.065–0.627] x 0.12 [0.045 × 0.316] x In residential care x x x 209.463 [19.483–2251.944] x p < 0.05; *p < 0.001; x the variable is not in the equation or the OR not within the confidence interval International Journal of Mental Health and Addiction References Barratt, M. 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International Journal of Mental Health and Addiction Affiliations Katalin Felvinczi1 & Annemieke Benschop2 & Róbert Urbán1 & Marie Claire Van Hout3 & Katarzyna Dąbrowska4 & Evelyn Hearne5 & Susana Henriques6 & Zsuzsa Kaló1 & Gerrit Kamphausen7 & Joana Paula Silva6 & Łukasz Wieczorek4 & Bernd Werse7 & Michal Bujalski4 & Zsolt Demetrovics1 & Dirk Korf2 Katalin Felvinczi1 & Annemieke Benschop2 & Róbert Urbán1 & Marie Claire Van Hout3 & Katarzyna Dąbrowska4 & Evelyn Hearne5 & Susana Henriques6 & Zsuzsa Kaló1 & Gerrit Kamphausen7 & Joana Paula Silva6 & Łukasz Wieczorek4 & Bernd Werse7 & Michal Bujalski4 & Zsolt Demetrovics1 & Dirk Korf2 1 Institute of Psychology, ELTE Eötvös Loránd University, Izabella utca 46, Budapest H-1064, Hungary 2 Bonger Institute, University of Amsterdam, Amsterdam, Netherlands 3 Public Health Institute, Liverpool John Moores University, Liverpool, UK 4 Institute of Psychiatry and Neurology, Warsaw, Poland 5 School of Health Sciences, Waterford Institute of Technology, Waterford, Ireland 6 Centre for Research and Studies in Sociology, University Institute of Lisbon, Lisbon, Portugal 7 Goethe-Universität, Frankfurt, Germany Katalin Felvinczi1 & Annemieke Benschop2 & Róbert Urbán1 & Marie Claire Van Hout3 & Katarzyna Dąbrowska4 & Evelyn Hearne5 & Susana Henriques6 & Zsuzsa Kaló1 & Gerrit Kamphausen7 & Joana Paula Silva6 & Łukasz Wieczorek4 & Bernd Werse7 & Michal Bujalski4 & Zsolt Demetrovics1 & Dirk Korf2 1 Institute of Psychology, ELTE Eötvös Loránd University, Izabella utca 46, Budapest H-1064, Hungary 2 Bonger Institute, University of Amsterdam, Amsterdam, Netherlands 3 Public Health Institute, Liverpool John Moores University, Liverpool, UK 4 Institute of Psychiatry and Neurology, Warsaw, Poland 5 School of Health Sciences, Waterford Institute of Technology, Waterford, Ireland 6 Centre for Research and Studies in Sociology, University Institute of Lisbon, Lisbon, Portugal 7 Goethe-Universität, Frankfurt, Germany 7 Goethe-Universität, Frankfurt, Germany
https://openalex.org/W4319748032	https://www.frontiersin.org/articles/10.3389/fvets.2023.1070482/pdf	English	null	The attitude-behaviour gap in biosecurity: Applying social theories to understand the relationships between commercial chicken farmers' attitudes and behaviours	Frontiers in veterinary science	2,023	cc-by	13,136	The attitude-behaviour gap in biosecurity: Applying social theories to understand the relationships between commercial chicken farmers’ attitudes and behaviours OPEN ACCESS EDITED BY Eva King, The University of Queensland, Australia REVIEWED BY Veronica Sri Lestari, Hasanuddin University, Indonesia Christopher Koliba, University of Vermont, United States CORRESPONDENCE Hai-ni Pao paohaini@gmail.com SPECIALTY SECTION This article was submitted to Veterinary Humanities and Social Sciences, a section of the journal Frontiers in Veterinary Science RECEIVED 14 October 2022 ACCEPTED 06 January 2023 PUBLISHED 09 February 2023 CITATION Pao H-n, Jackson E, Yang T-s, Tsai J-s, Hwang Y-t, Sung WHT and Pfeifer DU (2023) The attitude-behaviour gap in biosecurity: Applying social theories to understand the relationships between commercial chicken farmers’ attitudes and behaviours. Front. Vet. Sci. 10:1070482. doi: 10.3389/fvets.2023.1070482 Hai-ni Pao1, Elizabeth Jackson2, Tsang-sung Yang3, Jyan-syung Tsai4, Yi-ting Hwang5, Watson H. T. Sung6 and Dirk U. Pfeifer1,7 1Veterinary Epidemiology, Economics and Public Health Group, Department of Pathobiology and Population Sciences, Royal Veterinary College, Hatﬁeld, United Kingdom, 2School of Management and Marketing, Curtin University, Perth, WA, Australia, 3Independent Researcher, New Taipei City, Taiwan, 4Department of Finance and Cooperative Management, National Taipei University, New Taipei City, Taiwan, 5Department of Statistics, National Taipei University, New Taipei City, Taiwan, 6Agricultural Bank of Taiwan, New Taipei City, Taiwan, 7Centre for Applied One Health Research and Policy Advice, Jockey Club College of Veterinary Medicine and Life Sciences, City University, Kowloon, Hong Kong SAR, China CITATION Pao H-n, Jackson E, Yang T-s, Tsai J-s, Hwang Y-t, Sung WHT and Pfeifer DU (2023) The attitude-behaviour gap in biosecurity: Applying social theories to understand the relationships between commercial chicken farmers’ attitudes and behaviours. Front. Vet. Sci. 10:1070482. doi: 10.3389/fvets.2023.1070482 Introduction: Traditionally, it is believed that people’s behaviours align with their attitudes; however, during COVID-19 pandemic, an attitude-behaviour gap in relation to preventive measures has been observed in recent studies. As such, the mixed-methods research was used to examine the relationships between farmers’ biosecurity attitudes and behaviours in Taiwan’s chicken industry based on the cognitive consistency theory. COPYRIGHT © 2023 Pao, Jackson, Yang, Tsai, Hwang, Sung and Pfeifer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 09 February 2023 DOI 10.3389/fvets.2023.1070482 TYPE Original Research PUBLISHED 09 February 2023 DOI 10.3389/fvets.2023.1070482 TYPE Original Research PUBLISHED 09 February 2023 DOI 10.3389/fvets.2023.1070482 The attitude-behaviour gap in biosecurity: Applying social theories to understand the relationships between commercial chicken farmers’ attitudes and behaviours cognitive consistency, commercial chicken farms, content analysis, mixed-methods research, biosecurity Frontiers in Veterinary Science 1. Introduction FIGURE 1 The underlying mechanisms of cognitive dissonance. When an individual’s behaviour is inconsistent with his attitude, the tension of cognitive dissonance will increase unless the individual changes his/her attitude or behaviour. Alternatively, the individual may try to gain new information or reduce the importance of this issue. Epidemic animal diseases threaten livestock productivity and the wider society (1). Zoonotic diseases that threaten human health (2) by direct or indirect contact with animals have become of great concern to the global community (3). Six in ten infectious human diseases are transmitted from animals (4), and three in four emerging infectious diseases are zoonotic (4, 5). National authorities have faced the challenge of developing integrated strategies for eﬀective prevention and control of animal diseases (6, 7). Siekkinen et al. (8) emphasised that on-farm biosecurity is a critical component of disease prevention and control. Recently, studies have applied both quantitative and qualitative approaches to understand farmers’ knowledge, attitudes, and underlying drivers related to on-farm biosecurity practises (9– 17). Studies also discovered how social cues, messages of infection risk and message delivery methods aﬀect farmers’ compliance with biosecurity (18–21). FIGURE 1 The underlying mechanisms of cognitive dissonance. When an individual’s behaviour is inconsistent with his attitude, the tension of cognitive dissonance will increase unless the individual changes his/her attitude or behaviour. Alternatively, the individual may try to gain new information or reduce the importance of this issue. FIGURE 1 The underlying mechanisms of cognitive dissonance. When an individual’s behaviour is inconsistent with his attitude, the tension of cognitive dissonance will increase unless the individual changes his/her attitude or behaviour. Alternatively, the individual may try to gain new information or reduce the importance of this issue. y In the agricultural literature, an increasing number of studies have applied behavioural change theories to explain farmers’ decision-making process in relation to on-farm biosecurity (17, 22–24). The cognitive consistency theories have been applied to understand the logic behind people’s decision-making process and the theories describe how meanings and judgments inﬂuence an individual’s feelings, actions, and adoption (25). However, gaps in current knowledge persist regarding the relationships between farmers’ attitudes and behaviour towards biosecurity. Farmers’ attitudes have been shown to aﬀect farm management performance (26). Farmers with awareness and concerns about water quality have better practises in water management. Casal et al. 1. Introduction (27) also stated that farmers’ perception of a given biosecurity measure is associated with their current practise on the farm. Attitudes may have higher impacts on relevant behaviours when farmers’ attitudes are consistent with their beliefs, which are constructed from high amounts of relevant information and personal experience (28). Enticott (29) also suggested that farmers’ attitudes have a statistically signiﬁcant positive association with their behaviours. In contrast, with the example of peach growers, Mankad (22) explained the consequence of cognitive dissonance. Amongst all existing cognitive consistency theories, the cognitive dissonance theory (30) bears great signiﬁcance in the eﬀort to better understand people’s attitudes to their behaviours. Figure 1 shows the underlying mechanisms of cognitive dissonance. When receiving new information about the invasion risk of Queensland fruit ﬂy from farmer group advisers, if this new information leads to conﬂicts with farmers’ current beliefs, it is likely that farmers will respond negatively by not attending forums in relation to this issue, ignoring this new information or considering that proactive biosecurity practises are not necessary. The outcome, of course, is a heightened invasion risk. Fischer et al. (37) also observed that, when governments enforce their people to adhere to certain COVID-19 preventive behaviours, cognitive dissonance will be induced. We suggested that this is very much the case when it comes to biosecurity policy or relevant management recommendations at farm level. As Pao et al. (17) suggested that the relationships between attitudes and behaviours amongst farmers are complex, conﬂicts between farmers’ biosecurity attitudes and behaviours will lead to their physical and psychological tension when conducting biosecurity practises. Taiwan is located in East Asia and its neighbouring countries include China, Japan, Korea, Vietnam and the Philippines etc. There are around 100 million chickens and 6,200 chicken farms in Taiwan. The fact that Taiwan has limited farmland has resulted in intensive poultry rearing systems (land use for poultry is 1,199 ha). To mitigate the risk of potential infectious disease transmission and outbreaks within the national poultry ﬂock, Taiwan’s government has established biosecurity procedures for poultry disease management at the farm level for two decades. Taiwan’s chicken farmers have received the Taiwan government’s biosecurity training education for more than a decade. However, despite this, there have been outbreaks caused by avian inﬂuenza H5 strains in Taiwan over the past decade (40). Pao et al. The attitude-behaviour gap in biosecurity: Applying social theories to understand the relationships between commercial chicken farmers’ attitudes and behaviours Methods: Content analysis of face-to-face interviews with 15 commercial chicken farmers identiﬁed their biosecurity responses to infectious disease threats. Results: The results indicated the mismatch of farmers’ attitudes and behaviours towards speciﬁc biosecurity measures, in that they act diferently than they think. The ﬁndings of the qualitative research allowed the research team to conduct the subsequent quantitative, conﬁrmatory assessment to investigate the mismatch of farmers’ attitudes and behaviours in 303 commercial broiler farmers. Survey data were analyzed to discover the relationships between farmers’ attitudes and behaviours in relation to 29 biosecurity measures. The results show a mixed picture. The percentage of the farmers who had the attitude-behaviour gap towards 29 biosecurity measures ranged from 13.9 to 58.7%. Additionally, at the 5% signiﬁcant level, there is an association between farmers’ attitudes and behaviours for 12 biosecurity measures. In contrast, a signiﬁcant association does not exist for the other 17 biosecurity measures. Speciﬁcally, out of the 17 biosecurity measures, the disconnection of farmers’ attitudes and behaviours was observed in three speciﬁc biosecurity measures such as using a carcass storage area. Discussion: Based on a fairly large sample of farmers in Taiwan, this study conﬁrms the existence of an attitude-behaviour gap in context and applies social theories to provide an in-depth understanding of how infectious diseases are managed in the animal health context. As the results demonstrate the necessity of tailoring biosecurity strategies to address the gap, it is time to reconsider the current approach by understanding farmers’ real attitudes and behaviours in relation to biosecurity for the success of animal disease prevention and control at the farm level. cognitive consistency, commercial chicken farms, content analysis, mixed-methods research, biosecurity Frontiers in Veterinary Science Frontiers in Veterinary Science 01 frontiersin.org Pao et al. 10.3389/fvets.2023.1070482 Abbreviations: HPAI, highly pathogenic notiﬁable avian inﬂuenza; LDCC, local disease control centre. Frontiers in Veterinary Science frontiersin.org 2. Materials and methods in the literature, including risk factors for the introduction and transmission of poultry diseases in Taiwan. A total of 29 biosecurity measures were considered important and should be conducted continuously. The answer of “Yes” means that the speciﬁc biosecurity measure was conducted for each batch. The framework and the relationships of variables used in the study are presented in Figure 2. The study involved two consecutive phases: qualitative research followed by quantitative research. As this study aimed to examine if an attitude-behaviour gap might exist in relation to biosecurity at the farm level, the cognitive consistency theory was used as the theoretical basis of the research. Based on the theory, the hypothesis was proposed: In addition, the section in relation to farmers’ attitudes towards diﬀerent biosecurity measures consisted of a ﬁve-point Likert scale. The scale ranged from 1 to 5; that is, 1 = “highly unimportant,” 2 = “unimportant,” 3 = “neutral,” 4 = “important” and 5 = “highly important.” The questionnaire provided options of “Neutral” and “No idea” to avoid the bias that respondents do not want to answer or do not understand the question (48). Their responses were further divided into two groups: (1) “Recognising the importance of biosecurity measures” that included the responses of “Highly important & Important” of the ﬁve-point Likert scale; (2) “Not recognising the importance of biosecurity measures” that included the responses of “Neutral and Unimportant and Highly unimportant” of the ﬁve-point Likert scale. • Null hypothesis (H0): farmers’ attitudes and behaviours in relation to a speciﬁc biosecurity measure are not demonstrated to be signiﬁcantly associated. • Alternative hypothesis (Ha): farmers’ attitudes and behaviours in relation to a speciﬁc biosecurity measure are demonstrated to be signiﬁcantly associated. The qualitative phase served as exploratory research so that the relationships of farmers’ biosecurity attitudes and behaviours could be examined to inform the subsequent quantitative research; the latter being the conﬁrmatory phase to formally assess the attitude- behaviour gap. The survey was approved by the Ethics and Welfare Committee of the Royal Veterinary College, University of London, the United Kingdom (Approval # URN 2015 0125H). A pilot study involving 13 participants familiar with Taiwan broiler production systems was completed prior to the main study. The study populations included commercial broiler farmers from a variety of ﬂock sizes and farm types but excluded backyard farmers due to very few numbers of these farmers after urbanisation for decades. 2.1. The qualitative research This research was part of a two-tier research initiative that investigated the mindset of farmers in pursuing on-farm biosecurity (17, 41). Upon approval from the Ethics and Welfare Committee of the Royal Veterinary College, University of London, the United Kingdom (approval #URN 2014 0116H), participating farmers were recruited through local disease control centres (LDCCs), the Poultry Association, and private feed companies. Theoretical sampling was chosen to select participants based on a speciﬁc characteristic: at least 10 years of chicken farming experience. An interview guide was prepared [(42), p. 16] with simple, short open-ended questions to encourage the participants to express their views for exploring the current situation of on- farm biosecurity practises in Taiwan. Interviews were conducted on farms for each participant’s convenience. The interviews were conducted in Mandarin within one and a half to 2 h, and data were translated to English and typed into Word (Windows XP). Descriptive analyses and qualitative content analysis were performed (43–45). Open coding of respondents’ responses was performed to develop major themes and subthemes. Data were double- checked and peer debrieﬁng was repeated to reﬁne the themes and improve the validity and reliability of the data (44, 46, 47). This research was part of a two-tier research initiative that investigated the mindset of farmers in pursuing on-farm biosecurity (17, 41). Upon approval from the Ethics and Welfare Committee of the Royal Veterinary College, University of London, the United Kingdom (approval #URN 2014 0116H), participating farmers were recruited through local disease control centres (LDCCs), the Poultry Association, and private feed companies. Theoretical sampling was chosen to select participants based on a speciﬁc characteristic: at least 10 years of chicken farming experience. An interview guide was prepared [(42), p. 16] with simple, short open-ended questions to encourage the participants to express their views for exploring the current situation of on- farm biosecurity practises in Taiwan. Interviews were conducted on farms for each participant’s convenience. The interviews were conducted in Mandarin within one and a half to 2 h, and data were translated to English and typed into Word (Windows XP). Descriptive analyses and qualitative content analysis were performed (43–45). Open coding of respondents’ responses was performed to develop major themes and subthemes. Data were double- checked and peer debrieﬁng was repeated to reﬁne the themes and improve the validity and reliability of the data (44, 46, 47). 1. Introduction (17) discovered that the decision of Taiwan’s commercial chicken farmers to adopt biosecurity measures was inﬂuenced by a variety of internal and external factors. As farmers have a diverse range of needs and restrictions when it comes to their farming enterprises, it is diﬃcult to ensure farmers continually recognise and implement biosecurity measures that will protect their own and livestock’s health. As such, this study hypothesised that a gap in farmers’ biosecurity attitudes and behaviours may exist and put farmers’ and livestock’s health at risk. For example, if farmers took desirable biosecurity actions despite not having a positive attitude towards the importance of biosecurity, they might change their behaviours if there is a lack of resources. In this study, the cognitive consistency theory was used to examine the associations between farmers’ attitudes and behaviours towards biosecurity, and relevant social theories were applied to explain the relationships between farmers’ attitudes and behaviours. Due to the pandemic of COVID-19, a considerable amount of research has shown that a variety of factors inﬂuence people’s intentions in practising COVID-19 preventive behaviours (31–35). There is also increasing debate over the importance of a socio- cultural approach to better understand infectious diseases (36). In addition, an attitude-behaviour gap in relation to COVID-19 preventive measures has been observed in recent studies (37–39). frontiersin.org 02 Pao et al. 10.3389/fvets.2023.1070482 2.1. The qualitative research Commercial broiler farmers were given a detailed explanation to ensure their understanding of the purpose of the study. Participants were assured that their contribution to the study was entirely voluntary, all data would be anonymous and stored securely, and any participant was free to withdraw from the study at any time without prejudice. Prior to the survey, verbal consent from each participant was obtained, and any information which potentially could lead to their identiﬁcation was removed. Data cleaning methods (49, 50) were applied to verify structural stability such as responses and non-response were labelled accurately. Invalid entries were also identiﬁed. Respondents had to complete at least 80% of each section in the questionnaire. All analyses were performed using the statistical software package SPSS 22.0 for Windows (51). The statistical analyses were based upon two- way contingency tests (Fisher’s exact test if expected frequency <5) using the Crosstabs procedures to test the associations between farmers’ biosecurity attitudes and their behaviours. For speciﬁc biosecurity measures that farmers’ attitudes and behaviours could not be demonstrated to be signiﬁcantly associated, McNemar’s test was further used to test the consistency in responses across two 2. Materials and methods Due to outbreaks of HPAI (highly pathogenic avian inﬂuenza) in waterfowl during the data collection period, the willingness of farmers to participate in the research was low; therefore, a convenience sampling method had to be adopted. This was based on the following approach: In addition to face-to-face interviews when the LDCC oﬃcials conducted on-farm inspections, the oﬃcials also distributed questionnaires to commercial broiler farmers when farmers gathered together for a meeting or training. Moreover, due to restrictions of movement during outbreaks of HPAI, responses from HPAI-infected farms and surrounding farms within a three- kilometre radius were also obtained over the phone in order to reduce sampling bias. Frontiers in Veterinary Science frontiersin.org 3. Results As the phenomena of the mismatch of farmers’ attitudes and behaviours in relation to biosecurity measures were discovered in the qualitative research, the questionnaire survey (n = 303) reconﬁrmed the mismatch of farmers’ attitudes and behaviours towards biosecurity in a larger population sample. Thirteen farmers expressed that they were willing to implement a certain amount of biosecurity measures, and they were conﬁdent in their current biosecurity practises as “I’ve done a lot [biosecurity measures]; thus, I do not worry about avian inﬂuenza disease.” (White-chicken broiler farmer, Interview 6). They also expressed their appreciation about what they had learned in biosecurity: variables which can examine whether attitudes variables had eﬀect on behaviour variables. variables which can examine whether attitudes variables had eﬀect on behaviour variables. Five farmers thought the eﬀectiveness of biosecurity is determined by their attitudes and precaution measures as “The key to the success of biosecurity is farmers” (White-chicken broiler farmer, Interview 1). However, due to Taiwan’s high-density farm systems and the circulated variants of avian inﬂuenza, two farmers perceived the risks of avian inﬂuenza as uncontrollable as “Bird ﬂu will happen deﬁnitely because the government cannot control it.” (Indigenous chicken farmer, Interview 6) and “From other farmers’ experiences, I believe that it is hard to prevent bird ﬂu.” (Indigenous chicken farmer, Interview 5). Frontiers in Veterinary Science 2.2. The quantitative research As recommended by Creswell (47) the results of the qualitative research were combined with recent knowledge collected from the literature review and opinions from Taiwan’s poultry and epidemiology experts to develop a questionnaire which consisted of simple and closed questions. The questions were based on factors related to on-farm biosecurity status considered relevant 03 frontiersin.org Pao et al. 10.3389/fvets.2023.1070482 FIGURE 2 The framework and the relationships of variables used in the study. The study involved two consecutive phases: qualitative research followed by quantitative research. The qualitative phase served as exploratory research so that the relationships of farmers’ biosecurity attitudes and behaviours could be examined to inform the subsequent quantitative research. FIGURE 2 The framework and the relationships of variables used in the study. The study involved two consecutive phases: qualitative research followed by quantitative research. The qualitative phase served as exploratory research so that the relationships of farmers’ biosecurity attitudes and behaviours could be examined to inform the subsequent quantitative research. frontiersin.org 3.1. The qualitative research Fifteen recommended potential participants who had at least 10 years of chicken farming experience were recruited by telephone to ensure their understanding of the purpose and the procedure of the study, including data anonymisation and security, and seven of them declined to be interviewed. Alternatively, seven farmers with at least 20 years of chicken farming experience were recruited. As the main form of chicken farming in Taiwan is family production with men being the primary decision-makers (52), only male farmers (n = 15) joined this study and they all reaﬃrmed that they were their farm’s leading decision-maker. Supplementary Table 1 provides an overview of the characteristics of the farmers interviewed. Almost all farm owners (14/15) have developed their biosecurity strategies to decrease the risks of disease outbreaks. Theoretical saturation of the data was reached after 15 interviews where the additional interview did not yield any new knowledge to address the research questions (53–55). Themes and subthemes emerging from the interview data are summarised in Table 1. ‘The more [biosecurity] courses they provide, the better I can learn. The private feed companies and the Poultry Associations will oﬀer free training courses in relation to biosecurity. [...] Veterinary schools will also notify us to attend their courses.’ (Indigenous chicken farmer, Interview 1) ‘Young or educated farmers will attend seminars or discuss with others to improve their knowledge about biosecurity. [...] Most white chicken farmers have done their best in biosecurity, and we have the fewest avian inﬂuenza cases.’ (White-chicken broiler farmer, Interview 1) Conversely, three farmers were not willing to do speciﬁc biosecurity measures due to the impracticality and ineﬀectiveness of the measures as “Experts suggest that it is crucial to set up a transition zone at the farm for preventing infectious diseases, but it is impossible 04 frontiersin.org Pao et al. 10.3389/fvets.2023.1070482 TABLE 1 Summary of themes emerging from the interview data (n = 15). TABLE 1 Summary of themes emerging from the interview data (n = 15). “Poor outcomes for anti-bird netting.” (White-chicken broiler farmer, Interview 1): “Poor outcomes for anti-bird netting.” (White-chicken broiler farmer, Interview 1): TABLE 1 Summary of themes emerging from the interview data (n = 15). Major themes (The phenomenon observed) n Subthemes n A positive attitude towards biosecurity together with desirable biosecurity actions 13 1. Having done a lot and trusting in those biosecurity measures 2. 3.1. The qualitative research Vaccine programme 8 9 Neither having a positive attitude towards biosecurity nor taking desirable biosecurity actions 3 1. A loading area 2. Vehicles shipping broilers for sale always empty upon arrival /Transport the chickens by themselves/ Shipping cages and collection buckets always empty upon arrival at the farm 3. A transition zone 4. Fixed supply of chicks 1 1 1 1 A positive attitude towards biosecurity without taking desirable biosecurity actions 5 1. Downtime control 2. Cleaning and disinfection of vehicles upon arrival at the farm 3. A contracted veterinarian 3 1 2 Without a positive attitude towards biosecurity but taking desirable biosecurity actions 4 1. Anti-bird netting 2. Cleaning and disinfection of shipping cages and buckets upon arrival at the farm 3 2 ‘The government enforces us to install anti-bird netting, but we have problems. Some birds come in, but they do not have ways to go out.’ (Indigenous chicken farmer, Interview 4) ‘The government has announced that, if there is no anti- bird netting, the farmer cannot get any compensation for their chickens culled (because of avian inﬂuenza). But many farmers think the anti-bird netting is useless to prevent avian inﬂuenza.’ (White-chicken broiler farmer, Interview 1) (2) Five farmers admitted that because of potential restrictions such as time and money, some biosecurity measures have not been carried out despite the recognised importance of those measures. For example, intensive livestock producers are legally required to employ a contracted veterinarian to help manage the risk of disease outbreaks. Although all participants knew that the requirement of a contracted veterinarian or an employed veterinarian was meant to help them, two farmers still expressed their unwillingness to enter into a contract with a veterinarian due to cost concerns as “Having a contracted veterinarian is only for the purpose of obeying the policy. We do not have money to employ veterinarians. [...] We seek for the veterinarian’s assistance only when we need a ﬁnal diagnosis.” (White-chicken broiler farmer, Interview 1). In addition, trucks delivering feed and chicks are the most concerning issues related to transmitting disease upon farmers’ expression, but they don’t have enough resources to conduct this biosecurity measure: ‘We know that trucks such as those delivering feed will transmit diseases, but we cannot do disinfection each time. We don’t have enough time and labour to do this.’ (Indigenous chicken farmer, Interview 3) for us to set up it. 3.2. The quantitative research ‘The government suggests that we should transport the chickens by ourselves; otherwise, we should ensure that, when trucks [shipping broilers for sale] enter our farms, the trucks or all shipping cages are empty, but this is impossible. We cannot transport the chickens by ourselves, and we don’t know whether the trucks or their cages are empty or not when they arrive. In addition, I don’t think it is necessary because after the trucks take the chickens away, our farms are empty.’ (Indigenous chicken farmer, Interview 2)’ Seven LDCCs in diﬀerent cities/counties of Taiwan were willing to participate in the study, and a total of 335 farmers responded to the survey. The seven cities/counties covered 61.87% (2,440/3,944) of all commercial broiler farms in Taiwan. Amongst the 2,440 commercial broiler farm owners, 13.73% of the owners (335/2,440) were recruited for the study. After removing invalid responses from the data set, 303 responses were retained for further analyses. Most broiler farmers participating in the study were more than 30 years old, and over 75% of the farmers had more than 10 years farming experience. Supplementary Table 2 shows the general characteristics of farms and farmers. The mean chicken number per farm was 29,663 ± 1,419 and the mean batch number of the farms (the number of ﬂocks produced per year) was 3.9 ± 0.1. About 80% of the farmers raised their chickens indoors and about 65% of them have senior high school degrees (or higher). Additionally, the phenomena of the mismatch of farmers’ attitudes and behaviours in relation to biosecurity were observed in two ways: (1) Four farmers complained about the practicalities of current biosecurity regulations although they still followed those regulations such as cleaning and disinfection of shipping cages and buckets upon arrival at the farm. In particular, farmers all knew that a farm without evaporative cooling systems shall establish anti-bird netting to prevent transmission of avian inﬂuenza from wild bird contact which was governed by the laws; however, disagree with the necessity of anti-bird netting as a biosecurity strategy due to several reasons such as the eﬀectiveness of the netting in preventing bird contact as Table 2, Supplementary Tables 3, 4 provide the abbreviates and descriptive statistics of farmers’ attitudes and behaviours in relation to biosecurity. Most farmers recognised the importance of the 15 biosecurity measures. 3.1. The qualitative research We just don’t have extra space. We also don’t believe it works for avian inﬂuenza.” (Indigenous chicken farmer, Interview 4). They also expressed their intention to ignore the perceived risks of infectious diseases, particularly for avian inﬂuenza: Frontiers in Veterinary Science frontiersin.org 3.2. The quantitative research Obvious variations in the adoption of speciﬁc biosecurity measures were observed; that is, while 96.7% of the 05 Pao et al. 10.3389/fvets.2023.1070482 TABLE 2 Descriptive statistics of farmers’ attitudes and behaviours in relation to biosecurity (n = 303). p y ( ) Code Variable Abbreviation Category (A) Farmers’ attitudes A1 The implementation of vaccination programmes PercVaccineProgramme 1. Recognising the importance of biosecurity measures (including the responses of “Highly important and Important” of the ﬁve-point Likert scale) 2. Not recognising the importance of biosecurity measures (including the responses of “Neutral& Unimportant &Highly unimportant” of the ﬁve-point Likert scale) A2 The disinfection of chicken houses PercDisinfectedChickHouse A3 Removal for manure and dead animals PercRemovedManure A4 The disinfection of personnel and vehicles PercDisinfectedPersonnel&Vehicle A5 The management of diseased chickens PercDiseasedChick A6 Entrance control of personnel and vehicles PercEnteranceControl A7 All-in-all-out measures PercAllInAllOut A8 The disinfection of equipment PercDisinfectedEquipment A9 The disinfection of shipping cages and buckets PercDisinfectedCage A10 Vermin and wild bird control PercVermitControl A11 Movement control of chickens between chicken houses PercChickControl A12 Ensured water and feed quality PercWater&FeedQuality A13 A barrier between clean area and dirty area in the farm PercTransitionZone A14 Fixed suppliers of chicks PercChickFixedSupply A15 A separate loading area in the farm PercLoadArea (B) Farmers’ behaviours B1 Poultry house completely disinfected after each production round DisinfectedChickHouse Yes†/No B2 A sanitary transition period after each production round SanitaryPeriod Yes†/No B3 Vaccination programmes implemented VaccineProgramme Yes†/No B4 “All-in- all-out” management strictly implemented in the farm AllInAllOut Yes†/No B5 Disinfectant, gloves, shoe covers or taking baths “strictly” used for the cleaning and disinfection of personnel before entering animal housing DisinfectedPersonnelEnterance Yes†/No B6 Cleaning and disinfection of vehicles upon arrival at the farm DisinfectedVehicle Yes†/No B7 Anti-bird netting placed for the poultry houses BirdNetting Yes†/No B8 Disinfectant, gloves, shoe covers or taking baths “strictly” used for the cleaning and disinfection of personnel upon arrival DisinfectedPersonnel Yes†/No B9 Vehicles shipping broilers for sale always empty upon arrival at the farm VehicleArrivalBroilerEmpty Yes†/No B10 Downtime control implemented while personnel arrive at the farm DowntimeControl Yes†/No B11 Using a transition zone in the farm TransitionZone Yes†/No B12 Broilers moved between poultry houses ChickMovedBetweenHouse No†/Yes B13 Using a separate loading area in the farm LoadArea Yes†/No (Continued) (Continued) 06 Frontiers in Veterinary Science frontiersin.org 10.3389/fvets.2023.1070482 Pao et al. Frontiers in Veterinary Science frontiersin.org 3.2. The quantitative research Code Variable Abbreviation Category B14 The disinfection of equipment strictly conducted while moving between diﬀerent poultry houses DisinfectedEquipmentBetweenHouse Yes†/No B15 The disinfection of equipment after use strictly conducted DisinfectedEquipmentAfterUse Yes†/No B16 Disinfection measures taken for equipment before entering the farm DisinfectedEquipmeentBeforeEntering Yes†/No B17 New needles or disinfect needles while vaccinating chicks between diﬀerent poultry houses DisinfectedNeedleBetweenHouse Yes†/No B18 Dead chickens taken out from poultry houses for 2 times or more than 2 times per day FrequencyChickDisposal Yes†/No B19 Cleaning and disinfection of shipping cages and buckets upon arrival at the farm CageDisinfectedArrivial Yes†/No B20 Diseased animals always handled after the healthy ones DiseasdChickHandledAfterHealthyChick Yes†/No B21 Manure removed ManureMoved Yes†/No B22 Cleaning and disinfection of shipping cages and buckets upon before entering animal housing CageEntering Yes†/No B23 Diseased animals isolated from healthy ones DiseasedChickIsolation Yes†/No B24 Shipping cages and collection buckets always empty upon arrival at the farm CageEmpty Yes†/No B25 Using a carcass storage area CarcassStored Yes†/No B26 Chicks originate from ﬁxed suppliers ChickFixedSupply Yes†/No B27 Using a manure storage area ManureStored Yes†/No B28 The quality of chickens’ drinking water checked by bacteriological analysis WaterQuality Yes†/No B29 The quality of feed checked by bacteriological analysis FeedQuality Yes†/No †The desirable action of a biosecurity behaviour. †The desirable action of a biosecurity behaviour. 29 biosecurity measures. The former was the variable of “B1 DisinfectedChickHouse”; the latter was the variable of “B28 WaterQuality” and “B29 FeedQuality.” 29 biosecurity measures. The former was the variable of “B1 DisinfectedChickHouse”; the latter was the variable of “B28 WaterQuality” and “B29 FeedQuality.” farmers implemented the measure of “B21 ManureMoved,” only 5.3% of the participant conducted the measure of “B29 FeedQuality.” Contingency tables described the associations between attitude variables and their relevant behaviour variables. Supplementary Table 5 provides the descriptive statistics of the associations between farmers’ biosecurity attitudes and the relevant biosecurity behaviours. As shown in Figure 3, there is a variety of the relationships between farmers’ biosecurity attitudes and relevant behaviours: • A positive attitude towards biosecurity without taking desirable biosecurity actions: The percentage of the farmers who tended not to translate their knowledge into relevant biosecurity actions despite recognising the importance of biosecurity measures ranged from 0.7 to 42.9% for the 29 biosecurity variables. The former was the variable of “B21 ManureMoved”; the latter was the variable of “B27 ManureStored.” • A positive attitude towards biosecurity together with desirable biosecurity actions: The percentage of the farmers who conducted desirable biosecurity actions together with a positive attitude towards biosecurity ranged from 3.3 to 73.9% for the 29 biosecurity measures. The former was the variable of “B29 FeedQuality”; the latter was the variable of “B1 DisinfectedChickHouse.” • Without a positive attitude towards biosecurity but taking desirable biosecurity actions: The percentage of the participants who still took desirable biosecurity actions despite not having a positive attitude towards the importance of biosecurity ranged from 2 to 25.1% for the 29 biosecurity variables. The former was the variable of “B29 FeedQuality”; the latter was the variable of “B12 ChickMovedBetweenHouse.” • Neither having a positive attitude towards biosecurity nor taking desirable biosecurity actions: The percentage of the farmers who had neither a positive attitude towards biosecurity nor took desirable biosecurity actions ranged from 0.3 to 17.2% for the As shown in Figure 3, the variable of “B1 DisinfectedChickHouse” had the fewest farmers who experienced the mismatch of their attitudes and behaviours (13.9%) while the 07 frontiersin.org Pao et al. 10.3389/fvets.2023.1070482 FIGURE 3 The variety of the relationships between farmers’ biosecurity attitudes and relevant behaviours. While some farmers tended not to translate their knowledge into the relevant biosecurity actions despite recognising the importance of biosecurity measures, a number of farmers still took desirable biosecurity actions despite not having a positive attitude towards the importance of biosecurity. FIGURE 3 The variety of the relationships between farmers’ biosecurity attitudes and relevant behaviours. Frontiers in Veterinary Science frontiersin.org 29 biosecurity measures. The former was the variable of “B1 DisinfectedChickHouse”; the latter was the variable of “B28 WaterQuality” and “B29 FeedQuality.” While some farmers tended not to translate their knowledge into the relevant biosecurity actions despite recognising the importance of biosecurity measures, a number of farmers still took desirable biosecurity actions despite not having a positive attitude towards the importance of biosecurity. translate their knowledge into the relevant biosecurity actions despite recognising the importance of biosecurity measures, a number of farmers still took desirable biosecurity actions despite not having a positive attitude towards the importance of biosecurity. variable of “B13 LoadArea” had the most farmers who experienced the mismatch of their attitudes and behaviours (58.7%). In addition, the proportion of missing values for the 29 biosecurity variables varied from 1.0 to 55.8%. Surprisingly, nine variables such as “B4 AllInAllOut” and “B7 BirdNetting” had a high proportion of missing values (more than 50%). That is, many farmers intended to not answer the nine questions in relation to their biosecurity practises. Additionally, the nine biosecurity measures with a high proportion of missing values also had the fewest farmers who appropriately implemented these biosecurity practises. In addition, the statistical associations between farmers’ biosecurity attitudes and their relevant biosecurity behaviours were listed in Table 3. The results of two-way contingency tests revealed that the statistical associations of biosecurity attitudes and behaviours in relation to 29 biosecurity measures can be divided into two groups: • Group 1. Reject the null hypothesis at the 5% signiﬁcant level (Farmers’ attitudes and behaviours in relation to a speciﬁc biosecurity measure were demonstrated to be signiﬁcantly associated): The associations of farmers’ biosecurity attitudes In summary, the percentage of the farmers who had the mismatch of their attitudes and behaviours towards the 29 biosecurity measures ranged from 13.9 to 58.7%. The results showed that, according to the participants’ self-report, while some farmers tended not to 08 frontiersin.org Pao et al. 10.3389/fvets.2023.1070482 TABLE 3 The statistical associations of farmers’ biosecurity attitudes and their relevant biosecurity behaviours. TABLE 3 The statistical associations of farmers’ biosecurity attitudes and their relevant biosecurity behaviours. Variables of biosecurity attitudes Variables of biosecurity behaviours p-value of the chi-square test of independence p-value of the McNemar’s test Group 1. 29 biosecurity measures. The former was the variable of “B1 DisinfectedChickHouse”; the latter was the variable of “B28 WaterQuality” and “B29 FeedQuality.” Reject the null hypothesis and accept the alternative hypothesis at the 5% signiﬁcant level A13 PercTransitionZone B11 TransitionZone 0.00001 A15 PercLoadArea B13 LoadArea 0.001 A8 PercDisinfectedEquipment B16 B14 B15 B17 Disinfected EquipmeentBeforeEntering DisinfectedEquipmentBetweenHouse DisinfectedEquipmentAfterUse DisinfectedNeedleBetweenHouse 0.000023 0.001 0.013 0.042 A10 PercVermitControl B7 BirdNetting 0.002 A4 PercDisinfectedPersonnel&Vehicle B6 B8 B5 DisinfectedVehicle DisinfectedPersonnel DisinfectedPersonnelEnterance 0.019 0.002 0.002 A11 PercChickControl B12 ChickMovedBetweenHouse 0.031 A14 PercChickFixedSupply B26 ChickFixedSupply 0.046 Group 2. A failure to reject the null hypothesis at the 5% signiﬁcant level A7 PercAllInAllOut B4 AllInAllOut 0.054 0.0251 A2 PercDisinfectedChickHouse B1 B2 DisinfectedChickHouse SanitaryPeriod 0.216 1 0.0001 <0.0001 A1 PercVaccineProgramme B3 VaccineProgramme 0.333 0.0001 A3 PercRemovedManure B25 B18 B27 B21 CarcassStored FrequencyChickDisposal ManureStored ManureMoved 0.103 0.165 0.449 0.495 0.0718 <0.0001 <0.0001 <0.0001 A9 PercDisinfectedCage B22 B19 B24 CageEntering CageDisinfectedArrivial CageEmpty 0.361 0.461 0.813 0.0153 0.0240 0.0138 A5 PercDiseasedChick B20 B23 DiseasdChickHandledAfterHealthyChick DiseasedChickIsolation 0.572 0.989 1 0.1614 A6 PercEnteranceControl B9 B10 VehicleArrivalBroilerEmpty DowntimeControl 0.776 0.863 <0.0001 <0.0001 A12 PercWater&FeedQuality B29 B28 FeedQuality WaterQuality 0.754 0.908 <0.0001 <0.0001 Null hypothesis (H0): Farmers’ attitudes and behaviours in relation to a speciﬁc biosecurity measure are demonstrated to be signiﬁcantly associated. Alternative hypothesis (Ha): Farmers’ attitudes and behaviours in relation to a speciﬁc biosecurity measure are not demonstrated to be signiﬁcantly associated. Null hypothesis (H0): Farmers’ attitudes and behaviours in relation to a speciﬁc biosecurity measure are demonstrated to be signiﬁcantly associated. Alternative hypothesis (Ha): Farmers’ attitudes and behaviours in relation to a speciﬁc biosecurity measure are not demonstrated to be signiﬁcantly associated. • Group 2. A failure to reject the null hypothesis at the 5% signiﬁcant level (Farmers’ attitudes and behaviours in relation to a speciﬁc biosecurity measure were not demonstrated to be signiﬁcantly associated): The associations of farmers’ biosecurity attitudes and behaviours were not observed in the other 17 biosecurity measures. The attitude variables included “A1 PercVaccineProgramme,” “A2 PercDisinfectedChickHouse,” “A3 PercRemovedManure,” “A5 PercDiseasedChick,” “A6 PercEnteranceControl,” “A7 PercAllInAllOut,” “A9 PercDisinfectedCage” and “A12 PercWater&FeedQuality.” The behaviour variables included “B1 DisinfectedChickHouse,” “B2 SanitaryPeriod,” “B3 VaccineProgramme,” “B4 AllInAllOut,” “B9 VehicleArrivalBroilerEmpty,” “B10 DowntimeControl,” “B18 FrequencyChickDisposal,” “B19 CageDisinfectedArrivial,” “B20 DiseasdChickHandledAfterHealthyChick,” “B21 ManureMoved,” “B22 CageEntering,” “B23 and behaviours were observed in 12 biosecurity measures. Better biosecurity attitudes had a positive association with their regular biosecurity behaviours. frontiersin.org Frontiers in Veterinary Science 29 biosecurity measures. The former was the variable of “B1 DisinfectedChickHouse”; the latter was the variable of “B28 WaterQuality” and “B29 FeedQuality.” The attitude variables included “A4 PercDisinfectedPersonnel&Vehicle,” “A8 PercDisinfectedEquipment,” “A10 PercVermitControl,” “A11 PercChickControl,” “A13 PercTransitionZone,” “A14 PercChickFixedSupply” and “A15 PercLoadArea.” The behaviour variables included “B5 DisinfectedPersonnelEnterance,” “B6 DisinfectedVehicle,” “B7 BirdNetting,” “B8 DisinfectedPersonnel,” “B11 TransitionZone,” “B12 ChickMovedBetweenHouse,” “B13 LoadArea,” “B14 EnteringDisinfectedEquipmentBetweenHouse,” “B15 DisinfectedEquipmentAfterUse,” “B16 DisinfectedEquipmeentBefore,” “B17 DisinfectedNeedleBetweenHouse,” and “B26 ChickFixedSupply.” and behaviours were observed in 12 biosecurity measures. Better biosecurity attitudes had a positive association with their regular biosecurity behaviours. The attitude variables included “A4 PercDisinfectedPersonnel&Vehicle,” “A8 PercDisinfectedEquipment,” “A10 PercVermitControl,” “A11 PercChickControl,” “A13 PercTransitionZone,” “A14 PercChickFixedSupply” and “A15 PercLoadArea.” The behaviour variables included “B5 DisinfectedPersonnelEnterance,” “B6 DisinfectedVehicle,” “B7 BirdNetting,” “B8 DisinfectedPersonnel,” “B11 TransitionZone,” “B12 ChickMovedBetweenHouse,” “B13 LoadArea,” “B14 EnteringDisinfectedEquipmentBetweenHouse,” “B15 DisinfectedEquipmentAfterUse,” “B16 DisinfectedEquipmeentBefore,” “B17 DisinfectedNeedleBetweenHouse,” and “B26 ChickFixedSupply.” 09 Frontiers in Veterinary Science frontiersin.org 10.3389/fvets.2023.1070482 Pao et al. DiseasedChickIsolation,” “B24 CageEmpty,” “B25 CarcassStored,” “B27 ManureStored,” “B28 WaterQuality,” and “B29 FeedQuality.” behaviours in relation to 12 biosecurity measures; however, there is no signiﬁcant association observed for the other 17 biosecurity measures. The statistical diﬀerences in the relationships between 15 biosecurity attitude variables and 29 behaviour variables were also observed; that is, while seven attitude variables had a statistical association with 12 behaviour variables, eight attitude variables had no association with 17 behaviour variables (Table 3). While our sample contained an insuﬃcient amount of evidence to conclude that farmers’ attitudes and behaviours are associated in relation to 17 biosecurity measures such as disinfection of shipping cages, we further identiﬁed that the disconnection of biosecurity attitudes and behaviours was observed in three speciﬁc biosecurity measures, including “B20 DiseasdChickHandledAfterHealthyChick,” “B23 DiseasedChickIsolation” and “B25 CarcassStored.” Our study demonstrates that a gap in farmers’ attitudes and behaviours in relation to a given biosecurity measure may exist in some contexts. The results of this hypothesis testing revealed that, at the 5% signiﬁcant level, the null hypothesis was rejected for 12 biosecurity measures such as disinfection of personnel and vehicle; however, for the other 17 biosecurity measures such as disinfection of shipping cages, there was no suﬃcient evidence to demonstrate the signiﬁcance. Furthermore, for the 17 biosecurity measures, McNemar’s test was applied to examine whether attitudes variables had eﬀect on behaviour variables. As shown in Table 3, the disconnection of biosecurity attitudes and behaviours was observed in three biosecurity measures. 4. Discussion The study was aimed at exploring and describing the mismatch in relation to farmers’ biosecurity attitudes and behaviours from farmers’ perspectives (56). Although biosecurity measures implemented on farms are mainly based on the experience and perceptions of farmers, a web of complex factors may inﬂuence farmers’ biosecurity behaviours (17). While farmers encounter multifaceted barriers to implementing on-farm biosecurity, the relationships between attitudes and behaviours amongst farmers are complex. As a phenomenological study, this study utilised an exploratory sequential mixed methods approach to provide ﬁrst- hand, in-depth evidence-based knowledge (47, 57). The qualitative phase included interpretations of the phenomena of the mismatch of farmers’ attitudes and behaviours as well as the identiﬁcation of the issues surrounding the phenomenon from farmers’ hands-on farming experience. Based on the knowledge gathered from the qualitative phase, the quantitative phase provided statistical analysis to describe the gap in farmers’ biosecurity attitudes and behaviours from a larger sample of the population for additional insights. Traditionally, it is believed that farmers’ behaviours align with their attitudes; however, this study conﬁrms the existence of an attitude-behaviour gap and applies social theories to provide an in-depth understanding of how infectious diseases are managed in the animal health context. The attitude change theory can be used to explain the ﬁndings of this study. Firstly, the mismatch of farmers’ attitudes and behaviours for anti-bird netting was observed in both the qualitative and quantitative phases. This phenomenon might result from “Forced compliance behaviour” (62) since this publicly forced biosecurity practises was used by the government for attitude change (63, 64). Consequently, although those farmers were enforced to instal anti-bird netting, they didn’t recognise the importance of this measure. Secondly, taking the “Indigenous chicken farmer, Interview 5” as an example, the “Eﬀort justiﬁcation paradigm” (65, 66) and “Belief disconﬁrmation paradigm” (67, 68) can provide further explanations for those farmers who neither had a positive attitude towards biosecurity nor took desirable biosecurity actions. Farmers need to balance the costs of biosecurity with the expected beneﬁts such as reducing the risks of infectious diseases (69). As such, this farmer who perceived the risks of avian inﬂuenza as uncontrollable did not have the willingness to spend money for setting up a loading zone. Since zero risk of avian inﬂuenza could not be guaranteed, he believed that the strength of a loading area is not enough to prevent avian inﬂuenza. 29 biosecurity measures. The former was the variable of “B1 DisinfectedChickHouse”; the latter was the variable of “B28 WaterQuality” and “B29 FeedQuality.” The attitude variables included “A3 PercRemovedManure” and “A5 PercDiseasedChick.” The behaviour variables included “B20 DiseasdChickHandledAfterHealthyChick,” “B23 DiseasedChickIsolation” and “B25 CarcassStored.” The results conﬁrm the existence of a gap in farmers’ attitudes and behaviours in some contexts. In order to connect the ﬁndings of the qualitative research with the ﬁndings of the quantitative research, a joint display (Table 4) was used to allow sample quotes of the qualitative phase to be compared to the statistical results of the quantitative phase (61). The joint display demonstrated the linking of the two phases of the study in spite of the linking nature of the sequential design in this mixed- methods research (60, 61). The qualitative interviews highlighted the existence of the mismatch of farmers’ attitudes and behaviours in relation to speciﬁc biosecurity measures, which parallels the ﬁndings of the survey that there is a gap in farmers’ attitudes and behaviours in relation to biosecurity, particular for the three speciﬁc biosecurity measures. Evidence from the qualitative and quantitative phases conﬁrms the existence of a gap in farmers’ attitudes and behaviours in some contexts. Frontiers in Veterinary Science frontiersin.org The quantitative phase The descriptive results (n = 303) The quantitative phase The descriptive results (n = 303) The phenomenon observed Sample quotes T A positive attitude towards biosecurity together with desirable biosecurity actions 1. We have done everything we can do [biosecurity]. We know biosecurity is crucial for the prevention of bird ﬂu.’ (White-chicken broiler farmer, Interview 8) 2. “I am not afraid of any disease because of intensive vaccination.” (White-chicken broiler farmer, Interview 5) 3. “Experiences are very important. I believe that my farming experience provided me with the necessary expertise to determine a chicken’s health condition and to decide on relevant vaccination and medication programmes for the prevention and control of infectious diseases” (Indigenous chicken farmer, Interview 4). 4. “Most farmers have personal experience with avian inﬂuenza outbreaks on their farm and we also share our experiences with other farmers. With required knowledge of avian inﬂuenza and biosecurity, I believe that we have implemented the most relevant biosecurity measures that are compatible with our current husbandry practises.” (White-chicken broiler farmer, Interview 6 ) 1 2 Neither having a positive attitude towards biosecurity nor taking desirable biosecurity actions 1. “Experts suggest that it is crucial to set up a transition zone at farm for preventing infectious diseases, but it is impossible for us to set up it. We just don’t have extra space. We also don’t believe it works for avian inﬂuenza.” (Indigenous chicken farmer, Interview 4) 2. “We don’t have a separate loading area at farm. Experts told us that even if there is a loading area, it does not guarantee that we won’t have bird ﬂu. Should I spend money to rebuild my farm if zero risk is not guaranteed?” (Indigenous chicken farmer, Interview 5) 3. “The government suggests that we should transport the chickens by ourselves; otherwise, we should ensure that, when trucks [shipping broilers for sale] enter our farms, the trucks or all shipping cages are empty, but this is impossible. We cannot transport the chickens by ourselves, and we don’t know whether the trucks or their cages are empty or not when they arrive. In addition, I don’t think it is necessary because after the trucks take the chickens away, our farms are empty.” (Indigenous chicken farmer, Interview 2)’ 4. “We will choose the source of chicks according to the quantity and quality of chicks. Frontiers in Veterinary Science The quantitative phase The descriptive results (n = 303) It is impossible and non-sense to ask us to ﬁx suppliers.” (Indigenous chicken farmer, Interview 4)’ 1 2 3 4 5 6 A positive attitude towards biosecurity without taking desirable biosecurity actions 1. “I’ve raised chickens for 40 years, but my son doesn’t want to take on my business. [..] It is hardly for me to do downtime control even though I know it is important. I don’t have enough time and labours. Only my wife and I manage this farm.” (Indigenous chicken farmer, Interview 7) 2. “We know that trucks such as those delivering feed will transmit diseases, but we cannot do disinfection each time. We don’t have enough time and labour to do this.” (Indigenous chicken farmer, Interview 3) 3. “Having a contracted veterinarian is only for the purpose of obeying the policy. We do not have money to employ veterinarians. [...] We seek for the veterinarian’s assistance only when we need a ﬁnal diagnosis.” (White-chicken broiler farmer, Interview 1) 1 2 3 Without a positive attitude towards biosecurity but taking desirable biosecurity actions 1. “We have to instal anti-bird netting for compensation if we have bird ﬂu, but we don’t think that anti-bird netting can prevent bird ﬂu.” (White-chicken broiler farmer, Interview 7) 2. “Due to avian inﬂuenza outbreaks, the government asks us to clean and disinfect shipping cages and buckets upon arrival at the farm. But we don’t think it can prevent us from infections. We still have concerns about avian inﬂuenza.” (Indigenous chicken farmer, Interview 5) 1 2 3 1. The percentage of the farmers who conducted desirable biosecurity actions together with a positive attitude towards biosecurity ranged from 3.3 to 73.9% for the 29 biosecurity measures. 2. 58.4% of the respondents had a positive attitude towards “the implementation of vaccine programme” and implemented “vaccine programme” for each batch. 1. The percentage of the farmers who had neither a positive attitude towards biosecurity nor took desirable biosecurity actions ranged from 0.3 to 17.2% for the 29 biosecurity measures. 1. The percentage of the farmers who had neither a positive attitude towards biosecurity nor took desirable biosecurity actions ranged from 0.3 to 17.2% for the 29 biosecurity measures. 2. 16.5% of the respondents didn’t have a positive attitude towards “using a loading area,” and they didn’t use a “Loading area” for each batch. 3. 2. 11.9% of the respondents didn’t have a positive attitude towards “anti-bird netting”, but they installed “anti-bird netting” for each batch. 1. The percentage of the farmers who didn’t have a positive attitude towards biosecurity but still took desirable biosecurity actions ranged from 2 to 25.1% for the 29 biosecurity measures. 4. Discussion While biosecurity information provided by the government is contrary to their beliefs, this farmer may tend to adhere to his beliefs. In this study, the qualitative data was ﬁrst collected and analysed, and speciﬁc phenomena were observed and further used to develop the quantitative survey to further explore our research question (58–60). The qualitative phase of the mixed-methods research allowed for a deep understanding of the lived experiences of the participants’ on-farm biosecurity practises. By using the results of the qualitative phase, a survey instrument was built to more accurately measure the relationships of farmer’ attitudes and behaviours in relation to biosecurity. In the quantitative research, the percentage of the farmers who had the mismatch of their attitudes and behaviours towards the 29 biosecurity measures ranged from 13.9 to 58.7%. Additionally, out of 29 biosecurity measures, there is a signiﬁcant association between farmers’ attitudes and On the other hand, the social exchange theory can also explain the phenomenon of mismatch between farmers’ biosecurity attitudes and behaviours. The social exchange theory suggests that social behaviour is the result of an exchange process to maximise beneﬁts and minimise costs. Taking downtime control as an example, since those farmers were not willing to pay the cost (when perceived 10 frontiersin.org Pao et al. 10.3389/fvets.2023.1070482 BLE 4 Joint display comparison of the ﬁndings from the qualitative and quantitative phases. 3. 19.5% of the respondents didn’t have a positive attitude towards “cleaning and disinfection of shipping cages and buckets upon arrival at the farm,” but they implemented this measure for each batch. The quantitative phase The descriptive results (n = 303) Furthermore, a group of farmers who intended not to recognise the importance of biosecurity was observed in the qualitative and the quantitative phases, and another group of farmers who had a low implementation level of biosecurity was observed in the quantitative phase, suggesting that the social desirability response bias may only have had a minor impact on this research. As such, the sample is likely to be representative of the target population. However, further research to explore unexamined latent variables such as economic variables, the exact meaning of the missing value and the perspectives of farmers with less farming experience is useful to understand their impacts on farmers’ attitudes and behaviours in relation to biosecurity. It is also crucial to further explore the underly mechanisms of the gap in farmers’ attitudes and behaviours to understand their biosecurity decision-making process. This research did not intend to identify any series of feedback loops from any given biosecurity behaviour to the attitudes. Instead, this research examined the associations of farmers’ biosecurity attitudes and behaviours with the cognitive consistency theory. Our ﬁndings suggest that continuous eﬀorts to ensure farmers’ implementation need to be established through a full understanding of and timely response to farmers’ needs. The ﬁndings of this study are likely to be helpful in shaping future biosecurity strategies for Taiwan’s broiler farmers. Further study is required to tailor biosecurity intervention programs for managing the conﬂicts of farmers’ biosecurity attitudes and behaviours for the success of animal disease prevention and control at the farm level. For example, the approach of targeting strategy in business may be applied to manage the diﬀerences in farmers’ attitudes and behaviours. Larsen et al. (75) suggested that there is a need in improving messaging on biosecurity and risks due to the increase in unsafe practises despite the increase in messaging about infection risks and good biosecurity while Damiaans et al. (76) and Higgins et al. (77) suggested that farmers ignore practising on-farm biosecurity due to a lack of biosecurity information. However, this is likely not the case in this study. Information channels were adequate which was recognised by the participating farmers, since authorities in Taiwan have provided biosecurity training education to the industry since 1998 (78). In addition, similar results can be found in the study of Pao et al. (17) as broiler farmers’ biosecurity information is assumed to be suﬃcient. The quantitative phase The descriptive results (n = 303) 10.6% of the respondents didn’t have a positive attitude towards “using a loading area,” and they didn’t use a “Loading area” for each batch. 4. 13.9% of the respondents didn’t have a positive attitude towards “vehicles shipping broilers for sale always empty upon arrival at the farm,” and they didn’t implement this measure as well. 5. 4.6% of the respondents didn’t have a positive attitude towards ‘shipping cages and collection buckets always empty upon arrival at the farm’, and they didn’t implement this measure as well. 6. 15.5% of the respondents didn’t have a positive attitude towards “ﬁxed suppliers of chicks,” and they didn’t implement this measure as well. 3. 10.6% of the respondents had a positive attitude towards “cleaning and disinfection of vehicles upon arrival at the farm”, but they didn’t implement this measure for each batch. be cost-eﬀective and time-eﬃcient. Additionally, Kristensen and Jakobsen (71) and Brennan and Christley (70) stated that while farmers implement biosecurity practises that are considered useless, other practises that are considered useful may be not carried out. Starting from this study, future studies could explore how direct and indirect factors inﬂuence the relationships between farmers’ attitude-behaviour. beneﬁts were less than potential costs or could not aﬀord the costs, especially when lacking resources), they tended not to translate their knowledge into actions even though they recognised the importance of biosecurity practises. As a result, the exchange of biosecurity behaviours could not be achieved. There are similar ﬁndings criticised by Brennan and Christley (70) that only a small number of farmers have adopted biosecurity practises that are considered to 11 frontiersin.org Pao et al. 10.3389/fvets.2023.1070482 Pao et al. and Lancaster (83) also stated that the knowledge-attitude-behaviour model ignores all the external factors constraining and inﬂuencing those choices. However, when there are concerns about participants’ biosecurity knowledge, especially those in underdeveloped countries, the evaluation of farmers’ biosecurity relevant knowledge still needs to be considered. The assessment of knowledge can be challenging due to unobservable complex constructs of metacognition, which could be aﬀected by participants’ verbal ability and memory capacity (84) although attitude-relevant knowledge can be evaluated by asking people to provide their subjective understandings of research objects (85). Furthermore, considering biosecurity involves a set of measures and relevant attitudes and behaviours, there might be potential restrictions to the length of survey time participants could spend for, despite the limitations of participants’ verbal ability and memory capacity. The quantitative phase The descriptive results (n = 303) An increasing number of studies have emphasised the importance of incorporating human biosecurity behaviours and decisions to understand the link between farmers’ risk attitudes, decision-making process, compliance with biosecurity and the spread of diseases (12, 72–74). As farmers’ biosecurity attitudes and behaviours are related to many factors such as their social-economic background and their experience and knowledge in relation to infectious diseases (17), tailoring biosecurity strategies for farmers is essential for managing the conﬂicts of farmers’ biosecurity attitudes and behaviours. The ﬁndings of this study suggest a lack of eﬀective policy implementation as a result of the government placing the most eﬀort into providing knowledge and enforcement of the policy. Inappropriate application of behavioural change theory will not result in the desired biosecurity behaviours amongst many farmers and may instead induce the mismatch of their attitudes and behaviours. It is time to reconsider the current approach by understanding farmers’ real attitudes and behaviours in relation to biosecurity for the success of animal disease prevention and control at the farm level. The samples for the qualitative and the quantitative phase were limited in size. While there is likely to be selection bias in that the sample overrepresented individuals being a farmer for at least 20 years in the qualitative research, the disconnection of farmers’ attitudes and behaviours in relation to the three biosecurity measures exist in our fairly large sample (N = 303) accounting for 12.4% of the population (2,440 commercial broiler farmers). Selection bias is likely to occur in the situation that the sample overrepresented individuals with a higher level of chicken farming standards in the quantitative research. In addition, people tend to adhere to social norms even when the behaviours are inconsistent with their real attitudes (30, 86). There is a possibility that some farmers tended to report desirable biosecurity-related attitudes and actions because this study did not investigate those variables onsite. However, despite the likely presence of social desirability response bias, whereby respondents tend to report better attitudes and better biosecurity status than they practise (87), this bias is likely to be reduced by the local oﬃcials’ familiarity and long-term partnerships with the research participants (88). Frontiers in Veterinary Science 3. Wei X, Lin W, Hennessy DA. Biosecurity and disease management in China’s animal agriculture sector. Food Policy. (2015) 54:52–64. doi: 10.1016/j.foodpol.2015.04.005 4. Taylor LH, Latham SM, Woolhouse ME. Risk factors for human disease emergence. Philos Trans R Soc Lond B Biol Sci. (2001) 356:983–9. doi: 10.1098/rstb.2001.0888 5. Vorou RM, Papavassiliou VG, Tsiodras S. Emerging zoonoses and vector- borne infections aﬀecting humans in Europe. Epidemiol Infect. (2007) 135:1231– 47. doi: 10.1017/S0950268807008527 Data availability statement All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author. Author contributions similar to Taiwan, most researchers still suggest that biosecurity knowledge needs to be delivered to farmers in China (89–91). It will be more likely to apply to China’s commercial broiler farmers due to the sharing of the same language and culture between Taiwan and China. H-nP did the research and writing of the article as part of her PhD’s thesis. DP, EJ, and WS were H-nP’s supervisors and were involved extensively throughout all phases of the research and writing of this article. T-sY provided support in collecting data and developing the critiqued aspects of data analysis. J-sT and Y-tH provided the critiqued aspects of data analysis and presentation. All authors have given approval for the article submitted for publication. Overall, this study provides an in-depth understanding of the gap in farmers’ biosecurity attitudes and their behaviours based on a fairly large sample of farm owners. The qualitative phase explored the phenomena of the mismatch of farmers’ biosecurity attitudes and behaviours while the subsequent quantitative research conﬁrmed the gap in farmers’ biosecurity attitudes and behaviours in relation to biosecurity in a sample of 303 farmers. The combination of the results of the qualitative and quantitative phases provides detailed insights of the gap in farmers’ attitudes and behaviours in relation to biosecurity. By integrating social theory and knowledge generated by the qualitative research with the ﬁndings of the subsequent quantitative research, this study applies social theories to explain how infectious diseases are managed in the animal health context. The ﬁndings of this study imply that the gap in farmers’ biosecurity attitudes and behaviours in Taiwan will put farmers’ and livestock’s health at risk. This would appear to be particularly important in the context of animal health as farmers are the frontier of on-farm disease management. Traditionally, it is believed that farmers’ behaviours align with their attitudes. As such, these ﬁndings suggest a lack of eﬀective policy implementation as a result of the government placing most eﬀort on providing knowledge and enforcement of policy. Inappropriate application of behavioural change theory will not result in the desired biosecurity behaviours amongst many farmers and may instead induce the mismatch of their attitudes and behaviours. It is time to reconsider the current approach by understanding farmers’ real attitudes and behaviours in relation to biosecurity for the success of animal disease prevention and control at the farm level. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2023. 1070482/full#supplementary-material Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 1. Thomas M, Bouma A, Ekker H, Fonken A, Stegeman J, Nielen M. Risk factors for the introduction of high pathogenicity Avian Inﬂuenza virus into poultry farms during the epidemic in the Netherlands in 2003. Prev Vet Med. (2005) 69:1– 11. doi: 10.1016/j.prevetmed.2004.12.001 The quantitative phase The descriptive results (n = 303) As such, attitude-relevant knowledge was not included in the evaluation of this study. On the other hand, in veterinary epidemiology, the eﬀects of knowledge on attitudes towards on-farm biosecurity are also ambiguous. Ellis-Iversen et al. (79) suggested that knowledge is a factor aﬀecting the performance of zoonotic disease control programmes in UK cattle farms; however, Delabbio et al. (80) and Palmer et al. (81) argued that biosecurity knowledge did not aﬀect farmers’ biosecurity practises in the US, Canada, and Australia. Studies also revealed that the distribution of educational documents did not enhance farmers’ response to disease outbreaks, especially those provided by the governments in the UK, and Australia (9, 81). Additionally, Fabrigar et al. (82) showed that amount of knowledge did not have a signiﬁcant impact on the attitude–behaviour consistency in some conditions. Stanhope As a phenomenological study, it is unlikely to generalise the ﬁndings of this study to other areas until the results of repeated studies from other populations are similar. A replication of this study for other species or in other countries could help understand the mechanisms of farmers’ on-farm biosecurity decision-making processes and allow the development of more reliable strategies for the improvement of on-farm biosecurity. The ﬁndings of this study can be applied to other developing countries in regions with similar production conditions, where biosecurity information has been delivered to farmers for many years, but there is still a need to improve the practise of biosecurity at the farm level (2). For example, Frontiers in Veterinary Science 12 frontiersin.org Pao et al. 10.3389/fvets.2023.1070482 Acknowledgments The authors thank the Executive Yuan and the Bureau of Animal and Plant Health Inspection and Quarantine in Taiwan for oﬀering a doctoral study scholarship to H-nP and Professor Chin-cheng Chou, the LDCC oﬃcials, the staﬀof the Poultry Association and all the participating chicken farmers for supporting the data collection. The authors are grateful to members of the Veterinary Epidemiology, Economics and Public Health group of the Royal Veterinary College, University of London, United Kingdom for their assistance in data analysis. Ethics statement The studies involving human participants were reviewed and approved by the Ethics and Welfare Committee of the Royal Veterinary College, University of London, United Kingdom. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. 2. Cui B, Wang LD, Ke J, Tian Y. Chinese poultry farmers’ decision-making for avian inﬂuenza prevention: a qualitative analysis. Zoonoses Public Health. (2019) 66:647–54. doi: 10.1111/zph.1 2617 Frontiers in Veterinary Science References Front Vet Sci. (2020) 7:577. doi: 10.3389/fvets.2020.00577 12. Bucini G, Merrill SC, Clark E, Moegenburg SM, Zia A, Koliba CJ, et al. Risk attitudes aﬀect livestock biosecurity decisions with ramiﬁcations for disease control in a simulated production system. Front Vet Sci. (2019) 6:196. doi: 10.3389/fvets.2019.00196 37. Fischer I, Avrashi S, Oz T, Fadul R, Gutman K, Rubenstein D, et al. The behavioural challenge of the COVID-19 pandemic: indirect measurements and personalized attitude changing treatments (IMPACT). R Soc Open Sci. (2020) 7:201131. doi: 10.1098/rsos.201131 13. 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https://openalex.org/W2895555782	https://vestnik.donstu.ru/jour/article/download/1341/1323	Russian	null	GENERAL SCALARIZATION METHOD OF DYNAMIC ELASTIC FIELDS IN TRANSVERSALLY ISOTROPIC MEDIA AND ITS NEW APPLICATIONS	Vestnik Donskogo gosudarstvennogo tehničeskogo universiteta	2,018	cc-by	8,581	I. P. Miroshnichenko1, V. P. Sizov2** 1 Don State Technical University, Rostov-on-Don, Russian Federation 2 Rostov Scientific Research Institute for Radiocommunication, Rostov-o 1 Don State Technical University, Rostov-on-Don, Russian Federation 2 Rostov Scientific Research Institute for Radiocommunication, Rostov-on-Don, Russian Federation Introduction. An efficient technique of tensor field scalariza- tion is successfully used while investigating tensor elastic fields of displacements, stresses and deformations in the lay- ered structures of different materials, including transversally isotropic composites. These fields can be expressed through the scalar potentials corresponding to the quasi-longitudinal, quasi-transverse, and transverse-only waves. Such scalariza- tion is possible if the objects under consideration are tensors relating to the subgroup of general coordinate conversions, when the local affine basis has one invariant vector that coin- cides with the material symmetry axis of the material. At this, the known papers consider structures where this vector coin- cides with the normal to the boundary between layers. Howev- er, other cases of the mutual arrangement of the material symmetry axis of the material and the boundaries between layers are of interest on the practical side. Introduction. An efficient technique of tensor field scalariza- tion is successfully used while investigating tensor elastic fields of displacements, stresses and deformations in the lay- ered structures of different materials, including transversally isotropic composites. These fields can be expressed through the scalar potentials corresponding to the quasi-longitudinal, quasi-transverse, and transverse-only waves. Such scalariza- tion is possible if the objects under consideration are tensors relating to the subgroup of general coordinate conversions, when the local affine basis has one invariant vector that coin- cides with the material symmetry axis of the material. At this, the known papers consider structures where this vector coin- cides with the normal to the boundary between layers. Howev- er, other cases of the mutual arrangement of the material symmetry axis of the material and the boundaries between layers are of interest on the practical side. Введение. При исследовании тензорных упругих полей перемещений, напряжений и деформаций в слоистых конструкциях из различных материалов, включая трансверсально-изотропные композиты, успешно применяется эффективный метод скаляризации тен- зорных полей. Данные поля могут быть выражены через скалярные потенциалы, соответствующие ква- зипродольным, квазипоперечным и чисто поперечным волнам. Такая скаляризация возможна, если рассмат- риваемые объекты являются тензорами относительно подгруппы общих преобразований координат, когда локальный аффинный базис имеет один инвариантный вектор, который совпадает с осью материальной сим- метрии материала. * Работа выполнена в рамках Госзадания Минобрнауки РФ № 9.9770.2017/БЧ и при частичной поддержке гранта РФФИ № 16-08-00740. E-mail: ipmir@rambler.ru * The research is done within the frame of government task of RF Ministry of Education and Science no. 9.9770.2017/ БЧ and is supported in part by RFFI grant no. 16-08-00740. Вестник Донского государственного технического университета. 2018. Т. 18, № 3. C. 258–264. ISSN 1992-5980 eISSN 1992-6006 Vestnik of Don State Technical University. 2018. Vol. 18, no. 3, pp. 258–264. ISSN 1992-5980 eISSN 1992-6006 Вестник Донского государственного технического университета. 2018. Т. 18, № 3. C. 258–264. ISSN 1992-5980 eISSN 1992-6006 Vestnik of Don State Technical University. 2018. Vol. 18, no. 3, pp. 258–264. ISSN 1992-5980 eISSN 1992-6006 Обобщенный метод скаляризации динамических упругих полей в трансверсально-изотропных средах и его новые применения* 1 2 онской государственный технический университет, г. Ростов-на-Дону, Российская Федерация остовский-на-Дону научно-исследовательский институт радиосвязи, г. Ростов-на-Дону, Российская Федерация The research is done within the frame of government task of RF Ministry of Education and Science no. 9.9770.2017/ БЧ and is supp RFFI grant no. 16-08-00740. I. P. Miroshnichenko1, V. P. Sizov2 При этом в известных работах рас- сматриваются конструкции, где этот вектор совпадает с нормалью к границе между слоями. Однако, для практики представляют интерес и другие случаи вза- имного расположения оси материальной симметрии материала и границы между слоями. Materials and Methods. The work objective is further devel- opment of the scalarization method application in the bounda- ry value problems of the dynamic elasticity theory for the cases of an arbitrary arrangement of the material symmetry axis relative to the boundary between layers. The present re- search and methodological apparatus are developed through the general technique of scalarization of the dynamic elastic fields of displacements, stresses and strains in the transversally isotropic media. Целью является дальнейшее развитие применения метода скаляризации в граничных задачах динамиче- ской теории упругости на случаи произвольного рас- положения оси материальной симметрии по отноше- нию к границе между слоями. http://vestnik.donstu.ru Методы исследования. Предлагаемый научно- методический аппарат разработан на основе использо- вания обобщенного метода скаляризации динамических упругих полей перемещений, напряжений и деформаций в трансверсально-изотропных средах. Research Results. New design ratios for the determination of the displacement fields, stresses and deformations in the trans- versally isotropic media are obtained for the cases of an arbi- trary arrangement of the material symmetry axes of the layer Результаты исследования. Получены новые расчетные соотношения для определения полей перемещений, напряжений и деформаций в трансверсально-изотропных средах на случаи произвольного расположения осей 258 Мирошниченко И. П. и др. Обобщенный метод скаляризации динамических упругих полей в трансверсально-изотропных средах Miroshnichenko I. P. and the others. General scalarization method of dynamic elastic fields in transversally isotropic media materials with respect to the boundaries between layers. Discussion and Conclusions. The present research and meth- odological apparatus are successfully used in determining the stress-strain state in the layered structures of transversally isotropic materials, and in analyzing the diagnosis results of the state of the plane-layered and layered cylindrical structures under operation. materials with respect to the boundaries between layers. Discussion and Conclusions. The present research and meth- odological apparatus are successfully used in determining the stress-strain state in the layered structures of transversally isotropic materials, and in analyzing the diagnosis results of the state of the plane-layered and layered cylindrical structures under operation. материальной симметрии материалов слоев по отно- шению к границам между слоями. Обсуждение и заключения. I. P. Miroshnichenko1, V. P. Sizov2 Расчетная схема Вестник Донского государственного технического университета. 2018. Т. 18, № 3. C. 258–264. ISSN 1992-5980 eISSN 1992-6006 Vestnik of Don State Technical University. 2018. Vol. 18, no. 3, pp. 258–264. ISSN 1992-5980 eISSN 1992-6006 Вестник Донского государственного технического университета. 2018. Т. 18, № 3. C. 258–264. ISSN 1992-5980 eISSN 1992-6006 Vestnik of Don State Technical University. 2018. Vol. 18, no. 3, pp. 258–264. ISSN 1992-5980 eISSN 1992-6006 Вестник Донского государственного технического университета. 2018. Т. 18, № 3. C. 258–264. ISSN 1992-5980 eISSN 1992-6006 Vestnik of Don State Technical University. 2018. Vol. 18, no. 3, pp. 258–264. ISSN 1992-5980 eISSN 1992-6006 Расчетные соотношения В соответствии с [4]: Расчетные соотношения оотношения ии с [4]: В соответствии с [4]: ( ) ( ) ( ) ( ) 1 2 1 ( ) 1 ( ) L L T T J J K N N K i i i J i J i i i T U D D D g w g v g                       ; ( ) ( ) ( ) ( ) 1 2 1 ( ) 1 ( ) L L T T J J K N N K i i i J i J i i i T U D D D g w g v g                       ; ( ) ( ) ( ) ( ) 1 2 3 ( ) 4 ( ) L L L L J J J ij ij i j i j J i j d g d d d      ( ) ( ) ( ) ( ) 1 2 3 ( ) 4 ( ) T T T T J J J ij J i j J i j i J j d g d d d w        2 ( ) ( ) 4 ( ) ( ) 2 [ ( ) ( ) ] K N N K J K N J N K i j i j i j i j J g a a v      . (1) (1) 2 ( ) ( ) 4 ( ) ( ) 2 [ ( ) ( ) ] K N N K J K N J N K i j i j i j i j J g a a v      . Расчетные соотношения Компоненты тензорных полей перемещений i U и напряжений ij  в системе координат, связан- ной с анизотропией материала , J K x x , могут быть записаны следующим образом: ( ) 2 ( ) ( ) ( ) ( ) ( ) 1 2 1 ( ) ( ) ( ) T L L L T T J T U i D D D g w g      ; ( ) ( ) ( ) ( ) ( ) 1 1 ( ) T T L L T K T U i D D w g    ; (2) (2) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 1 2 3 4 1 2 3 4 [ ( ) ] ( ) L T L L L L T T T T T JJ d d d d i d d d d w           ; ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 1 2 3 4 1 2 3 4 [ ( ) ] ( ) L T L L L L T T T T T JJ d d d d i d d d d w           ; ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 3 4 3 4 1 1 ( ) ( ) 2 2 T L L L L L L T T T JK d d i d d w       ; ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 ( ) ( ) L T L L T T T d d i d d    ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 1 2 3 4 1 2 3 4 [ ( ) ] ( ) L T L L L L T T T T T JJ d d d d i d d d d w           ; ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 3 4 3 4 1 1 ( ) ( ) 2 2 T L L L L L L T T T JK d d i d d w       ; ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 3 4 3 4 1 1 ( ) ( ) 2 2 T L L L L L L T T T JK d d i d d w       ; ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 1 4 1 4 ( ) ( ) L T L L T T T KK d d i d d w       . Расчетные соотношения ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 1 4 1 4 ( ) ( ) L T L L T T T KK d d i d d w       . Здесь потенциалы квазипродольных  и квазипоперечных w волн должны удовлетворять волновому уравнению с соответствующим волновым числом g ( ( ) L g или ( ) T g ) и имеют вид: Здесь потенциалы квазипродольных  и квазипоперечных w волн должны удовлетворять волновому уравнению с соответствующим волновым числом g ( ( ) L g или ( ) T g ) и имеют вид: Здесь потенциалы квазипродольных  и квазипоперечных w волн должны удовлетворять волновому уравнению с соответствующим волновым числом g ( ( ) L g или ( ) T g ) и имеют вид: ( ) ( ) 0 L L J K i x i x e e    ; ( ) ( ) 0 T T J K i x i x w w e e    , (3) 2 ( ) 0 g    . (4) ( ) ( ) 0 L L J K i x i x e e    ; ( ) ( ) 0 T T J K i x i x w w e e    , (3) 0e e    ; ( ) ( ) 0 T T J K i x i x w w e e    , (3) где 2 2 2 g  ; 2 ( ) 0 g w    . (4) (3) 0 i x i x w w e e    , где 2 2 2 g  ; 2 ( ) 0 g w    . (4) где 2 2 2 g  ; 2 ( ) 0 g w    . 2 ( ) 0 g w    . I. P. Miroshnichenko1, V. P. Sizov2 Предлагаемый научно- методический аппарат успешно использован при определении напряженно-деформированного состоя- ния в слоистых конструкциях, выполненных из транс- версально-изотропных материалов, и при анализе ре- зультатов диагностики состояния плоскослоистых и слоистых цилиндрических конструкций, находящихся в эксплуатации. Keywords: scalarization method, transversally isotropic me- dium, acoustic waves, composite materials. Ключевые слова: метод скаляризации, трансверсально- изотропная среда, акустические волны, композиционные материалы. For citation: I.P. Miroshnichenko, V.P. Sizov. General sca- larization method of dynamic elastic fields in transversally isotropic media and its new applications. Vestnik of DSTU, 2018, vol. 18, no.3, pp. 258−264. https://doi.org/10.23947/1992-5980-2018-18-3-258-264 Образец для цитирования: Мирошниченко, И. П. Обоб- щенный метод скаляризации динамических упругих полей в трансверсально-изотропных средах и его новые приме- нения / И. П. Мирошниченко, В. П. Сизов // Вестник Дон- ского гос. техн. ун-та. — 2018. — Т.18, №3. — С. 258−264. https://doi.org/10.23947/1992-5980-2018-18-3-258-264 Введение. При исследовании тензорных упругих полей перемещений, напряжений и деформаций в слоистых конструкциях из различных материалов, включая трансверсально-изотропные композиты [1–3], успешно применяется эффективный метод скаляризации тензорных полей, предложенный в [4], когда отмеченные поля могут быть выражены через скалярные потенциалы, соответствующие квазипро- дольным, квазипоперечным и чисто поперечным волнам соответственно. Такая скаляризация возможна, если рассматриваемые объекты являются тензорами относительно подгруппы общих преобразований координат, когда локальный аффинный базис имеет один инвариант- ный вектор, который совпадает с осью материальной симметрии материала. В [5] рассматриваются кон- струкции, где этот вектор совпадает с нормалью к границе между слоями. Однако для практики пред- ставляют интерес и другие случаи взаимного расположения оси материальной симметрии материала и границы между слоями. Цель исследования. Целью работы является дальнейшее развитие применения метода скаляриза- ции в граничных задачах динамической теории упругости на случаи произвольного расположения оси материальной симметрии по отношению к границе между слоями. Постановка задачи. Первоначально в системе координат с допустимым репером находятся ком- поненты тензоров перемещений и напряжений относительно этого репера. Затем, зная все компоненты тензорных полей в данной системе координат, можно путем перехода к системе координат, связанной с границей между слоями найти нормальные и касательные компоненты перемещений и напряжений, со- ответствующие площадке, лежащей на границе. Эти компоненты далее используются для удовлетворе- ния граничным условиям задачи [5]. Более подробно изложим вышесказанное на примере решения задач для плоскослоистых кон- струкций. Рассмотрим случай, когда главная ось симметрии трансверсально-изотропного материала со- ставляет угол  по отношению к нормали поверхности плоской границы между слоями (рис. 1). При этом считаем, что поле не зависит от координаты y . Рис. 1. Расчетная схема Рис. 1. Расчетная схема Механика 259 Рис. 1. Расчетные соотношения (4) (4) где 2 2 2 g  ; (4) Входящие в (2) коэффициенты D и d , определены в [5] и имеют вид: ( ) L Входящие в (2) коэффициенты D и d , определены в [5] и имеют вид: ( ) L ( ) 2 ( ) 13 44 1 ( ) ( ) ( ) 2 2 2 2 44 11 13 44 ( ) ( )( ) L L L L L g C C D h C g h C C C        ; ( ) ( ) ( ) ( ) ( ) 2 2 2 2 2 2 ( ) ( ) 44 13 11 2 1 ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 2 2 2 2 44 11 13 44 (2 ) ( ) ( )( ) L L L L L L L L L L L L L h C C g h C g g D D h h C g h C C C g h             ; ( ) 2 ( ) 13 44 1 ( ) ( ) ( ) 2 2 2 2 44 13 11 ( ) (2 ) ( ) T T T T T g C C D h C C g h C        ; ( ) 2 ( ) 13 44 1 ( ) ( ) ( ) 2 2 2 2 44 11 13 44 ( ) ( )( ) L L L L L g C C D h C g h C C C        ; ( ) ( ) ( ) ( ) ( ) 2 2 2 2 2 2 ( ) ( ) 44 13 11 2 1 ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 2 2 2 2 44 11 13 44 (2 ) ( ) ( )( ) L L L L L L L L L L L L L h C C g h C g g D D h h C g h C C C g h             ; ( ) 2 ( ) 13 44 1 ( ) ( ) ( ) 2 2 2 2 44 13 11 ( ) (2 ) ( ) T T T T T g C C D h C C g h C        ; (5) ( ) ( ) ( ) ( ) ( ) 2 2 1 1 3 1 2 ( ) L L L L L d g a h a D D    ; ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 3 4 2 5 2 5 1 (2 ) L L L L L L d g a h a D a D a D     ; ( ) ( ) ( ) ( ) 3 2 2 4 1 2 2 2 (2 ) L L L L d a D a D D    ; ( ) ( ) 4 2 1 2 L L d a D  ; ( ) 2 ( ) ( ) ( ) ( ) ( ) 1 1 1 3 1 ( ) (1 ) T T T T T T T h d a g D a g D g               ; http://vestnik.donstu.ru (5) 44 13 11 ( ) ( ) g  ( ) ( ) ( ) ( ) ( ) 2 2 1 1 3 1 2 ( ) L L L L L d g a h a D D    ; ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 3 4 2 5 2 5 1 (2 ) L L L L L L d g a h a D a D a D     ; ( ) ( ) ( ) ( ) 3 2 2 4 1 2 2 2 (2 ) L L L L d a D a D D    ; ( ) ( ) 4 2 1 2 L L d a D  ; ( ) 2 ( ) ( ) ( ) ( ) ( ) 1 1 1 3 1 ( ) (1 ) T T T T T T T h d a g D a g D g               ; 260 Мирошниченко И. Расчетные соотношения П. и др. Обобщенный метод скаляризации динамических упругих полей в трансверсально-изотропных средах Miroshnichenko I. P. and the others. General scalarization method of dynamic elastic fields in transversally isotropic media Мирошниченко И. П. и др. Обобщенный метод скаляризации динамических упругих полей в трансверсально-изотропных средах Miroshnichenko I. P. and the others. General scalarization method of dynamic elastic fields in transversally isotropic media ( ) 2 ( ) ( ) ( ) ( ) ( ) ( ) 2 3 1 4 5 1 ( ) (1 ) 2 T T T T T T T T h d a g D a g a g D g                ; ( ) 2 ( ) ( ) ( ) ( ) 3 2 4 1 ( ) 2 2 2 T T T T T T h d a g a g D g              ; ( ) ( ) 4 2 1 ( ) 1 2 T T T d a D g  ; (6) 1 11 2 2 a C a   ;   2 11 12 1 2 a C C   ;   3 11 12 13 44 4 1 2 a C C C C a      ;   4 11 12 44 1 2 a C C C    ; 5 11 33 13 44 2( 2 ) a C C C C     , (7) (6) (7) где ij C — модули упругости материала, записанные по свернутому индексу [6−7]. где ij C — модули упругости материала, записанные по свернутому индексу [6−7]. В формулах (5) и (6) для этих коэффициентов: В формулах (5) и (6) для этих коэффициентов: cos cos( ) z h g    ; sin cos( ) zg    ; cos( ) zg g  . В граничные условия динамических задач теории упругости входят компоненты перемещений z U , x U и напряжений zz , zx , записанные в системе координат z , x , связанной с границей (рис. 1). Расчетные соотношения В граничные условия динамических задач теории упругости входят компоненты перемещений z U , x U и напряжений zz , zx , записанные в системе координат z , x , связанной с границей (рис. 1). Системы координат z , x и Jx , K x связаны между собой соотношениями [8]: Системы координат z , x и Jx , K x связаны между собой соотношениями [8]: sin cos K J x x   ; cos sin K J x x x    (8) sin cos K J z x x   ; cos sin K J x x x    (8) (8) или: cos sin K x x z   ; sin cos Jx x z   . (9) ия (8) и (9), по формулам [5]: cos sin K x x z   ; sin cos Jx x z   . (9) Используя соотношения (8) и (9), по формулам [5]: (9) Используя соотношения (8) и (9), по формулам [5]: m i m i x U U x   ; m p ij mp i j x x x x       , (10) (10) где коэффициенты с чертой относятся к системе координат z , x (назовем их «новыми» координатами), а без черты к системе координат Jx , K x («старые» координаты). где коэффициенты с чертой относятся к системе координат z , x (назовем их «новыми» координатами), а без черты к системе координат Jx , K x («старые» координаты). Запишем компоненты полей перемещений z U , x U и напряжений zz , zx , входящих в граничные условия через компоненты (2): cos sin z J K U U U     ; sin cos x J K U U U     ; (11) (11) 2 2 cos sin2 sin zz JJ JK KK        ; 2 1 sin 2 ( ) (1 2sin ) 2 zx JJ KK JK        . (12) 2 2 cos sin2 sin zz JJ JK KK        ; 2 1 sin 2 ( ) (1 2sin ) 2 zx JJ KK JK        . Расчетные соотношения (17) (17) Аналогично, используя (2), (14) и (15), из (12) получим соотношения для компонент напряжений, входящих в граничные условия задач: Аналогично, используя (2), (14) и (15), из (12) получим соотношения для компонент напряжений, входящих в граничные условия задач: ( ) ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 2 2 1 2 3 4 3 4 1 4 2 2 2 cos 1 cos sin sin {cos [ ( ) ] sin 2 ( ) sin ( )} 2 cos ( ) cos ( ) cos ( ) L L L L L L L L L L L zz z z z d d d d g d d g d d g                     ( ) ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 2 2 1 2 3 4 3 4 1 4 2 2 2 cos 1 cos sin sin {cos [ ( ) ] sin 2 ( ) sin ( )} 2 cos ( ) cos ( ) cos ( ) L L L L L L L L L L L zz z z z d d d d g d d g d d g                     ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 1 2 3 4 3 4 2 2 cos cos sin 1 cos {cos ( ) sin 2 ( ) cos( ) cos( ) 2 cos ( ) cos ( ) T T T T T T T T T T z z z z i g d d d d g i g d d g                  ( ) 2 ( ) ( ) ( ) 2 1 4 2 cos cos sin 2 ( )} cos( ) cos ( ) T T T T z z i g d d g w        ; ( ) ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 2 2 1 2 3 4 3 4 1 4 2 2 2 cos 1 cos sin sin {cos [ ( ) ] sin 2 ( ) sin ( )} 2 cos ( ) cos ( ) cos ( ) L L L L L L L L L L L zz z z z d d d d g d d g d d g                     ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 1 2 3 4 3 4 2 2 cos cos sin 1 cos {cos ( ) sin 2 ( ) cos( ) cos( ) 2 cos ( ) cos ( ) T T T T T T T T T T z z z z i g d d d d g i g d d g                  ( ) ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 2 2 3 4 4 3 4 2 2 2 1 cos sin 1 cos sin { sin 2 [ ( ) ] (1 2sin )( } 2 2 cos ( ) cos ( ) cos ( ) L L L L L L L L L zx z z z d d d g d g d d g                   ( ) ( ) 2 2 2 ( ) ( ) ( ) ( ) ( ) 2 2 2 2 3 4 3 4 2 2 1 cos cos sin sin 1 cos { sin 2 ( ) (1 2sin ) ( )} ; 2 cos( ) cos( ) 2 cos ( ) cos ( ) T T T T T T T z z i d d d g i d d g w                  (18) http://vestnik.donstu.ru (18) Таким образом, компоненты перемещений и напряжений, входящие в граничные условия (систе- ма координат z , x связана с границей), определяются соотношениями (16)–(18), где проекции волново- го вектора g в системах координат z , x и Jx , K x связаны соотношениями (15). Расчетные соотношения ( ) ( ) 0 L L x z i g x i g z e e  ; ( ) ( ) 0 T T x z i g x i g z w w e e  . ( ) ( ) 0 L L x z i g x i g z e e  ; ( ) ( ) 0 T T x z i g x i g z w w e e  . Расчетные соотношения (15) (15) sin( ) cos( )   П (15) (14) й sin( ) cos( )   Подставляя (15) в (14), получим выражения для перемещений в «новых» координатах: Подставляя (15) в (14), получим выражения для перемещений в «новых» координатах: ( ) ( ) ( ) ( ) ( ) 1 2 1 cos cos cos ( ) ( cos ) cos( ) cos( ) L L L T T z z z U i g D D g D w            ; ( ) ( ) ( ) ( ) ( ) ( ) 1 2 1 1 cos sin sin cos sin( )cos sin [ ( ) ] [ ] cos( ) cos( ) cos( ) cos( ) L L L L T T x z z U i g D D D g D w                  ( ) ( ) ( ) ( ) ( ) ( ) 1 2 1 1 cos sin sin cos sin( )cos sin [ ( ) ] [ ] cos( ) cos( ) cos( ) cos( ) L L L L T T x z z U i g D D D g D w                  (16) тветствии с (13) и (15): ( ) ( ) ( ) ( ) ( ) ( ) 1 2 1 1 cos sin sin cos sin( )cos sin [ ( ) ] [ ] cos( ) cos( ) cos( ) cos( ) L L L L T T x z z U i g D D D g D w                  (16) где, в соответствии с (13) и (15): (16) где, в соответствии с (13) и (15): где, в соответствии с (13) и (15): где, в соответствии с (13) и (15): ( ) ( ) 0 L L x z i g x i g z e e  ; ( ) ( ) 0 T T x z i g x i g z w w e e  . Расчетные соотношения (12) 2 1 sin 2 ( ) (1 2sin ) 2 zx JJ KK JK        . (12) (12) В «новых» координатах потенциальные функции (3) имеют вид: В «новых» координатах потенциальные функции (3) имеют вид: В «новых» координатах потенциальные функции (3) имеют вид: ( ) ( ) ( sin cos ) ( cos sin ) 0 L L i x z i x z e e        ; ( ) ( ) ( sin cos ) ( cos sin ) 0 T T i x z i x z w w e e        , ( ) ( ) ( sin cos ) ( cos sin ) 0 L L i x z i x z e e        ; ( ) ( ) ( sin cos ) ( cos sin ) 0 T T i x z i x z w w e e        , а перемещения (11) запишутся следующим образом: ( ) T а перемещения (11) запишутся следующим образом: ) у у р ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 1 2 1 1 1 ( ) ( ) cos [ ( ) ( ) ] sin [ ( ) ] T T L L L T T L L T T z T T U i D D D g w i D D g w g g              (13) ( ) ( ) ( ) 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) 1 2 1 1 1 ( ) ( ) sin [ ( ) ( ) ] cos [ ] T T T L L T T L L L x T T U i D D D g w i D D w g g            (14) (13) (14) Здесь волновые числа , , g определены относительно «старого» репера (рис. 2). Здесь волновые числа , , g определены относительно «старого» репера (рис. 2). 261 Вестник Донского государственного технического университета. 2018. Т. 18, № 3. C. 258–264. Расчетные соотношения ISSN 1992-5980 eISSN 1992-6006 Vestnik of Don State Technical University. 2018. Vol. 18, no. 3, pp. 258–264. ISSN 1992-5980 eISSN 1992-6006 Рис. 2. Схема «взаимосвязи» волновых чисел Рис. 2. Схема «взаимосвязи» волновых чисел Рис. 2. Схема «взаимосвязи» волновых чисел Рис. 2. Схема «взаимосвязи» волновых чисел Волновые числа , , которые являются проекциями вектора g на «старый» репер Jx , K x и волновые числа x g , zg которые являются проекциями вектора g на «новый репер» z , x связаны меж- ду собой следующими соотношениями: cos cos sin( ) cos( ) x z g g       ; sin sin sin( ) cos( ) x z g g       ; sin( ) cos( ) x z g g g    . Расчетные соотношения General scalarization method of dynamic elastic fields in transversally isotropic media (1) (2) (3) (4) c c C c c       , (19) где ( ) ( ) ( ) ( ) 11 12 11 12 (1) (2) 21 22 21 22 31 32 31 32 (3) (4) 41 42 41 42 0 0 0 0 0 0 0 0 0 0 0 0 L z T z x L z T z i g z i g z i g x i g z i g z e c c c c C C c c c c e C e c c c c e C C c c c c e                                                                ; (19) где ( ) ( ) ( ) ( ) 11 12 11 12 (1) (2) 21 22 21 22 31 32 31 32 (3) (4) 41 42 41 42 0 0 0 0 0 0 0 0 0 0 0 0 L z T z x L z T z i g z i g z i g x i g z i g z e c c c c C C c c c c e C e c c c c e C C c c c c e                                                                ; где ( ) ( ) T L x x x g g g   ; (20) ( ) ( ) ( ) (20) ( ) ( ) T L x x x g g g   ; (20) ( ) ( ) ( ) 11 1 2 cos cos ( ) cos( ) L L L z C i g D D      ; ( ) ( ) 12 1 cos ( cos ) cos( ) T T z C g D       ; ( ) ( ) ( ) 11 1 2 cos cos ( ) cos( ) L L L z C i g D D      ; ( ) ( ) ( ) ( ) 21 1 2 1 cos sin sin cos [ ( ) ] cos( ) cos( ) L L L L z C i g D D D          ; ( ) ( ) 22 1 sin( )cos sin [ ] cos( ) cos( ) T L z C g D         ; ( ) ( ) ( ) ( ) 21 1 2 1 cos sin sin cos [ ( ) ] cos( ) cos( ) L L L L z C i g D D D          ; ( ) ( ) 22 1 sin( )cos sin [ ] cos( ) cos( ) T L z C g D         ; ( ) ( ) ( ) ( ) 21 1 2 1 cos sin sin cos [ ( ) ] cos( ) cos( ) L L L L z C i g D D D          ; ( ) ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 2 2 31 1 2 3 4 3 4 1 4 2 2 2 cos 1 cos sin sin cos [ ( ) ] sin 2 ( ) sin ( ) 2 cos ( ) cos ( ) cos ( ) L L L L L L L L L L L z z z C d d d d g d d g d d g                   ( ) ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 2 2 31 1 2 3 4 3 4 1 4 2 2 2 cos 1 cos sin sin cos [ ( ) ] sin 2 ( ) sin ( ) 2 cos ( ) cos ( ) cos ( ) L L L L L L L L L L L z z z C d d d d g d d g d d g                   ( ) ( ) ( ) ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 32 1 2 3 4 3 4 2 2 cos cos sin 1 cos cos ( ) sin 2 ( ) cos( ) cos( ) 2 cos ( ) cos ( ) T T T T T T T T T T z z z z C i g d d d d g i g d d g                  ( ) 2 ( ) ( ) ( ) 2 2 1 4 2 cos sin sin ( ) cos( ) cos ( ) T T T T z z i g d d g        ; ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 1 2 3 4 3 4 2 2 cos cos sin 1 cos ( ) sin 2 ( ) os( ) cos( ) 2 cos ( ) cos ( ) T T T T T T T T T T z z z z i g d d d d g i g d d g                ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 1 2 3 4 3 4 2 2 cos cos sin 1 cos ( ) sin 2 ( ) os( ) cos( ) 2 cos ( ) cos ( ) T T T T T T T T T T z z z z i g d d d d g i g d d g                ( ) 2 ( ) ( ) ( ) 2 2 cos sin sin ( ) T T T T i g d d g      ; ( ) ( ) ( ) 2 2 ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 2 41 2 3 4 4 3 4 2 2 2 1 cos sin 1 cos sin sin 2 [ ( ) ] (1 2sin )( ; 2 2 cos ( ) cos ( ) cos ( ) L L L L L L L L L z z z C d d d g d g d d g                 ( ) 2 2 ( ) ( ) ( ) 2 42 2 3 4 2 1 cos cos sin sin 2 ( ) 2 cos( ) cos ( ) T T T T z C i d d d g          ( ) 2 ( ) ( ) 2 2 3 4 2 sin 1 cos (1 2sin ) ( ) cos( ) 2 cos ( ) T T T z i d d g         . Расчетные соотношения (21) (21) В случае, когда 0  элементы (21) матрицы C совпадают с соответствующими элементами этой матрицы для случая совпадения нормали к границе и направления оси симметрии материала, кото- рые представлены формулами (3.82) в [5]. Если 2   , то получается случай, когда ось симметрии материала касательна к поверхности границы и формулы (21) совпадают с выражениями (3.89)–(3.91) в [5]. Зная выражения для матриц C , можно построить решения различных задач, используя научно- методический аппарат, описанный в [5]. Выводы. Предлагаемый научно-методический аппарат успешно использован при определении напряженно-деформированного состояния в слоистых конструкциях, выполненных из трансверсально- изотропных материалов, и при анализе результатов диагностики состояния плоскослоистых и слоистых цилиндрических конструкций, находящихся в эксплуатации. Расчетные соотношения Таким образом, компоненты перемещений и напряжений, входящие в граничные условия (систе- ма координат z , x связана с границей), определяются соотношениями (16)–(18), где проекции волново- го вектора g в системах координат z , x и Jx , K x связаны соотношениями (15). Используемые в [5] матрицы C , характеризующие волновые свойства слоев [9–10], и которые являются основными элементами при построении конкретных решений граничных задач в слоистых конструкциях в данном случае, когда оси z , x образуют с компонентами репера J e , K e угол , имеют вид: 262 Мирошниченко И. П. и др. Обобщенный метод скаляризации динамических упругих полей в трансверсально-изотропных средах Miroshnichenko I. P. and the others. Об авторах: Miroshnichenko, Igor P., Мирошниченко Игорь Павлович, Мирошниченко Игорь Павлович, заведующий кафедрой «Основы конструирования машин» Донского государственного технического университета (РФ, 344000, г. Ростов-на-Дону, пл. Гагарина, 1), кандидат технических наук, доцент, ORCID: https://orcid.org/0000-0001-9450-2500 Ipmir@rambler.ru Библиографический список Механика 1. Тарнопольский, Ю. М. Пространственно-армированные композиционные материалы / Ю. М. Тарнопольский, И. Г. Жигун, В. А. Поляков. — Москва : Машиностроение. — 1987. — 224 с. 1. Тарнопольский, Ю. М. Пространственно-армированные композиционные материалы / Ю. М. Тарнопольский, И. Г. Жигун, В. А. Поляков. — Москва : Машиностроение. — 1987. — 224 с. 2. Справочник по композиционным материалам. Кн.1 / Под ред. Б. Э. Геллера. — Москва : Машино- строение. — 1988. — 448 с. 1. Тарнопольский, Ю. М. Пространственно армированные композиционные материалы / Ю. М. Тарнопольский, И. Г. Жигун, В. А. Поляков. — Москва : Машиностроение. — 1987. — 224 с. 2. Справочник по композиционным материалам. Кн.1 / Под ред. Б. Э. Геллера. — Москва : Машино- строение. — 1988. — 448 с. Ю. М. Тарнопольский, И. Г. Жигун, В. А. Поляков. Москва : Машиностроение. 1987. 224 с. 2. Справочник по композиционным материалам. Кн.1 / Под ред. Б. Э. Геллера. — Москва : Машино- строение. — 1988. — 448 с. 2. Справочник по композиционным материалам. Кн.1 / Под ред. Б. Э. Геллера. — Москва : Машино- строение. — 1988. — 448 с. р 3. Васильев, В. В. Композиционные материалы. Справочник / В. В. Васильев, В. Д. Протасов, В. В. Болотин и др. — Москва : Машиностроение. — 1990. — 512 с. р 3. Васильев, В. В. Композиционные материалы. Справочник / В. В. Васильев, В. Д. Протасов, В. В. Болотин и др. — Москва : Машиностроение. — 1990. — 512 с. 263 Вестник Донского государственного технического университета. 2018. Т. 18, № 3. C. 258–264. ISSN 1992-5980 eISSN 1992-6006 Vestnik of Don State Technical University. 2018. Vol. 18, no. 3, pp. 258–264. ISSN 1992-5980 eISSN 1992-6006 4. Сизов, В. П. О скаляризации динамических упругих полей в трансверсально-изотропных сре- дах / В. П. Сизов // Известия РАН. Механика твердого тела. — 1988. — № 5. — С. 55–58. 5. Сизов, В. П. Возбуждение упругих волн в слоистых анизотропных конструкциях. Монография / В. П. Сизов, И. П. Мирошниченко. — Saarbrucken : LAP LAMBERT Academic Publishing. — 2012. — 270 с. 6. Сиротин, Ю. И. Основы кристаллофизики / Ю. И. Сиротин, М. П. Шаскольская. — Москва : Наука. — 1979. — 640 с. 6. Сиротин, Ю. И. Основы кристаллофизики / Ю. И. Сиротин, М. П. Шаскольская. — Москва : Наука. — 1979. — 640 с. 7. Федоров, Ф. И. Теория упругих волн в кристаллах / Ф. И. Федоров. — Москва : Наука. — 1965. Библиографический список — 386 с. 7. Федоров, Ф. И. Теория упругих волн в кристаллах / Ф. И. Федоров. — Москва : Наука. — 1965. — 386 с. 8. Корн, Г. Справочник по математике для научных работников и инженеров / Г. Корн, Т. Корн. — Москва : Наука. — 1968. — 720 с. 8. Корн, Г. Справочник по математике для научных работников и инженеров / Г. Корн, Т. Корн. — Москва : Наука. — 1968. — 720 с. 9. Бреховских, Л. М. Волны в слоистых средах / Л. М. Бреховских. — Москва : АН СССР. — 1957. — 502 с. 9. Бреховских, Л. М. Волны в слоистых средах / Л. М. Бреховских. — Москва : АН СССР. — 1957. — 502 с. 10. Виноградова, М. Б. Теория волн / М. Б. Виноградова, О. В. Руденко, А. П. Сухоруков. — Москва : Наука. — 1979. — 384 с. 10. Виноградова, М. Б. Теория волн / М. Б. Виноградова, О. В. Руденко, А. П. Сухоруков. — Москва : Наука. — 1979. — 384 с. Received 18.06 .2018 Submitted 19.06.2018 Scheduled in the issue 05.07.2018 Поступила в редакцию 18.06 .2018 Поступила в редакцию 18.06 .2018 Сдана в редакцию 19.06.2018 Сдана в редакцию 19.06.2018 Запланирована в номер 05.07.2018 Запланирована в номер 05.07.2018 4. Сизов, В. П. О скаляризации динамических упругих полей в трансверсально-изотропных сре- дах / В. П. Сизов // Известия РАН. Механика твердого тела. — 1988. — № 5. — С. 55–58. Вестник Донского государственного технического университета. 2018. Т. 18, № 3. C. 258–264. ISSN 1992-5980 eISSN 1992-6006 Vestnik of Don State Technical University. 2018. Vol. 18, no. 3, pp. 258–264. ISSN 1992-5980 eISSN 1992-6006 Sizov, Valery P., , y , Leading research scholar, Rostov Scientific Research In- stitute for Radiocommunication (130, Nansena St., Ros- tov-on-Don, 344038, RF), Dr.Sci. (Eng.), professor, ORCID: https://orcid.org/0000-0003-4816-0145 р ведущий научный сотрудник Ростовского-на-Дону научно-исследовательского института радиосвязи (РФ, 344038, г. Ростов-на-Дону, ул. Нансена 130), доктор технических наук, профессор, ORCID: https://orcid.org/0000-0003-4816-0145 ORCID: https://orcid.org/0000-0003-4816-0145 http://vestnik.donstu.ru 264
https://openalex.org/W4309874207	https://egusphere.copernicus.org/preprints/2022/egusphere-2022-82/egusphere-2022-82.pdf	English	null	Evidence of localised Amazon rainforest dieback in CMIP6 models	Earth system dynamics	2,022	cc-by	10,139	ence of Amazon rainforest dieback in CMIP6 models Although these models typically project increases in area-mean forest carbon across Amazonia under 5 CO2-induced climate change, five of the seven models also produce abrupt reductions in vegetation carbon which indicate localised dieback events. The Northern South America region (NSA), which contains most of the rainforest, is especially vulnerable in the models. These dieback events, some of which are mediated by fire, are preceded by an increase in the amplitude of the seasonal cycle in near surface temperature, which is consistent with more extreme dry seasons. Based-on the ensemble mean of the detected dieback events we estimate that 7+/-5 % of the NSA region will experience abrupt downward 10 shifts in vegetation carbon per oC of global warming above 1.5oC. 10 https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. Evidence of Amazon rainforest dieback in CMIP6 models Isobel Parry1, Paul Ritchie1, and Peter Cox1 1College of Engineering, Mathematics and Physical Sciences, University of Exeter, Exeter, UK, EX4 4QE Correspondence: Isobel Parry (ip294@exeter.ac.uk) Evidence of Amazon rainforest dieback in CMIP6 models Isobel Parry1, Paul Ritchie1, and Peter Cox1 1College of Engineering, Mathematics and Physical Sciences, University of Exeter, Exeter, UK, EX4 4QE Correspondence: Isobel Parry (ip294@exeter.ac.uk) ence of Amazon rainforest dieback in CMIP6 models Isobel Parry1, Paul Ritchie1, and Peter Cox1 1College of Engineering, Mathematics and Physical Sciences, University of Exeter, Exeter, UK, E Correspondence: Isobel Parry (ip294@exeter.ac.uk) Abstract. Amazon forest dieback is seen as a potential tipping point under climate change. These concerns are partly based- on an early coupled climate-carbon cycle simulation, that produced unusually strong drying and warming in Amazonia. In contrast, the 5th generation Earth System Models (CMIP5) produced few examples of Amazon dieback under climate change. Here we examine results from seven 6th generation models (CMIP6) which include vegetation dynamics, and in some cases interactive forest fires. Although these models typically project increases in area-mean forest carbon across Amazonia under 5 CO2-induced climate change, five of the seven models also produce abrupt reductions in vegetation carbon which indicate localised dieback events. The Northern South America region (NSA), which contains most of the rainforest, is especially vulnerable in the models. These dieback events, some of which are mediated by fire, are preceded by an increase in the amplitude of the seasonal cycle in near surface temperature, which is consistent with more extreme dry seasons. Based-on the ensemble mean of the detected dieback events we estimate that 7+/-5 % of the NSA region will experience abrupt downward 10 shifts in vegetation carbon per oC of global warming above 1.5oC. Abstract. Amazon forest dieback is seen as a potential tipping point under climate change. These concerns are partly based- on an early coupled climate-carbon cycle simulation, that produced unusually strong drying and warming in Amazonia. In contrast, the 5th generation Earth System Models (CMIP5) produced few examples of Amazon dieback under climate change. Here we examine results from seven 6th generation models (CMIP6) which include vegetation dynamics, and in some cases interactive forest fires. Although these models typically project increases in area-mean forest carbon across Amazonia under 5 CO2-induced climate change, five of the seven models also produce abrupt reductions in vegetation carbon which indicate localised dieback events. The Northern South America region (NSA), which contains most of the rainforest, is especially vulnerable in the models. These dieback events, some of which are mediated by fire, are preceded by an increase in the amplitude of the seasonal cycle in near surface temperature, which is consistent with more extreme dry seasons. Based-on the % 5 interactive forest fires. 1 Introduction A ‘tipping point’ commonly refers to small changes to input levels causing a system to abruptly transition to some alternative (often less desirable) stable state (Lenton et al., 2008). Future tipping points pose a risk to both natural ecosystems and, by extension, human activities, as they produce abrupt system wide changes that are often difficult or even impossible to reverse 15 (Lenton et al., 2013). The Amazon rainforest is one example in the climate system that is at risk of experiencing a tipping event, with the possibility of abrupt forest dieback in response to rising global temperatures (Cox et al., 2004). Amazon dieback has the potential to accelerate global warming through reducing the Amazon’s ability to act as a carbon sink, and releasing carbon dioxide that would lead to additional global warming (Cox et al., 2000). Tipping points may play an important role in the future extension, human activities, as they produce abrupt system wide changes that are often difficult or even impossible to reverse 15 (Lenton et al., 2013). The Amazon rainforest is one example in the climate system that is at risk of experiencing a tipping event, with the possibility of abrupt forest dieback in response to rising global temperatures (Cox et al., 2004). Amazon dieback has the potential to accelerate global warming through reducing the Amazon’s ability to act as a carbon sink, and releasing carbon dioxide that would lead to additional global warming (Cox et al., 2000). Tipping points may play an important role in the future 15 of our changing climate (Jørgensen et al., 2014; Lenton et al., 2013), with previous analysis of CMIP5 models suggesting that 20 multiple regional abrupt transitions could occur for global warming levels less than 2 degree Celsius (Drijfhout et al., 2015). There are several factors which could contribute to a decline in vegetation in the Amazon, including a lengthened dry season, increased fire frequency, and reduced precipitation (Malhi et al., 2009). The number of extreme hot and dry days in the Amazon is predicted to increase with global warming (Vogel et al., 2020) and the length and intensity of the dry season expected to intensify (Malhi et al., 2009). In this paper These recent studies (Boulton et al., 2022; Luo and Keenan, 2022) focus on fairly short observational records. In this paper we look instead at projections from the latest CMIP6 Earth System Models for evidence of Amazon dieback, and identify a 50 precursor which is based-on longer-term temperature records. we look instead at projections from the latest CMIP6 Earth System Models for evidence of Amazon dieback, and identify a 50 precursor which is based-on longer-term temperature records. we look instead at projections from the latest CMIP6 Earth System Models for evidence of Amazon dieback, and identify a 50 precursor which is based-on longer-term temperature records. 1 Introduction Further drying in the Amazon is anticipated from the slowdown of the Atlantic Meridional 25 Overturning circulation due to ice melt causing an influx of fresh water into the North Atlantic (Lenton et al 2019) Moisture intensify (Malhi et al., 2009). Further drying in the Amazon is anticipated from the slowdown of the Atlantic Meridional 25 Overturning circulation due to ice melt causing an influx of fresh water into the North Atlantic (Lenton et al., 2019). Moisture 1 1 https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. stress resulting from severe droughts in the Amazon is likely to result in a degree of tree mortality (Phillips et al., 2009). As the rainforest dries it becomes more vulnerable to fire which, coupled with the increased frequency of fires seen in the Amazon over recent years, could lead the rainforest to pass a tipping point and result in vegetation dieback (Malhi et al., 2009; Aragão et al., 2018). Anthropogenic deforestation also contributes to this by reducing dry season rainfall and decreasing the resilience 30 of the forest to climate change, potentially leading to permanent forest loss in some regions of the Amazon (Zemp et al., 2017). Mechanisms which result in the drying of the Amazon rainforest can therefore be considered to be the main cause of vegetation dieback. Dieback tipping events are primarily thought of as bifurcation type tipping points (Ritchie et al 2021) which occur when 30 Dieback tipping events are primarily thought of as bifurcation type tipping points (Ritchie et al., 2021), which occur when external climatic factors reduce the resilience of a state (e.g. forest) and ultimately cause the system to tip into a new contrasting 35 state (e.g. savannah) (Scheffer et al., 2009). For some bifurcation type tipping points there are generic features of a system that can be detected to indicate the approach of a tipping point (Scheffer et al., 2009; Dakos et al., 2008). The most common example is ‘critical slowing down’, where a system becomes increasingly slow at recovering from small perturbations as negative feedbacks become overwhelmed by positive ones. Critical slowing down can be observed by increases to the autocorrelation external climatic factors reduce the resilience of a state (e.g. forest) and ultimately cause the system to tip into a new contrasting 35 state (e.g. savannah) (Scheffer et al., 2009). For some bifurcation type tipping points there are generic features of a system that can be detected to indicate the approach of a tipping point (Scheffer et al., 2009; Dakos et al., 2008). The most common example is ‘critical slowing down’, where a system becomes increasingly slow at recovering from small perturbations as negative feedbacks become overwhelmed by positive ones. Critical slowing down can be observed by increases to the autocorrelation and variance in a state variable (Scheffer et al., 2009; Dakos et al., 2008). A recent study which looked at such signals in 40 satellite-retrieved vegetation greenness, reports evidence of reducing resilience of the Amazon rainforest since 2005 (Boulton et al., 2022). However, previous research into projections of Amazonian vegetation dieback has also suggested that generic early warning signals (EWS) such as these fail in the Amazon, but that more system specific indicators may be found (Boulton et al., 2013). and variance in a state variable (Scheffer et al., 2009; Dakos et al., 2008). A recent study which looked at such signals in 40 satellite-retrieved vegetation greenness, reports evidence of reducing resilience of the Amazon rainforest since 2005 (Boulton et al., 2022). However, previous research into projections of Amazonian vegetation dieback has also suggested that generic early warning signals (EWS) such as these fail in the Amazon, but that more system specific indicators may be found (Boulton et al., 2013). For example, the interannual variability of the atmospheric CO2 concentration, as a function of tropical temperature variability, 45 has been shown to be connected to the sensitivity of tropical carbon to climate change (Cox et al., 2013; Wenzel et al., 2014). This metric shows trends which are also consistent with reducing resilience of tropical forests (Wang et al., 2014; Luo and Keenan, 2022). These recent studies (Boulton et al., 2022; Luo and Keenan, 2022) focus on fairly short observational records. In this paper For example, the interannual variability of the atmospheric CO2 concentration, as a function of tropical temperature variability, 45 has been shown to be connected to the sensitivity of tropical carbon to climate change (Cox et al., 2013; Wenzel et al., 2014). This metric shows trends which are also consistent with reducing resilience of tropical forests (Wang et al., 2014; Luo and Keenan, 2022). These recent studies (Boulton et al., 2022; Luo and Keenan, 2022) focus on fairly short observational records. In this paper For example, the interannual variability of the atmospheric CO2 concentration, as a function of tropical temperature variability, 45 has been shown to be connected to the sensitivity of tropical carbon to climate change (Cox et al., 2013; Wenzel et al., 2014). This metric shows trends which are also consistent with reducing resilience of tropical forests (Wang et al., 2014; Luo and Keenan, 2022). These recent studies (Boulton et al., 2022; Luo and Keenan, 2022) focus on fairly short observational records. 2.2 Abrupt shift detection algorithm. Finally, 65 to remove detected abrupt shifts that might be due to a model’s high internal variability the mean annual rate of change of the abrupt shift must be at least three times larger than the variability of the rates of change in the unforced control run. Grid points where abrupt shifts were detected are subsequently sorted based on the direction of the abrupt shift (positive or negative) and the direction of overall trend (positive or negative). This results in four classifications of abrupt shifts, where kgC/m2 over a 15-year period and that this must contribute to at least 25% of the overall change in vegetation carbon. Finally, 65 to remove detected abrupt shifts that might be due to a model’s high internal variability the mean annual rate of change of the abrupt shift must be at least three times larger than the variability of the rates of change in the unforced control run. Grid points where abrupt shifts were detected are subsequently sorted based on the direction of the abrupt shift (positive or negative) and the direction of overall trend (positive or negative). This results in four classifications of abrupt shifts, where to remove detected abrupt shifts that might be due to a model s high internal variability the mean annual rate of change of the abrupt shift must be at least three times larger than the variability of the rates of change in the unforced control run. Grid points where abrupt shifts were detected are subsequently sorted based on the direction of the abrupt shift (positive or negative) and the direction of overall trend (positive or negative). This results in four classifications of abrupt shifts, where analysis focused on the dieback abrupt shifts corresponding to the overall trend and direction of the abrupt shift being negative. 70 This type of abrupt shift can be used as an analogy for a tipping event where a region changes equilibrium state from rainforest to savannah. analysis focused on the dieback abrupt shifts corresponding to the overall trend and direction of the abrupt shift being negative. 70 This type of abrupt shift can be used as an analogy for a tipping event where a region changes equilibrium state from rainforest to savannah. 2.1 CMIP6 models, experiment runs and data used. Climate models that incorporate dynamic vegetation and are from the 6th Phase of the Coupled Climate models that incorporate dynamic vegetation and are from the 6th Phase of the Coupled Model Intercomparison Project CMIP6 were utilised in this study. See Table 1 for the corresponding seven CMIP6 climate models. For the purpose of this study 55 we wanted to focus on the climatic drivers alone impacting vegetation and therefore make use of the 1pctCO2 runs. Data from the PIControl runs were also used to determine each model’s internal variability. Primarily, we use model output data of the vegetation carbon and surface temperature for the seven climate models in the NSA region. The amplitude of the temperature seasonal cycle in this study is defined as the difference between the maximum and minimum monthly mean for each year. All CMIP6 were utilised in this study. See Table 1 for the corresponding seven CMIP6 climate models. For the purpose of this study 55 we wanted to focus on the climatic drivers alone impacting vegetation and therefore make use of the 1pctCO2 runs. Data from the PIControl runs were also used to determine each model’s internal variability. Primarily, we use model output data of the vegetation carbon and surface temperature for the seven climate models in the NSA region. The amplitude of the temperature seasonal cycle in this study is defined as the difference between the maximum and minimum monthly mean for each year. All 55 2 2 https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. Table 1. CMIP6 models used within this study Model Institution EC-Earth3-Veg EC-Earth-Consortium GFDL-ESM4 NOAA-GFDL MPI-ESM1-2-LR Max-Planck-Institut für Meteorologie NorCPM1 EarthClim TaiESM1 AS-RCEC SAM0-UNICON Seoul National University UKESM1-0-LL Met Office Hadley Centre Table 1. CMIP6 models used within this study Model Institution EC-Earth3-Veg EC-Earth-Consortium GFDL-ESM4 NOAA-GFDL MPI-ESM1-2-LR Max-Planck-Institut für Meteorologie NorCPM1 EarthClim TaiESM1 AS-RCEC SAM0-UNICON Seoul National University UKESM1-0-LL Met Office Hadley Centre anomalies presented correspond to the yearly mean relative to the mean of the first ten years, aside from temperature anomalies 60 which correspond to the ten year running mean relative to the mean of the first ten years. anomalies presented correspond to the yearly mean relative to the mean of the first ten years, aside from temperature anomalies 60 which correspond to the ten year running mean relative to the mean of the first ten years. 60 2.2 Abrupt shift detection algorithm. The algorithm used to detect abrupt shifts is relatively simple by design. Three criteria must be be fulfilled for a grid point to be identified as containing an abrupt shift in the vegetation carbon. Namely, the vegetation carbon must change by at least 2 kgC/m2 over a 15-year period and that this must contribute to at least 25% of the overall change in vegetation carbon. Finally, 65 The algorithm used to detect abrupt shifts is relatively simple by design. Three criteria must be be fulfilled for a grid point to be identified as containing an abrupt shift in the vegetation carbon. Namely, the vegetation carbon must change by at least 2 kgC/m2 over a 15-year period and that this must contribute to at least 25% of the overall change in vegetation carbon. Finally, 5 to remove detected abrupt shifts that might be due to a model’s high internal variability the mean annual rate of change of the abrupt shift must be at least three times larger than the variability of the rates of change in the unforced control run. The algorithm used to detect abrupt shifts is relatively simple by design. Three criteria must be be fulfilled for a grid point to be identified as containing an abrupt shift in the vegetation carbon. Namely, the vegetation carbon must change by at least 2 The algorithm used to detect abrupt shifts is relatively simple by design. Three criteria must be b be identified as containing an abrupt shift in the vegetation carbon. Namely, the vegetation carbo be identified as containing an abrupt shift in the vegetation carbon. Namely, the vegetation ca kgC/m2 over a 15-year period and that this must contribute to at least 25% of the overall change in vegetation carbon. Finally, 65 to remove detected abrupt shifts that might be due to a model’s high internal variability the mean annual rate of change of the abrupt shift must be at least three times larger than the variability of the rates of change in the unforced control run. Grid points where abrupt shifts were detected are subsequently sorted based on the direction of the abrupt shift (positive or negative) and the direction of overall trend (positive or negative). This results in four classifications of abrupt shifts, where kgC/m2 over a 15-year period and that this must contribute to at least 25% of the overall change in vegetation carbon. 2.3 EWS for Amazon dieback. Our system specific EWS for Amazon dieback is to observe high sensitivities of the amplitude Our system specific EWS for Amazon dieback is to observe high sensitivities of the amplitude of the seasonal cycle to global y pi g p y g warming. This is defined as the gradient of a linear regression fit to the amplitude of the temperature seasonal cycle against 75 global warming. For a comparison between grid points with abrupt shifts and those that do not, the regression is fitted against the first 73 years of data corresponding to when CO2 has doubled from pre-industrial levels (noting that most abrupt shifts occur after a doubling of CO2). Area weighted histograms of the sensitivities are calculated to compare the distributions for areas with abrupt vegetation dieback (red) and areas with no abrupt shifts (purple). The mean of the seven model histograms warming. This is defined as the gradient of a linear regression fit to the amplitude of the temperature seasonal cycle against 75 global warming. For a comparison between grid points with abrupt shifts and those that do not, the regression is fitted against the first 73 years of data corresponding to when CO2 has doubled from pre-industrial levels (noting that most abrupt shifts occur after a doubling of CO2). Area weighted histograms of the sensitivities are calculated to compare the distributions for areas with abrupt vegetation dieback (red) and areas with no abrupt shifts (purple). The mean of the seven model histograms was taken to produce the compiled models histogram in Fig. 4h. The percentage risk of an abrupt dieback shift occurring 80 (Fig. 4i) is calculated as the percentage of abrupt dieback grid points to all grid points for the specified sensitivity. 3 https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 1. Abrupt shifts detected in the Amazon by the described algorithm and example time series for dieback shifts. (a-h) Maps of detected abrupt shifts. Red grid points indicate detected dieback shifts where the direction of the overall trend (T) and the abrupt shift (AS) are both negative. Green points indicate that the direction of trend and abrupt shift are both positive. Blue points indicate a positive abrupt shift but a negative trend, while orange points indicate a negative abrupt shift associated with a positive trend. 2.3 EWS for Amazon dieback. (h) Example time series for detected dieback shifts in each model, corresponding to grid points highlighted by black squares. he Amazon by the described algorithm and example time series for dieback shifts. (a-h) Maps of Figure 1. Abrupt shifts detected in the Amazon by the described algorithm and example time series for dieback shifts. (a-h) Maps of detected abrupt shifts Red grid points indicate detected dieback shifts where the direction of the overall trend (T) and the abrupt shift (AS) Figure 1. Abrupt shifts detected in the Amazon by the described algorithm and example time series for dieback shifts. (a-h) Maps of detected abrupt shifts. Red grid points indicate detected dieback shifts where the direction of the overall trend (T) and the abrupt shift (AS) are both negative. Green points indicate that the direction of trend and abrupt shift are both positive. Blue points indicate a positive abrupt shift but a negative trend, while orange points indicate a negative abrupt shift associated with a positive trend. (h) Example time series for detected dieback shifts in each model, corresponding to grid points highlighted by black squares. 3.1 Detection of abrupt shifts This may be due to the high natural variability that is inherent in this model and which is difficult to distinguish from climate forced abrupt shifts. Interestingly, UKESM1-0-LL displays no dieback events, despite showing large scale dieback in previous CMIP generations (Cox et al., 2004). Similarly, very few abrupt shifts are detected in the MPI-ESM1-2-LR model. clustering of dieback abrupt shifts in the north of the Amazon. GFDL-EMS4 also presents a coherent structure with abrupt shifts 95 clustered in central-west Amazonia. Contrast to this, EC-Earth3-Veg shows many abrupt shifts scattered across the Amazon basin. This may be due to the high natural variability that is inherent in this model and which is difficult to distinguish from climate forced abrupt shifts. Interestingly, UKESM1-0-LL displays no dieback events, despite showing large scale dieback in previous CMIP generations (Cox et al., 2004). Similarly, very few abrupt shifts are detected in the MPI-ESM1-2-LR model. 95 Some sample time series of detected dieback abrupt shifts across the models are shown in Fig. 1h. Between the models there 100 is some variation in the general shape of abrupt shift time series, however, most exhibit a change of state from one equilibrium to another that would be expected of a tipping event. Some sample time series of detected dieback abrupt shifts across the models are shown in Fig. 1h. Between the models there 100 is some variation in the general shape of abrupt shift time series, however, most exhibit a change of state from one equilibrium to another that would be expected of a tipping event. Some sample time series of detected dieback abrupt shifts across the models are shown in Fig. 1h. Between the models there 100 is some variation in the general shape of abrupt shift time series, however, most exhibit a change of state from one equilibrium to another that would be expected of a tipping event. 3.1 Detection of abrupt shifts We focus our analysis on detecting Amazon dieback abrupt shifts in seven state-of-the-art climate models, which all enable dynamic vegetation, from the 6th Phase of the Climate Model Intercomparison Project (CMIP6). Specifically, we are interested 85 in climate change induced dieback (rather than direct deforestation) and therefore consider the idealised scenario of CO2 increasing 1% per year starting from pre-industrial levels. We have defined that for an abrupt shift to be detected a 2kgC/m2 change must be observed within a 15-year period and this change must contribute to over a quarter of the change observed in the entire simulation run. We additionally require that the mean annual rate of change is more than 3 times as large as the 90 The algorithm categorises abrupt shifts into four types dependent on the directions of the abrupt shift (AS) itself and the overall trend (T; change in 5-year means between start and end of the run). Figure 1a–g depicts the types of abrupt shifts detected spatially for the CMIP6 models. Conventional dieback abrupt shifts (red; T < 0, AS < 0), which indicate a move towards a savannah state, are predominantly detected. Three models; NorCPM1, TaiESM1 and SAM0-UNICON all show a 4 4 https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 2. Plots showing the evolution of abrupt dieback shifts and vegetation carbon with global warming. (a) IPCC AR6 defined regions of the Amazon. (b) Plot showing the evolution of the percentage of the NSA region that has experienced a dieback shift with increasing global warming. The plume indicates the error in the averaged compiled models line (black). (c) Plot showing how the vegetation carbon anomaly relative to the mean of the first ten years evolves with global warming. The plume indicates the error in the averaged compiled model line (black). Figure 2. Plots showing the evolution of abrupt dieback shifts and vegetation carbon with global warming. (a) IPCC AR6 defined regions of the Amazon. (b) Plot showing the evolution of the percentage of the NSA region that has experienced a dieback shift with increasing global warming. The plume indicates the error in the averaged compiled models line (black). (c) Plot showing how the vegetation carbon anomaly relative to the mean of the first ten years evolves with global warming. 3.1 Detection of abrupt shifts The plume indicates the error in the averaged compiled model line (black). Figure 2. Plots showing the evolution of abrupt dieback shifts and vegetation carbon with global warming. (a) IPCC AR6 defined regions of the Amazon. (b) Plot showing the evolution of the percentage of the NSA region that has experienced a dieback shift with increasing global warming. The plume indicates the error in the averaged compiled models line (black). (c) Plot showing how the vegetation carbon anomaly relative to the mean of the first ten years evolves with global warming. The plume indicates the error in the averaged compiled model line (black). Figure 2. Plots showing the evolution of abrupt dieback shifts and vegetation carbon with global warming. (a) IPCC AR6 defined Figure 2. Plots showing the evolution of abrupt dieback shifts and vegetation carbon with global warming. (a) IPCC AR6 defined regions of the Amazon. (b) Plot showing the evolution of the percentage of the NSA region that has experienced a dieback shift with Figure 2. Plots showing the evolution of abrupt dieback shifts and vegetation carbon with glo regions of the Amazon. (b) Plot showing the evolution of the percentage of the NSA region that has experienced a dieback shift with increasing global warming. The plume indicates the error in the averaged compiled models line (black). (c) Plot showing how the vegetation carbon anomaly relative to the mean of the first ten years evolves with global warming. The plume indicates the error in the averaged compiled model line (black). clustering of dieback abrupt shifts in the north of the Amazon. GFDL-EMS4 also presents a coherent structure with abrupt shifts 95 clustered in central-west Amazonia. Contrast to this, EC-Earth3-Veg shows many abrupt shifts scattered across the Amazon basin. This may be due to the high natural variability that is inherent in this model and which is difficult to distinguish from climate forced abrupt shifts. Interestingly, UKESM1-0-LL displays no dieback events, despite showing large scale dieback in previous CMIP generations (Cox et al., 2004). Similarly, very few abrupt shifts are detected in the MPI-ESM1-2-LR model. clustering of dieback abrupt shifts in the north of the Amazon. GFDL-EMS4 also presents a coherent structure with abrupt shifts 95 clustered in central-west Amazonia. Contrast to this, EC-Earth3-Veg shows many abrupt shifts scattered across the Amazon basin. 3.2 Evolution and impact of abrupt shifts in the NSA region Henceforth, we restrict our analysis to the IPCC AR6 defined North South America (NSA) region, which contains the majority Henceforth, we restrict our analysis to the IPCC AR6 defined North South America (NSA) region, which contains the majority of the Amazon basin (see Fig. 2a), and features many of the detected abrupt dieback shifts (red points in Fig. 1). Figure 2b 105 shows how the fractional area of the NSA region to experience an abrupt dieback shift evolves for increasing global warming. Some models show clear jumps in the NSA area to experience an abrupt shift reflecting multiple gird points featuring an abrupt shift at a similar level of warming. Strikingly, TaiESM1 shows about 20% of the NSA region suffering an abrupt dieback event at about 1.7oC warming and by 3oC global warming about 40% of the NSA region would experience an abrupt shift. The bold yi g j y of the Amazon basin (see Fig. 2a), and features many of the detected abrupt dieback shifts (red points in Fig. 1). Figure 2b 105 shows how the fractional area of the NSA region to experience an abrupt dieback shift evolves for increasing global warming. Some models show clear jumps in the NSA area to experience an abrupt shift reflecting multiple gird points featuring an abrupt shift at a similar level of warming. Strikingly, TaiESM1 shows about 20% of the NSA region suffering an abrupt dieback event at about 1.7oC warming and by 3oC global warming about 40% of the NSA region would experience an abrupt shift. The bold black line represents the mean behaviour and the plume variability of all seven models. Although some of the large jumps from 110 individual models can still be identified, the model mean shows a smoother increase in fractional NSA area to undergo an abrupt 5 Figure 3. Time series showing how the temperature seasonal cycle amplitude evolves over time for grid points which experience abrupt dieback shifts. (a-c) Time series of selected dieback shifts for three models at grid points 5◦S 65◦W, 0◦60◦W and 0◦60◦respec- tively. The red dotted line indicates the midpoint of the 15 year period where the abrupt shift is detected by the algorithm. (d-f) The change in the amplitude of the temperature seasonal cycle with time and CO2 for these dieback shifts in each model. 3.2 Evolution and impact of abrupt shifts in the NSA region The solid lines represents the 10 year running average of the seasonal cycle amplitude, while the dotted lines are the yearly data. https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 3. Time series showing how the temperature seasonal cycle amplitude evolves over tim g how the temperature seasonal cycle amplitude evolves over time for grid points which experience Figure 3. Time series showing how the temperature seasonal cycle amplitude evolves over time for grid points which experience abrupt dieback shifts. (a-c) Time series of selected dieback shifts for three models at grid points 5◦S 65◦W, 0◦60◦W and 0◦60◦respec- tively. The red dotted line indicates the midpoint of the 15 year period where the abrupt shift is detected by the algorithm. (d-f) The change in the amplitude of the temperature seasonal cycle with time and CO2 for these dieback shifts in each model. The solid lines represents the 10 year running average of the seasonal cycle amplitude, while the dotted lines are the yearly data. me series of selected dieback shifts for three models at grid points 5◦S 65◦W, 0◦60◦W and 0◦60◦respec- ates the midpoint of the 15 year period where the abrupt shift is detected by the algorithm. (d-f) The change ture seasonal cycle with time and CO2 for these dieback shifts in each model. The solid lines represents the seasonal cycle amplitude, while the dotted lines are the yearly data. shift under global warming. There is not a singular temperature threshold, instead the risk of tipping increases (approximately linearly) between 1.3oC and 3oC of warming and reaches approximately 20% of the NSA region to undergo an abrupt dieback shift. shift under global warming. There is not a singular temperature threshold, instead the risk of tipping increases (approximately linearly) between 1.3oC and 3oC of warming and reaches approximately 20% of the NSA region to undergo an abrupt dieback shift. Interestingly, when evaluating at the regional scale many of the abrupt shifts do not appear to materialise, despite identifying 115 a significant number of local abrupt shifts, (see Fig. 2c). Only the cluster of abrupt shifts at approximately 1.3oC warming for GFDL-ESM4 appear in the total vegetation carbon anomaly for the NSA region. https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. 3.3 An EWS for Amazonian dieback risk of a grid point having an abrupt shift (defined as the ratio of gird points with an abrupt dieback shift to all grid points 140 for each sensitivity) generally increases for grid points with higher sensitivities to global warming as shown in Fig. 4i. The minimum risk of a grid point experiencing an abrupt shift is for a sensitivity close to 0, where the seasonal cycle amplitude is unaffected by global warming. As the sensitivity increases from 0.5 to 1.0K/K the risk of a grid point containing an abrupt shift increases approximately linearly from 10% to 60%. For sensitivities greater than 1.0K/K the risk remains between 60% risk of a grid point having an abrupt shift (defined as the ratio of gird points with an abrupt dieback shift to all grid points 140 for each sensitivity) generally increases for grid points with higher sensitivities to global warming as shown in Fig. 4i. The minimum risk of a grid point experiencing an abrupt shift is for a sensitivity close to 0, where the seasonal cycle amplitude is unaffected by global warming. As the sensitivity increases from 0.5 to 1.0K/K the risk of a grid point containing an abrupt shift increases approximately linearly from 10% to 60%. For sensitivities greater than 1.0K/K the risk remains between 60% and 80% providing a potential EWS.The risk also increases to 35% for negative sensitivities, however this is largely from the 145 EC-Earth3-Veg model in which it is not clear how many of the detected shifts are indeed abrupt. 3.3 An EWS for Amazonian dieback 4b,d,e,f) display clear thresholds in the sensitivity, such that above the thresholds only grid points with an abrupt shift feature. EC-Earth3-Veg (Fig. 4a) provides an exception, however due to the seasonal cycle amplitude sensitivity to global warming. The sensitivity is calculated up to a doubling of CO2 (most abrupt 130 shifts are detected after doubling of CO2) for all grid points and models (Fig. 4a-g). The grid point are colour coded according to whether an abrupt shift occurs and its type. Four of the five models that contain abrupt shifts within the NSA region (GFDL- ESM4, NorCPM1, SAM0-UNICON, TaiESM1; Fig. 4b,d,e,f) display clear thresholds in the sensitivity, such that above the thresholds only grid points with an abrupt shift feature. EC-Earth3-Veg (Fig. 4a) provides an exception, however due to the high stochasticity of the model some of the detected abrupt shifts are likely to be simply natural variation. Promisingly, MPI- 135 ESM1-2-LR and UKESM1-0-LL (Fig. 4c,g) do not possess grid points with high sensitivities and could therefore offer an explanation for not exhibiting any abrupt shifts in the NSA region. Taking an ensemble mean of all the models, shows that grid points without an abrupt shift tend to have sensitivities centred around zero, whereas abrupt shift grid points are positively skewed to higher sensitivities (see Fig. 4h). This means that the around zero, whereas abrupt shift grid points are positively skewed to higher sensitivities (see Fig. 4h). This means that the risk of a grid point having an abrupt shift (defined as the ratio of gird points with an abrupt dieback shift to all grid points 140 for each sensitivity) generally increases for grid points with higher sensitivities to global warming as shown in Fig. 4i. The minimum risk of a grid point experiencing an abrupt shift is for a sensitivity close to 0, where the seasonal cycle amplitude is unaffected by global warming. As the sensitivity increases from 0.5 to 1.0K/K the risk of a grid point containing an abrupt shift increases approximately linearly from 10% to 60%. For sensitivities greater than 1.0K/K the risk remains between 60% and 80% providing a potential EWS.The risk also increases to 35% for negative sensitivities, however this is largely from the 145 EC-Earth3-Veg model in which it is not clear how many of the detected shifts are indeed abrupt. 3.2 Evolution and impact of abrupt shifts in the NSA region Furthermore, the CMIP6 models do not even agree on the sign change in vegetation carbon. 115 Interestingly, when evaluating at the regional scale many of the abrupt shifts do not appear to materialise, despite identifying 115 a significant number of local abrupt shifts, (see Fig. 2c). Only the cluster of abrupt shifts at approximately 1.3oC warming for GFDL-ESM4 appear in the total vegetation carbon anomaly for the NSA region. Furthermore, the CMIP6 models do not even agree on the sign change in vegetation carbon. 6 6 3.3 An EWS for Amazonian dieback Three identified abrupt shifts, which all occur around a doubling of CO2 in different models, are shown in Fig. 3a–c and all 120 show a change of equilibrium state after the abrupt shift. Initially, vegetation carbon may increase due to the CO2 fertilization effect (c.f. Fig. 3a,b), however there exists a critical threshold in the CO2 concentration at which increased temperature and drying overwhelm the positive effect of CO2 and results in an abrupt dieback shift. Fig. 3d–f show the trend in the temperature seasonal cycle associated with these three grid points. An increasing trend is observed in the amplitude of the seasonal cycle in the lead up to an abrupt shift for each point, suggesting that this increase in variability may be an EWS for abrupt dieback 125 events. This behaviour can be expected in the lead up to a vegetation dieback shift because the length and intensity of the dry season has been shown to increase due to drying in the Amazon (Vogel et al., 2020; Malhi et al., 2009). Figure 4 investigates the robustness of using the temperature seasonal cycle amplitude as an EWS for an impending dieback event. Specifically, we compare the distributions of grid points possessing an abrupt shift and those without for the temperature i seasonal cycle amplitude sensitivity to global warming. The sensitivity is calculated up to a doubling of CO2 (most abrupt 130 shifts are detected after doubling of CO2) for all grid points and models (Fig. 4a-g). The grid point are colour coded according to whether an abrupt shift occurs and its type. Four of the five models that contain abrupt shifts within the NSA region (GFDL- ESM4, NorCPM1, SAM0-UNICON, TaiESM1; Fig. 4b,d,e,f) display clear thresholds in the sensitivity, such that above the thresholds only grid points with an abrupt shift feature. EC-Earth3-Veg (Fig. 4a) provides an exception, however due to the seasonal cycle amplitude sensitivity to global warming. The sensitivity is calculated up to a doubling of CO2 (most abrupt 130 shifts are detected after doubling of CO2) for all grid points and models (Fig. 4a-g). The grid point are colour coded according to whether an abrupt shift occurs and its type. Four of the five models that contain abrupt shifts within the NSA region (GFDL- ESM4, NorCPM1, SAM0-UNICON, TaiESM1; Fig. Additionally, we use the idealised 1% per year increase of CO2 run to focus on abrupt dieback shifts caused solely by anthropogenic climate change, though abrupt dieback can also be caused through land use changes such as deforestation. 165 We find evidence of clustered localised abrupt dieback shifts in over half the CMIP6 models analysed, however, this analysis is limited by the number of CMIP6 models containing dynamic vegetation. Additionally, we use the idealised 1% per year increase of CO2 run to focus on abrupt dieback shifts caused solely by anthropogenic climate change, though abrupt dieback can also be caused through land use changes such as deforestation. 165 https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. more localised. Thus, despite the CMIP6 models failing to agree on the overall impact of vegetation carbon in the Amazon, 155 abrupt shifts remain a threat to local communities and ecosystems. Our analysis shows that typically the sensitivity of the amplitude of the temperature seasonal cycle to global warming is higher for grid points subsequently featuring an abrupt dieback shift, compared with grid points with no abrupt dieback (Fig. 4). This therefore offers the possibility of using this sensitivity as a system specific early-warning signal for future dieback in the more localised. Thus, despite the CMIP6 models failing to agree on the overall impact of vegetation carbon in the Amazon, 155 abrupt shifts remain a threat to local communities and ecosystems. more localised. Thus, despite the CMIP6 models failing to agree on the overall impact of vegetation carbon in the Amazon, 155 abrupt shifts remain a threat to local communities and ecosystems. Our analysis shows that typically the sensitivity of the amplitude of the temperature seasonal cycle to global warming is higher for grid points subsequently featuring an abrupt dieback shift, compared with grid points with no abrupt dieback (Fig. 4). This therefore offers the possibility of using this sensitivity as a system specific early-warning signal for future dieback in the more localised. Thus, despite the CMIP6 models failing to agree on the overall impact of vegetation carbon in the Amazon, 155 abrupt shifts remain a threat to local communities and ecosystems. Our analysis shows that typically the sensitivity of the amplitude of the temperature seasonal cycle to global warming is higher for grid points subsequently featuring an abrupt dieback shift, compared with grid points with no abrupt dieback (Fig. 4). This therefore offers the possibility of using this sensitivity as a system specific early-warning signal for future dieback in the Amazon. The increase in risk observed for negative sensitivities also could mean that any changes in moisture and temperature 160 cycles in the Amazon suggest an increased risk of an abrupt shift occurring. We find evidence of clustered localised abrupt dieback shifts in over half the CMIP6 models analysed, however, this analysis is limited by the number of CMIP6 models containing dynamic vegetation. 4 Discussion The effects of abrupt shifts observed in the NSA region may be limited if anthropogenic climate change is restricted to below 1.5 degrees, as set out in the aims of the Paris Agreement (UNFCCC, 2015). Exceeding 1.5°C warming is likely to result in sharp increases in the areas experiencing abrupt shifts. Despite large areas of the Amazon experiencing tipping events 150 with warming, the abrupt shifts observed in Fig. 2 may be considered localised events. These are largely balanced out by the 7 7 https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. gure 4. Plots demonstrating the efficacy of the temperature seasonal cycle amplitude as an EWS and how Figure 4. Plots demonstrating the efficacy of the temperature seasonal cycle amplitude as an EWS and how it may be used to indicate the risk of an oncoming abrupt dieback shift. (a-g) Histograms with bins of width 0.2 showing the percentage area of the NSA region that have different sensitivities of the temperature seasonal cycle amplitude to global warming. The bar colors correspond to the type of abrupt shift detected at the grid point with a specific sensitivity, as in Figure 1, where purple bars indicate grid points where no abrupt shifts are detected. (h) The mean of histograms a-g. (i) A bar chart showing how the percentage risk of a grid point, in any of the 7 analysed models, experiencing a dieback shift changes with increasing sensitivity of the seasonal cycle amplitude to global warming increase in vegetation carbon seen elsewhere in the NSA region, likely resulting from CO2 fertilization, where an increase in photosynthesis rate results in an increase in biomass (Cox et al., 2004; Huntingford et al., 2013). This appears to indicate that large scale regional dieback, as observed in previous generations of models, are not present in CMIP6 and the impacts will be 8 https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-82 Preprint. Discussion started: 23 March 2022 c⃝Author(s) 2022. CC BY 4.0 License. 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Cox, P., Betts, R., Collins, M., Harris, P., Huntingford, C., and Jones, C.: Amazonian forest dieback under climate-carbon cycle projections Cox, P., Betts, R., Jones, C., Spall, S., and Totterdell, I.: Acceleration of global warming due to carbon-cycle feedbacks in a coupled climate model, Nature, 408, 184–187, https://doi.org/10.1038/35041539, 2000. 5 Conclusions Anthropogenic climate change could result in localised tipping events occurring in the Amazon rainforest, as observed in several CMIP6 models. The dieback events detected would have severe consequences for local communities and ecosystems. This study suggests that 7+/-5 % of the Northern South America region would experience abrupt downward shifts in vegetation carbon per oC of global warming above 1.5oC. 170 carbon per C of global warming above 1.5 C. 170 Further research is needed to assess the risk of tipping events under climate change and to identify forewarning methods that can be applied to observational data. However, our results indicate that the sensitivity of the amplitude of the temperature seasonal cycle to global warming is a promising potential early warming signal for local Amazon forest dieback. Further research is needed to assess the risk of tipping events under climate change and to identify forewarning methods that can be applied to observational data. However, our results indicate that the sensitivity of the amplitude of the temperature seasonal cycle to global warming is a promising potential early warming signal for local Amazon forest dieback. Data availability. The CMIP6 model output datasets analysed during this study are available online at [https://esgf-node.llnl.gov/search/cmip6/] Data availability. The CMIP6 model output datasets analysed during this study are available online at [https://esgf-node.llnl.gov/search/cmip6/] Data availability. 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https://openalex.org/W2027489101	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0040411&type=printable	English	null	Streptococcus pyogenes SpyCEP Influences Host-Pathogen Interactions during Infection in a Murine Air Pouch Model	PloS one	2,012	cc-by	9,960	Abstract Streptococcus pyogenes is a major human pathogen worldwide, responsible for both local and systemic infections. These bacteria express the subtilisin-like protease SpyCEP which cleaves human IL-8 and related chemokines. We show that localization of SpyCEP is growth-phase and strain dependent. Significant shedding was observed only in a strain naturally overexpressing SpyCEP, and shedding was not dependent on SpyCEP autoproteolytic activity. Surface-bound SpyCEP in two different strains was capable of cleaving IL-8. To investigate SpyCEP action in vivo, we adapted the mouse air pouch model of infection for parallel quantification of bacterial growth, host immune cell recruitment and chemokine levels in situ. In response to infection, the predominant cells recruited were neutrophils, monocytes and eosinophils. Concomitantly, the chemokines KC, LIX, and MIP-2 in situ were drastically increased in mice infected with the SpyCEP knockout strain, and growth of this mutant strain was reduced compared to the wild type. SpyCEP has been described as a potential vaccine candidate against S. pyogenes, and we showed that surface-associated SpyCEP was recognized by specific antibodies. In vitro, such antibodies also counteracted the inhibitory effects of SpyCEP on chemokine mediated PMN recruitment. Thus, a- SpyCEP antibodies may benefit the host both directly by enabling opsonophagocytosis, and indirectly, by neutralizing an important virulence factor. The animal model we employed shows promise for broad application in the study of bacterial pathogenesis. ation: Chiappini N, Seubert A, Telford JL, Grandi G, Serruto D, et al. (2012) Streptococcus pyogenes SpyCEP Influences Host-Pathog ection in a Murine Air Pouch Model. PLoS ONE 7(7): e40411. doi:10.1371/journal.pone.0040411 Editor: Paul Sumby, The Methodist Hospital Research Institute, United States of America Copyright: 2012 Chiappini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by internal funding from Novartis Vaccines and Diagnostics, whose role, through the employment of Nico Chiappini, Anja Seubert, John L. Telford, Guido Grandi, Davide Serruto, Immaculada Margarit and Robert Janulczyk, involved conceiving, designing and performing the experiments and analyzing the data. Competing Interests: Nico Chiappini, Anja Seubert, John L. Telford, Guido Grandi, Davide Serruto, Immaculada Margarit and Robert Janulczyk are employees of Novartis Vaccines and Diagnostics. There are no patents, products in development or marketed products to declare. John L. Streptococcus pyogenes SpyCEP Influences Host- Pathogen Interactions during Infection in a Murine Air Pouch Model Nico Chiappini, Anja Seubert, John L. Telford, Guido Grandi, Davide Serruto, Immaculada Margarit, Robert Janulczyk* Research Centre, Novartis Vaccines and Diagnostics, Siena, Italy Research Centre, Novartis Vaccines and Diagnostics, Siena, Italy Abstract Telford, Guido Grandi and Immaculada Margarit are holders of company stock options. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: Robert.Janulczyk@Novartis.com peptidase ScpA [4], a well-characterized virulence factor that interferes with host defences by cleaving and inactivating the chemotactic C5a peptide [6]. Analogously, IL-8 treated with culture supernatants from SpyCEP-expressing S. pyogenes showed a reduced capacity to induce PMN transmigration in vitro [3]. In soft-tissue infection models, histopathology showed increased PMN infiltration following infection with SpyCEP mutant strains when compared to the wild type (w.t.) strain [4,7]. SpyCEP mutant strains also had impaired virulence in soft-tissue, subcu- taneous, and intranasal (i.n.) infection models [4,8,9], while increased virulence was seen for a SpyCEP mutant strain in a sepsis model [7], and in one case with a soft tissue infection model [10]. Expression of SpyCEP is negatively regulated by the two- component signal transduction system CovRS and, in some strains, by another two-component system (Sil) and the cognate peptide pheromone [4,10,11]. Hypervirulent clinical isolates with CovRS mutations show overexpression of SpyCEP [11]. PLoS ONE \| www.plosone.org Bacterial strains and mutagenesis S. pyogenes M1 strains 3348 (Istituto Superiore di Sanita`, Rome, Italy) and SF370 (University of Siena, Italy) were grown in Todd- Hewitt broth supplemented with 0.5% yeast extract (THY medium) or on THY agar plates with 5% sheep blood, at 37uC in 5% CO2 atmosphere. The growth of S. pyogenes was monitored by optical density at 600 nm (OD600) using an Ultrospec 10 cell density meter (Amersham Biosciences). Escherichia coli DH5a, and BL21(DE3) (Invitrogen) were used for cloning, plasmid propaga- tion and protein purification. E. coli were grown at 37uC in Luria- Bertani (LB) liquid medium with agitation or on LB agar plates. Antibiotics were added to the medium at the following final concentrations: 0.5 mg/ml or 1 mg/ml erythromycin (Erm) with S. pyogenes, and 100 mg/ml ampicillin with E. coli. The recombinant proteins rSpyCEP and rSpyCEP* were purified as previously described [14]. The in-frame deletion mutant 3348DspyCEP was previously described [14]. The SF370DspyCEP mutant was obtained in a similar way, using the same construct pJRS233::DspyCEP. To obtain strains 3348spyCEP* and SF370spy- CEP* by gene replacement mutagenesis, the previously obtained construct pET21b+spyCEP(D151A) [14] was used as a template for PCR using primers spyCEPF1 (TCGGATCCG- GAAGCGTTTTCTTGGTGATG) and spyCEPR1 (CCCTCGAGTGGAG TCCCTTCTTTAGGGG). The ampli- con obtained was cloned into pJRS233 [18] using BamHI and XhoI restriction sites, generating the new construct pJRS233spyCEP. S. pyogenes were transformed by electroporation, and plasmid insertion/duplication and excision were performed essentially as described [18,19]. Transformants were selected by growth on THY(Erm) plates at 30uC, and integration events were selected at the non-permissive temperature 37uC, and verified by PCR. After S. pyogenes M1 strains 3348 (Istituto Superiore di Sanita`, Rome, Italy) and SF370 (University of Siena, Italy) were grown in Todd- Hewitt broth supplemented with 0.5% yeast extract (THY medium) or on THY agar plates with 5% sheep blood, at 37uC in 5% CO2 atmosphere. The growth of S. pyogenes was monitored by optical density at 600 nm (OD600) using an Ultrospec 10 cell density meter (Amersham Biosciences). Escherichia coli DH5a, and BL21(DE3) (Invitrogen) were used for cloning, plasmid propaga- tion and protein purification. E. coli were grown at 37uC in Luria- Bertani (LB) liquid medium with agitation or on LB agar plates. Antibiotics were added to the medium at the following final concentrations: 0.5 mg/ml or 1 mg/ml erythromycin (Erm) with S. pyogenes, and 100 mg/ml ampicillin with E. coli. In vitro IL-8 cleavage Animal treatments were performed in compliance with Italian laws, and approved by the institutional review board (Animal Ethical Committee, AEC nr 200819) of Novartis Vaccines and Diagnostics, Siena, Italy. The procedures were authorized by the Italian Ministry of Health (decree 111/2008-B). All the in vitro cleavage assays were comprised of 10 mg/ml of recombinant IL-8 (PeproTechH) in 50 ml PBS, including addition- al recombinant proteins, extracts or bacteria (see below). Cleavage reactions were performed for 2 hours at 37uC. Proteins were then separated by SDS-PAGE on 18% Tris-Glycine gels (Invitrogen). IL-8 was detected by silver staining with Silver QuestTM (Invitrogen). For spike experiments, 3348DspyCEP cell wall extracts were incubated with IL-8 and 0–5 ng of rSpyCEP, and compared with reactions containing 3348 cell wall extracts (with native SpyCEP) and IL-8. For IL-8 cleavage with live bacteria, 10 ml of w.t. or mutant cultures were grown to OD600 = 0.4 (exponential phase), washed, and concentrated 200 fold in PBS. Alternatively, bacteria were grown o/n, diluted to OD600 = 0.4 in PBS, and concentrated 200 fold (stationary phase). 40 ml of bacterial suspension (,109 CFU) were incubated with IL-8 and 10 mg/ml of chloramphenicol in a total reaction volume of 50 ml. Serial dilutions of live bacteria were performed in 4 fold steps starting with 107 (strain 3348) or 108 CFU (strain SF370). After incubation and centrifugation, supernatants were collected and analyzed by SDS-PAGE as above. SpyCEP Action in a Murine Air Pouch Model LPXTG cell wall attachment motif. Recombinant expression of SpyCEP fragments showed that the protease domain alone is not sufficient for cleavage of IL-8 in vitro, but that at least the proximal part of the C-terminal needs to be present [12,13]. We recently showed that the protein exhibits atypical auto processing and that one of the residues in the catalytic triad is located in an N-terminal fragment of the protein, which is released upon autocatalytic hydrolysis. Interestingly, the fragment remains non-covalently associated with the rest of the protein, and thus preserves proteolytic activity [14]. In vitro cleavage assays with recombinant SpyCEP (rSpyCEP) showed that not only IL-8 but several other chemokines are cleaved [8,10,12,14]. SpyCEP is also considered a vaccine candidate following its identification by independent high throughput screening strategies [15–17], although the mechanism of protection is incompletely understood. In this study we asked a series of related questions regarding the role of SpyCEP in virulence and as a vaccine constituent. Is SpyCEP secreted/shed or surface anchored? Can surface-attached SpyCEP cleave IL-8? What are the specific contributions of SpyCEP to virulence and host response in vivo? Answers to these questions, together with an understanding of potential antibody interference with SpyCEP action, will provide a better understanding of the potential role of SpyCEP in a vaccine against S. pyogenes. 5 passages permitting allelic exchange by homologous recombi- nation and excision of the plasmid, ErmS colonies were selected by replica plating. PCR sequencing on gDNA from the selected clones confirmed the base substitution 452A.C in spyCEP and excluded secondary mutations in the locus. Preparation and analysis of bacterial extracts Supernatant and cell wall proteins were obtained from 10 ml cultures grown until exponential and stationary phases. For cell wall extracts, bacterial pellets were washed in PBS, resuspended in 200 ml of 100 mM KPO4 (pH 6.1), 40% (w/v) sucrose, 200 U mutanolysin (Sigma), 1x Complete Mini EDTA-free protease inhibitor cocktail (Roche), and incubated at 37uC for 1 h. Proteins in the culture supernatant were precipitated by trichloroacetic acid at a final concentration of 10% (v/v) and incubation at 4uC for 16 h. The precipitate was washed in acetone and resuspended in 200 ml PBS. Proteins were separated by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) using MES 12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose mem- branes with the iBlotH system (Invitrogen). Immunodetection was performed using 1:30,000 (3348 w.t. and mutant strains) or 1:10,000 (SF370 w.t. and mutant strains) rabbit polyclonal a- rSpyCEP* serum, and 1:20,000 goat a-rabbit IgG (H+L)-HRP conjugate (Bio-Rad). Blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Introduction The Gram-positive bacterium Streptococcus pyogenes is a strictly human pathogen. It causes a wide range of local and systemic infections, ranging from relatively common and trivial diseases such as tonsillitis and erysipelas, to life-threatening conditions such as streptococcal toxic shock syndrome, septicemia, and necrotizing fasciitis. The estimated global burden of disease due to S. pyogenes pharyngitis is 616 million cases annually. The corresponding estimate of invasive infections is 663,000, causing 163,000 deaths each year [1]. SpyCEP (S. pyogenes cell envelope protease, also called ScpC) was originally described in a clinical isolate from a case of necrotizing fasciitis, where broth culture supernatants contained interleukin 8 (IL-8)-degrading activity [2]. Subsequent partial purification of the protease and genetic manipulation of S. pyogenes strains resulted in the identification of a subtilisin-like protease responsible for the IL-8 cleavage [3,4]. IL-8 is an important chemokine participating in the chemotactic recruitment of polymorphonuclear cells (PMN) to a site of injury or infection [5]. SpyCEP is highly conserved and paralogous to the C5a The domain architecture of SpyCEP comprises a subtilisin-like protease domain in the N-terminal part of the protein, and a C- terminal region of unknown function followed by a canonical PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40411 1 SpyCEP Action in a Murine Air Pouch Model Bacterial strains and mutagenesis The recombinant proteins rSpyCEP and rSpyCEP* were purified as previously described [14]. The in-frame deletion mutant 3348DspyCEP was previously described [14]. The SF370DspyCEP mutant was obtained in a similar way, using the same construct pJRS233::DspyCEP. To obtain strains 3348spyCEP* and SF370spy- CEP* by gene replacement mutagenesis, the previously obtained construct pET21b+spyCEP(D151A) [14] was used as a template for Air pouch experiments and analysis Eleven week old female CD1 mice (Charles River Laboratories) were used for the animal experiments in accordance with institutional and Italian guidelines for animal research. Dorsolat- eral air pouches were inflated by subcutaneous injection of 3 ml air on day 1 and day 4. On day 6, 16107 CFU of exponential phase (OD600 = 0.4) S. pyogenes diluted in 1 ml PBS were injected into the pouch. Strain SF370 was also used at a higher dose (16108). In all experiments, the inocula were subjected to viable counts by plating. At 2, 4 or 24 h after infection, the animals were euthanized, and an air pouch lavage was performed by repeated injection/aspiration of 2 ml PBS. 200 ml of lavage material from the air pouches were frozen at 280uC to promote cell lysis and permit release of intracellular bacteria. Bacterial load was determined by viable counts of thawed samples. Surface extracts and bacterial growth supernatants from 3348DspyCEP, SF370DspyCEP, 3348spyCEP, SF370spyCEP and w.t. strains were analyzed by Western blot with a-SpyCEP antibodies (Figure 1B). Purified recombinant SpyCEP (rSpyCEP) and the inactive form rSpyCEP* were used as controls. Both the knockout strains showed the absence of a band corresponding to SpyCEP. In repeated experiments, an estimated 50–70% of total SpyCEP produced in strain 3348 was found associated with the bacterial surface in exponential phase. In contrast, by late stationary phase most (.90%) of the SpyCEP was found in the culture supernatant of strain 3348. Surface extracts from SF370 instead showed the presence of SpyCEP and SpyCEP* only in exponential phase. Significantly longer exposure and a threefold higher amount of primary antibodies were needed to visualize the proteins, suggesting an overall lower expression than in 3348. These data are consistent with a repression of spyCEP expression by the functional CovRS system in SF370. In the 3348spyCEP* and SF370spyCEP* strains we observed the unprocessed form of SpyCEP, and a second band corresponding to the size of processed SpyCEP. Previous work has shown that SpyCEP is incapable of undergoing autocatalytic processing in vitro [14]. The presence of a second band suggests that the cleavage site may be somewhat susceptible to cleavage by unknown protease(s) present in the extracts. Nevertheless, a protein thus processed should be Cell recruitment. the cellular fraction of the lavage was obtained by centrifugation for 7 min (3206 g, 4uC). The supernatant was collected for subsequent analysis (see below). Results Localization and activity of SpyCEP varies with growth- phase Statistical analysis Statistical analysis was performed with GraphPad Prism software for Windows (version 5.00; GraphPad Software). Data sets were first analyzed for normal distribution and variance in order to choose appropriate statistical tests. Transmigration was performed on 3 mm pore transwell plates (Millipore), with 56104 PMN/well in the upper chamber. KC or IL-8 were diluted in RPMI-PSG, 10% FCS, ranging from 0– 1 mg/ml (KC) and 0–30 ng/ml (IL-8) using 3 fold dilution steps. The proteins rSpyCEP or rSpyCEP* were added at a concentra- tion of 50 ng/ml, and samples (including the positive control without protease) were incubated at 37uC for 2 h. Antibody functional inhibition of rSpyCEP was assessed by an initial preincubation of rSpyCEP with (1:500) rabbit polyclonal a- SpyCEP or a-Spy0269 (control) antibodies for 20 minutes at room temperature. Subsequently, the treated IL-8/KC samples were transferred to the lower chamber wells and the plates were incubated for 90 min at 37uC. PMN in the lower chamber were fixed in 2% PFA for 30 min, and counted by flow cytometry using a LSR II SOS (Becton Dickinson). PMN transwell migration PMN from heparinized whole mouse blood were purified by adding 5 volumes of erythrocyte lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH = 7.3), incubated for 8 min on ice, and the reaction was terminated by adding 45 volumes of cold PBS. Neutrophils were then purified by magnetic assisted cell sorting using mouse a-Ly6G-PE (BD Pharmingen), a-PE microbe- ads and MACS LS columns (Miltenyi Biotec) according to the manufacturer’s instructions. Human PMN were isolated as described [20]. In a separate experiment, in vitro digestion of the 13 chemokines (standard provided with the kit) was performed by coincubation with 0.5 mg/ml of rSpyCEP or rSpyCEP* at 37uC for 4 and 24 h. Localization and activity of SpyCEP varies with growth- phase Mutagenesis of spyCEP was performed in two M1 strains with diverse genetic backgrounds. 3348 is a strain with relatively high virulence in mice, and sequencing of the covRS locus showed the presence of several mutations inactivating the system (data not shown). Transcriptome analysis of invasive and non-invasive disease isolates has shown that several known virulence factors show a particular transcriptional profile associated with invasive- ness, and that this is related to mutations in CovRS [21–23]. 3348 shows high expression of SpyCEP and SLO, together with a low expression of SpeB (data not shown), which is consistent with an invasive phenotype. SF370 was used to sequence the first complete genome of S. pyogenes [24], has an intact CovRS system, and is practically avirulent in traditional (i.p/i.n.) mouse challenge models (unpublished results). We obtained in-frame deletions of spyCEP in the SF370 strain, and the genotype was verified by sequencing of the locus. Independently, gene replacement was conducted, where the wild type (w.t.) spyCEP gene was replaced by spyCEP. The latter encodes a mutated form of SpyCEP which is proteolytically inactive (D151A substitution) [14]. Sequencing of the spyCEP locus verified that only the intended mutation was present after gene replacement. The w.t. and genetically manipulated loci are shown (Figure 1A). Growth curves of w.t. and isogenic mutant strains were compared and showed no discernible differences (Figure S1). SpyCEP surface detection by flow cytometry SpyCEP surface detection by flow cytometry Cell wall-attached SpyCEP in 3348, 3348DspyCEP, 3348spy- CEP, SF370, SF370DspyCEP and SF370spyCEP was analyzed by flow cytometry. Bacteria grown to exponential phase were centrifuged at 30006 g, washed in PBS and resuspended in 250 ml newborn calf serum (NCS) for 20 minutes at 25uC with shaking. Bacteria were then incubated at 4uC for 1 hour with mouse a-Alum (mock immunization with Alum) or mouse a- SpyCEP sera diluted 1:200 in PBS, BSA 0.1% (PBSA). Samples were washed twice in PBSA and labeled using goat a-mouse phycoreythrin (PE)-conjugate secondary antibodies (Jackson Im- muno Research) diluted 1:50 for 30 minutes at 4uC. Bacteria were fixed in 2% paraformaldehyde (PFA) and washed in PBS. Data were collected using a BD FACS CANTO II (BD Bioscience) by acquiring 10,000 events. Data analysis was performed with Flow- Jo software (v.8.6, TreeStar Inc.) PCR using primers spyCEPF1 (TCGGATCCG- GAAGCGTTTTCTTGGTGATG) and spyCEPR1 (CCCTCGAGTGGAG TCCCTTCTTTAGGGG). The ampli- con obtained was cloned into pJRS233 [18] using BamHI and XhoI restriction sites, generating the new construct pJRS233spyCEP. S. pyogenes were transformed by electroporation, and plasmid insertion/duplication and excision were performed essentially as described [18,19]. Transformants were selected by growth on THY(Erm) plates at 30uC, and integration events were selected at the non-permissive temperature 37uC, and verified by PCR. After PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40411 2 SpyCEP Action in a Murine Air Pouch Model to the manufacturer’s instruction using a mouse 13-plex custom panel. Air pouch experiments and analysis (B) Western blot analysis of cell wall extracts or supernatants from the 3348 and SF370 w.t. and mutant strains. SpyCEP was detected using rabbit polyclonal a-SpyCEP serum. 10 ng of rSpyCEP or rSpyCEP were added as controls (ctr). (C–D) IL-8 cleavage assay with washed live bacteria. SDS-PAGE and silver staining were performed after digestion of IL-8 (10 mg/ml) in the presence of 10 mg/ml of chloramphenicol. Uncleaved and cleaved IL-8 are indicated by full and trace arrowhead respectively. Control lane (ctr) is IL-8 alone. (C) Strains were grown to exponential or stationary phase, resuspended in PBS at comparable bacterial densities (,109 CFU), and then incubated with IL-8. (D) Exponential phase bacteria 3348 w.t. (107 CFU) and SF370 w.t. (108 CFU) were serially diluted in PBS using 4 fold steps before incubation with IL-8. doi:10.1371/journal.pone.0040411.g001 cysteine protease inhibitor E64 (25 mM), and the localization experiment was repeated. However, extracts from bacteria grown in the presence/absence of E64 showed no differences in SpyCEP localization or amount (data not shown). proteolytically inactive due to the absence of a functional catalytic site (see below). ( ) We hypothesized that surface-associated SpyCEP is active, and cleavage of IL-8 may occur independently of shed and/or secreted SpyCEP. W.t. and mutant bacteria were washed and incubated with IL-8. Complete cleavage of IL-8 by 3348 from exponential phase was observed, while there was no cleavage with 3348Dspy- CEP or 3348spyCEP* (Figure 1C). Similar results were obtained for the SF370 w.t. and mutants, suggesting that even a limited amount of surface-associated SpyCEP is sufficient for IL-8 cleavage. In stationary phase incomplete cleavage of IL-8 was seen for 3348, and no cleavage for SF370, consistent with the data on surface localization described above. To exclude the possibility that the activity observed depended on shed SpyCEP, the reactions were performed in the presence of chloramphenicol to block protein synthesis during the incubation. Moreover, in a control experi- ment Western blot analysis of precipitated supernatants from the reaction showed no detectable presence of SpyCEP (data not shown) - further strengthening that cleavage of IL-8 was by cell wall-attached SpyCEP only. By repeating the above experiment with serial dilutions of bacteria, the relative efficacy of SpyCEP- dependent cleavage in 3348 and SF370 was assessed. Air pouch experiments and analysis Cells were resuspended in 1 ml of PBS with 1:1000 of Live/Dead- AquaFluor (Invitrogen) for 30 min at 4uC. Cells were then fixed in 2% PFA, centrifuged as above, and resuspended in 25 ml staining solution for 30 min at 4uC with a combination of the following cell marker antibodies: a-Ly6C-FITC, a-CD11b-PE-Cy7, a-Ly6G- PE, a-CD11c-APCeFluor780, a-Ly6C+a-Ly6G (GR1)-PER-CP (from BD Pharmingen) and a-F4/80-PacificBlue, a-MHC-II- A700 (from eBioscience). The stained cells were analyzed using a LSR II SOS (BD) and BD DIVA software (BD Bioscience). In particular, we identified neutrophils (Ly6Ghigh, GR1high), eosino- phils (Ly6Gint, F4/80int, SSChigh), monocytes (CD11bhigh, CD11c2, Ly6Chigh, GR1+), dendritic cells (CD11c+, MHC-II+), and macrophages (CD11b+, F4/80high). Chemokine analysis. chemokine concentrations in the air pouch lavage were measured at 2, 4 and 24 h after infection. Lavage material from the air pouch was centrifuged at 3206 g, 4uC for 7 min and supernatants were filtered through 0.22 mm filters to remove bacteria. Chemokine concentrations were measured by MILLIPLEXTM MAP kit (MilliporeTM) according PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40411 3 SpyCEP Action in a Murine Air Pouch Model Figure 1. Localization and activity of SpyCEP. (A) Schematic representation of SF370 wild type and genetically manipulated spy0416/spyCEP loci. The same mutagenesis was conducted in the 3348 strain. The base substitution 452A.C is indicated (). (B) Western blot analysis of cell wall extracts or supernatants from the 3348 and SF370 w.t. and mutant strains. SpyCEP was detected using rabbit polyclonal a-SpyCEP serum. 10 ng of rSpyCEP or rSpyCEP were added as controls (ctr). (C–D) IL-8 cleavage assay with washed live bacteria. SDS-PAGE and silver staining were performed after digestion of IL-8 (10 mg/ml) in the presence of 10 mg/ml of chloramphenicol. Uncleaved and cleaved IL-8 are indicated by full and trace arrowhead respectively. Control lane (ctr) is IL-8 alone. (C) Strains were grown to exponential or stationary phase, resuspended in PBS at comparable bacterial densities (,109 CFU), and then incubated with IL-8. (D) Exponential phase bacteria 3348 w.t. (107 CFU) and SF370 w.t. (108 CFU) were serially diluted in PBS using 4 fold steps before incubation with IL-8. doi:10.1371/journal.pone.0040411.g001 Figure 1. Localization and activity of SpyCEP. (A) Schematic representation of SF370 wild type and genetically manipulated spy0416/spyCEP loci. The same mutagenesis was conducted in the 3348 strain. The base substitution 452A.C is indicated (). Air pouch experiments and analysis Compared to SF370, a 10 fold lower bacterial density of strain 3348 was sufficient to obtain a similar cleavage profile, suggesting a pronounced difference in SpyCEP expression/activity between the two strains (Figure 1D). PLoS ONE \| www.plosone.org SpyCEP specifically reduces chemokine levels in situ during infection In order to analyze the presence of chemokines in situ, 13 different chemokines in the air pouch lavage samples were measured (Figure 4). At 2 h post-infection, only KC (the murine functional homologue of IL-8) and IL-6 were detected, and KC levels in mice infected with 3348DspyCEP were significantly higher than in w.t. infected mice (data not shown). At 4 h post-infection, five chemokines (KC, LIX, MIP-2, MCP-1 and IL-6) were clearly elevated (Figure 4A) compared to samples from mock-infected animals, in which most chemokines were undetectable and in no case higher than 20 pg/ml. KC and MIP-2 were seen at concentrations above 10,000 pg/ml in the samples from mice infected with the 3348DspyCEP strain, while the samples from mice infected with the w.t. strain had at least 10 fold lower mean concentrations. The differences were statistically significant for both KC and MIP-2. At 24 h post-infection, the above chemokines were all still present at high concentrations (Figure 4B), and differences between w.t. and mutant were even more pronounced (10–1000 fold) and significant also for LIX. Figure 2. S. Pyogenes infection in a murine air pouch model. (A) Schematic of the in vivo air pouch model. For each experiment, 8 CD1 mice per group were infected with 16107 CFU of 3348, 3348DspyCEP or PBS. After air pouch inflation and bacterial infection, the lavage material was collected and fractionated for bacterial viable counts, leukocyte counts and chemokine analysis for each animal. (B) Bacterial multiplication factor (total CFU/inoculum CFU) in the lavage from individual mice. Time points 4 and 24 h represent aggregate data from two experiments. Horizontal bars are geometric means. Statistical significance () was tested by Mann-Whitney U, P = 0.014). doi:10.1371/journal.pone.0040411.g002 p ( ) g In order to examine the specificity of SpyCEP against the chemokines used in our assay, the panel of 13 chemokines was also examined by incubation in vitro with rSpyCEP or rSpyCEP. Subsequent measurement of chemokine levels showed that KC, LIX and MIP-2 levels decreased (100, 0 and 3 fold, respectively, at 4 h, and 200, 10 and 3 fold at 24 h) in samples incubated with rSpyCEP, while incubation with rSpyCEP caused no such decrease (data not shown). The other 10 chemokines were unaffected by the treatment. In situ recruitment of leukocytes Eukaryotic cells from the air pouch lavage of mice infected with 3348 and 3348DspyCEP were collected, stained for viability, fixed, and then stained with seven cell markers. The gating strategy involved successive identification of cells by morphology, vitality, and the presence/absence of specific cell markers. An overview of the cell types identified by combinations of markers is shown (Figure 3A). At 2 h, the number and type of live cells identified were similar to that in mock-infected mice, suggesting that cell recruitment in response to the infection was yet to occur (data not shown). At 4 h post-infection, the numbers of cell-like particles in the lavage from infected mice were in the range 105–106, and most cells were viable (78%). At 24 h post-infection, 106 or more cell- like particles were identified, but only 0–1.3% represented live cells. While the total number of cell-like particles was higher (2–4 fold) at 24 h compared to 4 h, in terms of live cells there were 100 fold less. Concomitantly, the bacterial load in situ (see above) was up to 10 fold higher at 24 h than at 4 h. This suggests that the host cells failed to suppress the infection, and that the large number of cell-like particles mostly represent cellular debris. Consequently, conclusions about cell recruitment are only feasible at the 4 h time point. In absolute numbers, the predominant live cell population SpyCEP Action in a Murine Air Pouch Model consisted of neutrophils, followed by eosinophils and macrophages (Figure 3B). Eosinophils are not known to have any role in the host response against S. pyogenes infection, and their presence in quantity was surprising. All cell types from the lavage except for dendritic cells were clearly elevated in infected mice compared to controls (mock infection with PBS). Mice infected with 3348DspyCEP showed a tendency to have more neutrophils and monocytes in situ compared to mice infected with the w.t. strain. This trend was consistent across three independent experiments. However, variability between individual samples was high and the trend was not statistically significant (Mann-Whitney U test, P = 0.1). Figure 2. S. Pyogenes infection in a murine air pouch model. (A) Schematic of the in vivo air pouch model. For each experiment, 8 CD1 mice per group were infected with 16107 CFU of 3348, 3348DspyCEP or PBS. After air pouch inflation and bacterial infection, the lavage material was collected and fractionated for bacterial viable counts, leukocyte counts and chemokine analysis for each animal. (B) Bacterial multiplication factor (total CFU/inoculum CFU) in the lavage from individual mice. Time points 4 and 24 h represent aggregate data from two experiments. Horizontal bars are geometric means. Statistical significance () was tested by Mann-Whitney U, P = 0.014). doi:10.1371/journal.pone.0040411.g002 Figure 2. S. Pyogenes infection in a murine air pouch model. (A) Schematic of the in vivo air pouch model. For each experiment, 8 CD1 mice per group were infected with 16107 CFU of 3348, 3348DspyCEP or PBS. After air pouch inflation and bacterial infection, the lavage material was collected and fractionated for bacterial viable counts, leukocyte counts and chemokine analysis for each animal. (B) Bacterial multiplication factor (total CFU/inoculum CFU) in the lavage from individual mice. Time points 4 and 24 h represent aggregate data from two experiments. Horizontal bars are geometric means. Statistical significance () was tested by Mann-Whitney U, P = 0.014). doi:10.1371/journal.pone.0040411.g002 SpyCEP specifically reduces chemokine levels in situ during infection As this experiment involves the simultaneous presence of competing substrates we conclude that among the 13 chemokines in the panel, only KC, LIX and MIP-2 are substrates for SpyCEP. that 3348DspyCEP had a statistically significant decrease in CFU counts compared to the w.t. strain (.50% less bacteria in the mutant) at 4 h. PLoS ONE \| www.plosone.org A murine air pouch model to study the role of SpyCEP in S. pyogenes infection in situ To investigate whether SpyCEP interferes with innate immune responses during S. pyogenes infection in situ, we adapted a murine air pouch model which has been extensively used to study inflammation [27,28]. In brief, a dorsolateral air pouch was established by repeated injections of air. Bacteria were then injected into the air pouch, and 2, 4 or 24 h later a lavage with PBS was performed (Figure 2A). The lavage fluid was used for three types of analysis: the bacterial load was examined by viable counts, the presence and identity of immune cells were determined by flow cytometry, and chemokine levels were quantified by a multiplex bead ELISA. In initial experiments, dose-ranging was performed. We aimed to use the lowest possible dose that guaranteed bacterial survival and/or growth. In pilot experiments with 3348 and SF370 we established an infectious dose for 3348, while infection with SF370 resulted in .99% clearance of bacteria (Figure S2), and the strain was omitted from further study. Mice (n = 8 per group) were infected with 16107 CFU of w.t. 3348 or spyCEP knockout strains. Mock infection with PBS (n = 3 per group) was performed as a negative control, and viable counts on lavages (2, 4 and 24 h) showed no CFU. At 2 h post-infection, bacterial counts were similar to or lower than the starting inoculum (multiplication factor ,1, Figure 2B). After 4 h, mice infected with 3348 and 3348DspyCEP had higher or equal bacterial loads in situ when compared to the starting inoculum. At 24 h, all mice except one showed considerable growth of bacteria (multiplication factor of 10–1000). The experiment was repeated for the two later time points, and aggregate data showed The streptococcal cysteine protease SpeB is a well-studied virulence factor that acts upon a variety of substrates and is regulated by CovRS [25,26]. We examined SpeB levels in supernatants from bacteria in stationary phase, and noted dramatically higher levels of SpeB in SF370 compared to 3348 (data not shown). We hypothesized that the low quantity of SpyCEP in SF370 was related to SpeB activity. Consequently, bacteria were grown in the presence/absence of the specific PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40411 4 SpyCEP Action in a Murine Air Pouch Model Specific antibodies counteract rSpyCEP inhibition of PMN migration in vitro Cell recruitment in situ. (A) Gating strategy to identify neutrophils (NPh; Ly6Ghigh, GR1high), dendritic cells (DC; CD11c+, MHC- II+), macrophages (MPh; CD11b+, F4/80high), monocytes (Mo; CD11bhigh, CD11c2, Ly6Chigh, GR1+) and eosinophils (Eos; Ly6Gint, F4/80int, SSChigh) 4 h post-infection. (B) Counts of each cell population identified in the lavage 4 h post-infection with 3348 (w.t.), 3348DspyCEP (D) or PBS. Data shown are means plus SEM of one representative experiment using 8 mice per group, except for the PBS control (n = 3). Statistical significance was tested by Mann-Whitney U: P,0.05 (), P,0.01 (). doi:10.1371/journal.pone.0040411.g003 Figure 4. Increased levels of KC, LIX and MIP-2 during infe with 3348DspyCEP. Thirteen different chemokines were measu the individual mouse air pouch lavages collected at 4 h (A) and 2 post-infection with 3348 (white bars) and 3348DspyCEP (black Results are shown as means and SEM of one representative expe using 8 mice per group. Statistical significance was tested by Whitney U: P,0.01 (), P,0.001 (). doi:10.1371/journal.pone.0040411.g004 Figure 4. Increased levels of KC, LIX and MIP-2 during infection with 3348DspyCEP. Thirteen different chemokines were measured in the individual mouse air pouch lavages collected at 4 h (A) and 24 h (B) post-infection with 3348 (white bars) and 3348DspyCEP (black bars). Results are shown as means and SEM of one representative experiment using 8 mice per group. Statistical significance was tested by Mann- Whitney U: P,0.01 (), P,0.001 (**). doi:10.1371/journal.pone.0040411.g004 I i i i h S CEP i d i i i i Figure 3. Cell recruitment in situ. (A) Gating strategy to identify neutrophils (NPh; Ly6Ghigh, GR1high), dendritic cells (DC; CD11c+, MHC II+), macrophages (MPh; CD11b+, F4/80high), monocytes (Mo; CD11bhigh CD11c2, Ly6Chigh, GR1+) and eosinophils (Eos; Ly6Gint, F4/80int, SSChigh 4 h post-infection. (B) Counts of each cell population identified in the lavage 4 h post-infection with 3348 (w.t.), 3348DspyCEP (D) or PBS. Data shown are means plus SEM of one representative experiment using 8 mice per group, except for the PBS control (n = 3). Statistica significance was tested by Mann-Whitney U: P,0.05 (), P,0.01 (*). doi:10.1371/journal.pone.0040411.g003 Figure 4. Increased levels of KC, LIX and MIP-2 during infection with 3348DspyCEP. Thirteen different chemokines were measured in the individual mouse air pouch lavages collected at 4 h (A) and 24 h (B) post-infection with 3348 (white bars) and 3348DspyCEP (black bars). Results are shown as means and SEM of one representative experiment using 8 mice per group. Specific antibodies counteract rSpyCEP inhibition of PMN migration in vitro g In order to study PMN transmigration in a clean system, we first investigated whether rSpyCEP would constitute a suitable replacement for native SpyCEP on bacteria or in culture supernatant. IL-8 was incubated with serial dilutions of cell wall extract from the 3348 strain or from the isogenic mutant 3348DspyCEP where rSpyCEP had been added (Figure 5A). This ensured that potential inhibitory or activating constituents derived from the cell wall digestion procedure were equally present. The experiment covered a range of SpyCEP/rSpyCEP quantities, which induced non-detectable to complete IL-8 cleavage. The breakpoint (approximately equal proportions of cleaved and uncleaved IL-8) occurred at the same dilution for native SpyCEP and rSpyCEP, suggesting that the recombinant enzyme is fully active and can be used as a substitute for bacterial extracts or supernatant preparations. PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40411 5 SpyCEP Action in a Murine Air Pouch Model Immunization with rSpyCEP induces protective immunity in murine models of S. pyogenes infection [15,16,29]. As SpyCEP is a virulence factor, the mechanism of protection may include Previous studies have shown that IL-8 dependent transmig of PMN in vitro is inhibited by SpyCEP from bacterial supern [3]. In a similar setup, we observed that pretreatment of K IL-8 with rSpyCEP reduces murine and human PMN re ment, respectively. In the case of KC and murine PM reduction of up to 8 fold was observed (Figure 5B), while fo the reduction was in the order of 2–3 fold (Figure Pretreatment with the inactive rSpyCEP was used as a c and had no effect on human PMN transmigration, indicatin this functional interference is strictly dependent on prote activity of rSpyCEP. In the case of murine PMN, rSpy showed a small increase in PMN migration over the whol concentration range compared to the control. We speculat some impurities from the rSpyCEP* purification might re this minor activation of murine PMN. In a short preincu step, polyclonal antisera specific for rSpyCEP or for an unr S. pyogenes surface protein (Spy0269) were added to the s (Figure 5D). The control samples (with no IL-8) suggested th addition of serum causes a slight increase in transmigr perhaps due to presence of complement or chemokines. N theless, the presence of a-rSpyCEP abrogated or alleviate effect of SpyCEP on PMN transmigration, while a-Sp antibodies had no effect. This suggests that immunization Figure 3. Specific antibodies counteract rSpyCEP inhibition of PMN migration in vitro Statistical significance was tested by Mann- Whitney U: P,0.01 (), P,0.001 (). doi:10.1371/journal.pone.0040411.g004 Previous studies have shown that IL-8 dependent transmigration of PMN in vitro is inhibited by SpyCEP from bacterial supernatants [3]. In a similar setup, we observed that pretreatment of KC and IL-8 with rSpyCEP reduces murine and human PMN recruit- ment, respectively. In the case of KC and murine PMN, a reduction of up to 8 fold was observed (Figure 5B), while for IL-8 the reduction was in the order of 2–3 fold (Figure 5C). Pretreatment with the inactive rSpyCEP was used as a control and had no effect on human PMN transmigration, indicating that this functional interference is strictly dependent on proteolytic activity of rSpyCEP. In the case of murine PMN, rSpyCEP* showed a small increase in PMN migration over the whole KC concentration range compared to the control. We speculate that some impurities from the rSpyCEP* purification might result in this minor activation of murine PMN. In a short preincubation step, polyclonal antisera specific for rSpyCEP or for an unrelated S. pyogenes surface protein (Spy0269) were added to the system (Figure 5D). The control samples (with no IL-8) suggested that the addition of serum causes a slight increase in transmigration, perhaps due to presence of complement or chemokines. Never- theless, the presence of a-rSpyCEP abrogated or alleviated the effect of SpyCEP on PMN transmigration, while a-Spy0269 antibodies had no effect. This suggests that immunization with rSpyCEP generates antibodies which can interfere functionally with the native S. pyogenes virulence factor. Figure 3. Cell recruitment in situ. (A) Gating strategy to identify neutrophils (NPh; Ly6Ghigh, GR1high), dendritic cells (DC; CD11c+, MHC- II+), macrophages (MPh; CD11b+, F4/80high), monocytes (Mo; CD11bhigh, CD11c2, Ly6Chigh, GR1+) and eosinophils (Eos; Ly6Gint, F4/80int, SSChigh) 4 h post-infection. (B) Counts of each cell population identified in the lavage 4 h post-infection with 3348 (w.t.), 3348DspyCEP (D) or PBS. Data shown are means plus SEM of one representative experiment using 8 mice per group, except for the PBS control (n = 3). Statistical significance was tested by Mann-Whitney U: P,0.05 (), P,0.01 (). doi:10.1371/journal.pone.0040411.g003 Immunization with rSpyCEP induces protective immunity in murine models of S. pyogenes infection [15,16,29]. As SpyCEP is a virulence factor, the mechanism of protection may include opsonization and/or functional interference with SpyCEP activity. PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40411 6 Figure 5. Discussion Most of the studies on SpyCEP have focused on the secreted or shed form that is found in the supernatant of isolates that express high levels of the protease. Our results show that in exponential phase a considerable fraction of the protease is securely anchored to the bacterial surface, can be recognized by specific antibodies, and retains the ability to cleave chemokines. SpyCEP harbors a typical cell wall attachment motif, and surface localization through covalent attachment by the housekeeping sortase A would be expected. On the contrary, a considerable amount of the protease is consistently found in the supernatant of broth cultures during stationary phase. Since the protease undergoes autocatalytic processing, it was possible that shedding may depend, directly or indirectly, upon this activity. However, our data showed that shedding is independent of SpyCEP autoproteolytic activity. We speculate that shedding is related to spyCEP overexpression in the 3348 strain, and possibly contributes to the CovRS2 hypervirulent phenotype [22]. Figure 6. Specific antibodies recognize SpyCEP on bacteria. Exponential (A) and stationary (B) phase bacteria were labeled with pooled mouse a-Alum (white) or mouse a-SpyCEP (gray) sera. Secondary antibodies were rabbit a-mouse phycoerythrin conjugates, and the fluorescence (PE-A) is shown on the x axis. Strains 3348 (w.t.), 3348DspyCEP (D) and 3348spyCEP () are displayed on the z axis. doi:10.1371/journal.pone.0040411.g006 Figure 5. Activity of rSpyCEP and PMN transmigration. (A) Comparison of rSpyCEP and native SpyCEP activity on IL-8. SDS-PAGE (18% Tris-Glycine) and silver staining after 2 h of digestion with 5 ng, 1 ng, 0.2 ng, 0.04 ng or 0 ng (ctr lane) of rSpyCEP+3348DspyCEP extracts (left). Cell wall extracts containing comparable amounts of native SpyCEP from 3348 extracts (right). Full and trace arrowheads indicate intact and cleaved IL-8, respectively. Lower panel: control Western blot showing relative SpyCEP amounts compared to 5 ng of rSpyCEP (ctr). (B,C) SpyCEP effect on PMN transmigration. (B) Murine or (C) human PMN migration in response to KC or IL-8 (white), respectively, in the presence of rSpyCEP (black) or rSpyCEP (grey). (D) Counteraction of rSpyCEP activity by specific antibodies. Human PMN migration using IL-8 (white); IL-8, rSpyCEP and a-SpyCEP (black); and IL-8, rSpyCEP, a-Spy0269 (grey). Data represent means plus SEM of one representative experiment using triplicates. Statistical significance was tested by unpaired Student T, () P,0.05, () P,0.01, (*), P,0.001. Figure 6. Specific antibodies recognize SpyCEP on bacteria. SpyCEP Action in a Murine Air Pouch Model SpyCEP Action in a Murine Air Pouch Model SpyCEP Action in a Murine Air Pouch Model SpyCEP, at least in exponential phase, was found attached to the bacterial surface, and active against IL-8 (see above). Moreover, the action of SpyCEP on chemokines and PMN recruitment was countered by the presence of specific antibodies. We next asked whether specific antibodies against rSpyCEP can recognize native SpyCEP on the bacterial surface. Bacteria in exponential and stationary phase were incubated with a-SpyCEP antisera, or sera from mice that were mock-immunized with Alum (neg. control). The 3348 w.t. and spyCEP strains in exponential phase growth showed an appreciable shift in fluorescence intensity with a-SpyCEP antibodies compared to negative control samples (Figure 6). Knockout strains were similar to the negative control. In stationary phase, similar results were observed for strain 3348 and its mutants. With SF370, backgrounds were higher, and no appreciable shifts were observed compared to control samples (data not shown) in either phase of growth. In conclusion, recognition of SpyCEP at the bacterial surface requires a relatively high level of expression, such as in the overexpressing strain 3348. Specific antibodies counteract rSpyCEP inhibition of PMN migration in vitro Activity of rSpyCEP and PMN transmigration. (A) Comparison of rSpyCEP and native SpyCEP activity on IL-8. SDS-PAGE (18% Tris-Glycine) and silver staining after 2 h of digestion with 5 ng, 1 ng, 0.2 ng, 0.04 ng or 0 ng (ctr lane) of rSpyCEP+3348DspyCEP extracts (left). Cell wall extracts containing comparable amounts of native SpyCEP from 3348 extracts (right). Full and trace arrowheads indicate intact and cleaved IL-8, respectively. Lower panel: control Western blot showing relative SpyCEP amounts compared to 5 ng of rSpyCEP (ctr). (B,C) SpyCEP effect on PMN transmigration. (B) Murine or (C) human PMN migration in response to KC or IL-8 (white), respectively, in the presence of rSpyCEP (black) or rSpyCEP* (grey). (D) Counteraction of rSpyCEP activity by specific antibodies. Human PMN migration using IL-8 (white); IL-8, rSpyCEP and a-SpyCEP (black); and IL-8, rSpyCEP, a-Spy0269 (grey). Data represent means plus SEM of one representative experiment using triplicates. Statistical significance was tested by unpaired Student T, () P,0.05, () P,0.01, (*) P,0 001 1. Carapetis JR, Steer AC, Mulholland EK, Weber M (2005) The global burden of group A streptococcal diseases. Lancet Infect Dis 5: 685–694. Supporting Information Figure S1 Growth curves for 3348, 3348DspyCEP, 3348spyCEP, SF370, SF370DspyCEP and SF370spyCEP* strains in THY. Bacteria were grown until early exponential phase (OD = 0.2) and then diluted 1:50 in fresh THY medium. The growth was followed by recording OD600 at 30 min intervals. (A) 3348 and mutants. (B) SF370 and mutants. (TIF) Figure S1 Growth curves for 3348, 3348DspyCEP, 3348spyCEP, SF370, SF370DspyCEP and SF370spyCEP strains in THY. Bacteria were grown until early exponential phase (OD = 0.2) and then diluted 1:50 in fresh THY medium. The growth was followed by recording OD600 at 30 min intervals. (A) 3348 and mutants. (B) SF370 and mutants. (TIF) Figure S2 SF370 infection in a murine air pouch model. After air pouch inflation, CD1 female mice were infected with 16108 CFU of SF370 (n = 6, two mice were excluded due to in- fighting prior to infection), SF370DspyCEP (n = 8) or PBS (n = 3). 4 or 24 h post infection lavage material from each mouse was serially diluted and bacterial viable counts were performed. Multiplication factor (total CFU/inoculum CFU) in the lavage from individual mice is shown. Horizontal bars are geometric means. Some general observations from our model of infection can also be made. One of the most prominent chemokines in the air pouch lavage was IL-6, a proinflammatory chemokine with prognostic value for severity of infection [7,35,36]. In our case, the high levels of IL-6 suggest that substantial general inflammation was taking place in situ, while we did not observe any difference between w.t. and mutant. However, the levels of IL-6 were in the uppermost end of the range covered by our assay, where values are distinctly less precise. A rather surprising finding was the high number of eosinophils recruited to the site. Eosinophils are not known to have any role in host defense against S. pyogenes infection, and neither are they particularly numerous among circulating leukocytes. Eosinophils are recruited during mycobacterial infection, partic- ularly in lung granulomas, and it has been suggested that they play a direct role in TLR2-mediated innate immunity against mycobacteria [37]. We speculate that eosinophils may also have a role in S. pyogenes localized infection. Acknowledgments We are indebted to Giuliano Bensi for his expertise with animal models and for his support in setting up the air pouch model. We thank the animal facility at NVD Siena for their invaluable technical assistance during the animal experiments, in particular Marco Tortoli and Elena Amantini. We thank Sandra Nuti, Simona Tavarini and Chiara Sammicheli for their availability and excellent technical support with flow cytometry exper- iments. We thank Vincenzo NardiDei for providing purified rSpyCEP and rSpyCEP. We thank Marilena Gallotta for providing the mutant strain 3348DspyCEP and the construct pJRS233:: DspyCEP. We thank Fabiana Falugi for providing the construct pET21b+spyCEP(D151A). We thank Kate Seib for critical reading of the manuscript. SpyCEP Action in a Murine Air Pouch Model overall outcome of infection in our model. We believe that this model of local infection holds great promise for the study of host- pathogen interactions, as it permits quantification of many interrelated infection parameters. Compared to traditional lethal challenges the model is fast, relatively harmless to the animals, and does not require complicated animal manipulation techniques. It should be applicable also to study virulence factors of other bacterial species, and we are currently investigating whether it can also serve as a model to investigate protective immunity. We have shown that SpyCEP can cleave chemokines not only in its shed form, but also on the bacterial surface. Moreover, we have shown that specific antibodies to SpyCEP can counteract the inhibitory effects on PMN recruitment in vitro. Such antibodies can also interact with SpyCEP at the bacterial surface. Importantly, SpyCEP acts on chemokines in situ, and absence of the protein puts bacteria at a relative disadvantage. In the context of a vaccine against S. pyogenes infection, the inclusion of SpyCEP as a component may exert a dual effect – the promotion of opsonophagocytosis and a relative reduction of virulence 2. Hidalgo-Grass C, Dan-Goor M, Maly A, Eran Y, Kwinn LA, et al. (2004) Effect of a bacterial pheromone peptide on host chemokine degradation in group A streptococcal necrotising soft-tissue infections. Lancet 363: 696–703. SpyCEP Action in a Murine Air Pouch Model Previous work has suggested that mutants lacking spyCEP are less virulent in animal models, although there are conflicting results [4,7–11]. S. pyogenes is an obligate human pathogen, and animal models of S. pyogenes infection are challenging to implement. We were interested in developing a model suitable for studying the interplay between bacteria and host, specifically the contribution of SpyCEP to host cell recruitment to the site of infection. Subcutaneous teflon chamber implants have been used with mice to study local responses to infection and host cell recruitment, but this model requires a surgical procedure and a post-surgical recovery period [30]. The air pouch route of infection has been used for S. pyogenes as a tissue damage model (ulceration and/or histopathology) or a lethal challenge model [31]. A similar model has been used by immunologists for several decades to evaluate immunomodulatory drugs and acute inflam- mation, where the main readout is inflammatory parameters [32]. In a typical setup, an air pouch is inflated in the mouse by repeated subcutaneous injections of air over time, an irritant is injected together with a putative modulator or control, and a lavage is then collected and analyzed [33,34]. We adapted this protocol and replaced the irritant with a bacterial infection. In this work we show that the chemokines KC, LIX and MIP-2 are highly susceptible to SpyCEP action in vivo. In the strain lacking SpyCEP, levels of the above chemokines were 10–1,000 fold higher compared to the wild type strain. These three chemokines all promote PMN recruitment. In fact, mice infected with the mutant strain repeatedly showed higher levels of neutrophils and monocytes compared to w.t. infections, although the difference was not statistically significant (P = 0.1). We speculate that even the lower levels of chemoattractants in mice infected with the w.t. may be sufficient for near maximal recruitment to the site of infection. While it is clear from our work and that of others that cleaved IL-8 and KC fail to recruit PMN, we cannot rule out that LIX or MIP- 2 might retain part or all of their chemoattractant properties even when cleaved, or, that other chemoattractants are involved (e.g. C5a). As for bacterial survival, at 4 h post-infection, mice infected with 3348DspyCEP showed bacterial growth retardation compared to mice infected with the 3348 w.t. strain, consistent with an elevated number of professional phagocytes in the former case. Author Contributions Conceived and designed the experiments: RJ NC AS IM DS. Performed the experiments: NC. Analyzed the data: NC RJ GG IM DS JLT AS. Contributed reagents/materials/analysis tools: AS. Wrote the paper: RJ NC IM. Provided critical review of manuscript: GG DS JLT AS. More extensive studies, including different infection doses, histopathology, the study of systemic dissemination from the site etc., could eventually clarify the extent to which SpyCEP affects Discussion Exponential (A) and stationary (B) phase bacteria were labeled with pooled mouse a-Alum (white) or mouse a-SpyCEP (gray) sera. Secondary antibodies were rabbit a-mouse phycoerythrin conjugates, and the fluorescence (PE-A) is shown on the x axis. Strains 3348 (w.t.), 3348DspyCEP (D) and 3348spyCEP (*) are displayed on the z axis. doi:10.1371/journal.pone.0040411.g006 doi:10.1371/journal.pone.0040411.g005 PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40411 7 SpyCEP Action in a Murine Air Pouch Model SpyCEP Action in a Murine Air Pouch Model Kuo CF, Wu JJ, Lin KY, Tsai PJ, Lee SC, et al. (1998) Role of streptococcal pyrogenic exotoxin B in the mouse model of group A streptococcal infection. Infect Immun 66: 3931–3935. J 15. 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(2008) A chemokine- degrading extracellular protease made by group A Streptococcus alters pathogenesis by enhancing evasion of the innate immune response. Infect Immun 76: 978–985. 27. Delano DL, Montesinos MC, D’Eustachio P, Wiltshire T, Cronstein BN (2005) An interaction between genetic factors and gender determines the magnitude of the inflammatory response in the mouse air pouch model of acute inflammation. Inflammation 29: 1–7. 11. Turner CE, Kurupati P, Jones MD, Edwards RJ, Sriskandan S (2009) Emerging role of the interleukin-8 cleaving enzyme SpyCEP in clinical Streptococcus pyogenes infection. J Infect Dis 200: 555–563. 28. Nickerson-Nutter CL, Medvedeff ED (1996) The effect of leukotriene synthesis inhibitors in models of acute and chronic inflammation. Arthritis Rheum 39: 515–521. J 12. Fritzer A, Noiges B, Schweiger D, Rek A, Kungl AJ, et al. (2009) Chemokine degradation by the Group A streptococcal serine proteinase ScpC can be reconstituted in vitro and requires two separate domains. Biochem J 422: 533– 542. 29. Turner CE, Kurupati P, Wiles S, Edwards RJ, Sriskandan S (2009) Impact of immunization against SpyCEP during invasive disease with two streptococcal species: Streptococcus pyogenes and Streptococcus equi. Vaccine 27: 4923–4929. 13. Kaur SJ, Nerlich A, Bergmann S, Rohde M, Fulde M, et al. (2010) The CXC chemokine-degrading protease SpyCep of Streptococcus pyogenes promotes its uptake into endothelial cells. J Biol Chem 285: 27798–27805. 30. Kazmi SU, Kansal R, Aziz RK, Hooshdaran M, Norrby-Teglund A, et al. (2001) Reciprocal, temporal expression of SpeA and SpeB by invasive M1T1 group a streptococcal isolates in vivo. Infect Immun 69: 4988–4995. 14. Zingaretti C, Falugi F, Nardi-Dei V, Pietrocola G, Mariani M, et al. (2010) Streptococcus pyogenes SpyCEP: a chemokine-inactivating protease with unique structural and biochemical features. FASEB J 24: 2839–2848. 31. References PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40411 8 SpyCEP Action in a Murine Air Pouch Model SpyCEP Action in a Murine Air Pouch Model Appl Environ Microbiol 63: 3539–3547. J 37. Driss V, Legrand F, Hermann E, Loiseau S, Guerardel Y, et al. (2009) TLR2- dependent eosinophil interactions with mycobacteria: role of alpha-defensins. Blood 113: 3235–3244. 20. Seubert A, Monaci E, Pizza M, O’Hagan DT, Wack A (2008) The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoat- PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40411 9
https://openalex.org/W4239960337	https://jcovm.uobaghdad.edu.iq/index.php/Iraqijvm/article/download/403/355	English	null	Extraction and Titration of Leukotoxins from Fusobacterium necrophorum Isolates Recovered from Bovine Liver Abscesses in Sulaimaniyah Region	The Iraqi journal of veterinary medicine/Al-maǧallaẗ al-ṭibbiyyaẗ al-bayṭariyyaẗ al-’irāqiyyaẗ	2,012	cc-by	3,060	Extraction and Titration of Leukotoxins from Fusobacterium necrophorum Isolates Recovered from Bovine Liver Abscesses in Sulaimaniyah Region Suha Ali Hussein1 and Essam F. Al-Jumaily2 1Dept. of microbiology, College of Veterinary Medicine, University of Sulaimani, Sulaimaniyah, Kurdistan Region- Iraq (suhaali68@yahoo.com). 2Biotechnology Dept., Genetic Engineering and Biotechnology Institute for Post Graduate Studies, Baghdad University, Baghdad- Iraq. Summary This study was conducted to extract and titrate the leukotoxin of Fusobacterium necrophorum isolates recovered from 75 abscesses found in 24 livers of slaughtered cattle in Sulaimaniyah region. The culture supernatants of these isolates were subjected to the tetrazolium dye reduction test which revealed that the leukotoxin titer values of 34 F. necrophorum subsp. necrophorum isolates ranged from 128 to 1024 (with a leukotoxin titer mean of 516±46), whereas the leukotoxin titer values of the 11 F. necrophorum subsp. funduliforme isolates ranged from 0 to 128 (with a leukotoxin titer mean of 73±12). K d F b t i h l k t i t ti ey words: Fusobacterium necrophorum; leukotoxin extraction. استخالص ومعايرة لوكوتوكسينات جراثيم Fusobacterium necrophorum المعزولة من خراجات كبد االبقار في منطقة السليمانية سهى علي حسين1 و عصام فاضل الجميلي2 1 فشع االدٍاء الوجهشٌت، كلٍت الطب البٍطشي، جاهعت السلٍواًٍت، إقلٍن كشدسخاى العشاق 4 ،قسن الخقاًت االدٍائٍت هعهذ الهٌذست الىساثٍت والخقاًت االدٍائٍت ، جاهعت بغذاد، العشاق الخالص ـة صووـج هـزٍ الـذساس ـ ت السخخالص وهعاٌشة اللىكىحىكسٌٍاث ل عضالث جشاثٍن F. necrophorum عضلج هي 75 خشاج وجذث فً أكبـاد24 بقشة ربذج فً هذافظت السلٍواًٍت . اخضعج الشواشخ الضسعٍت لهزٍ العضالث الى فذص اخخضال صبغت الخخشاصولٍىم والزي اظهش أى قٍن هعٍا س اللىكىحىكسٍي الخً كاًج حخشاوح بٍي 141 الى1142 (بوعذل هعٍاس لىكىحىكسٍي (46 ±516 لـ 42 عضلت هي جشاثٍنF. necrophorum subsp. necrophorum بٌٍوا كاًج حخشاوح بٍي 1 الى141 (بوعذل هعٍاس لىكىحىكسٍي(12 ±73 لـ 11 عضلت هي جشاثٍنF. necrophorum subsp. Funduliform. استخالص ومعايرة لوكوتوكسينات جراثيم Fusobacterium necrophorum المعزولة من خراجات كبد االبقار في منطقة السليمانية سهى علي حسين1 و عصام فاضل الجميلي2 1 فشع االدٍاء الوجهشٌت، كلٍت الطب البٍطشي، جاهعت السلٍواًٍت، إقلٍن كشدسخاى العشاق 4 ،قسن الخقاًت االدٍائٍت هعهذ الهٌذست الىساثٍت والخقاًت االدٍائٍت ، جاهعت بغذاد، العشاق ا صووـج هـزٍ الـذساس ـ ت السخخالص وهعاٌشة اللىكىحىكسٌٍاث ل عضالث جشاثٍن F. necrophorum عضلج هي 75 خشاج وجذث فً أكبـاد24 بقشة ربذج فً هذافظت السلٍواًٍت . اخضعج الشواشخ الضسعٍت لهزٍ العضالث الى فذص اخخضال صبغت الخخشاصولٍىم والزي اظهش أى قٍن هعٍا س اللىكىحىكسٍي الخً كاًج حخشاوح بٍي 141 الى1142 (بوعذل هعٍاس لىكىحىكسٍي (46 ±516 لـ 42 عضلت هي جشاثٍنF. necrophorum subsp. necrophorum بٌٍوا كاًج حخشاوح بٍي 1 الى141 (بوعذل هعٍاس لىكىحىكسٍي(12 ±73 لـ 11 عضلت هي جشاثٍنF. necrophorum subsp. Funduliform. Proceeding of the Eleventh Veterinary Scientific Conference, 2012; 110 -114 Proceeding of the Eleventh Veterinary Scientific Conference, 2012; 110 -114 y ( ) 2. Leukotoxin assay 2. Leukotoxin assay Bacterial isolates identified to be F. necrophorum were investigated for their ability of leukotoxin production as follows: Bacterial isolates identified to be F. necrophorum were investigated for their ability of leukotoxin production as follows: Introduction Fusobacterium necrophorum, a gram-negative, anaerobic bacterium and a normal inhabitant of the rumen, is the primary etiologic agent of bovine liver abscesses (1, 2). It is classified into two subspecies, F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme (3, 4). The pathogenicity of F. necrophorum is attributed mainly to its leukotoxin which seems to be an important virulence factor in the pathogenesis of hepatic and interdigital necrobacillosis and it is indicated to be cytotoxic to leukocytes, macrophages, ruminal epithelial cells, and possibly hepatocytes (5, 6). Although the pathogenicity and virulence factors of F. necrophorum have been studied widely for many years, attempts to develop an effective vaccine against liver abscess have not been successful commercially (7). However, previous studies have indicated that antileukotoxin immunity reduced the incidence of hepatic abscesses and interdigital necrobacillosis (5). The aim of the present study was to extract and titrate the leukotoxins of F. necrophorum isolated locally from liver abscesses of slaughtered cattle in Sulaimaniyah region. 111 1. Bacterial isolates F. necrophorum isolates were recovered from 57 abscesses found in livers of 42 slaughtered cattle in the abattoir of Sulaimaniyah governorate. The isolates were recovered by obtaining swab samples from the inner walls of the liver abscesses which were opened under sterile conditions with a sterile scalpel. The swabs were streaked on brain heart infusion supplemented with 5% horse blood. These media were then incubated in an anaerobic jar with an AnaeroGen gas pack at 39ºC (8) for 48-72 hours. Growing colonies that revealed gram negative rod characteristics were re-cultured on brain heart infusion. These media were then incubated aerobically and anaerobically for 48 hours in order to check out the exact growing conditions they need (facultative anaerobe growing condition or strictly anaerobe growing condition). The facultative anaerobic bacteria were excluded. Strictly anaerobic bacterial colonies were picked up and inoculated into sterilized thioglycollate and brain heart infusion broths. The thyioglycollate broth cultures were sealed with rubber stoppers and incubated overnight at 39 ˚C whereas the brain heart infusion broth cultures were incubated anaerobically using the pyrogallol-carbonate seal according to (9, 10). The overnight cultures of both broths were tested for morphology, staining characteristics (using Gram’s stain) and sedimentation of the cultures in the broth. Following that, the colonies that revealed Gram’s negative, rod-shaped bacterial characteristics were re-streaked on brain heart infusion agar supplemented with 5% horse blood and incubated anaerobically in an anaerobic jar with an AnaeroGen gas pack for 48 hours. The recovered colonies were identified according to conventional bacteriological and biochemical procedures according to the instructions of the materials manufacturers and as described by (11-14). y ( ) 2. Leukotoxin assay a. Production of leukotoxin The production of leukotoxin was performed according to (15) in which purified colonies of each isolate were inoculated into 10 ml of a brain heart infusion broth and then incubated for 7 hours at 39ºC under an anaerobic condition using pyrogallol-carbonate seal. b. Extraction of leukotoxin-containing culture supernatant b. Extraction of leukotoxin-containing culture supernatant Extraction of leukotoxin-containing culture supernatant was performed according to (8) in which the culture supernatants of a 7 hours old growth of F. necrophorum isolates were obtained by centrifugation at 15000g for 30 minutes at 4ºC in a cooled centrifuge then the supernatants were filtered through 0.22µm membrane filter. These samples were stored at - 28ºC until used for the leukotoxicity assay. c. Preparation and Determination of the concentration and viability of leukotoxin-target cells Bovine polymorphonuclear neutrophils (PMN leukocytes) were used as target cells for leukotoxin of F. necrophorum organisms. The target cells were prepared according to (16) while the concentration and viability of the PMN leukocytes were determined according to (17) by the trypan blue dye exclusion method. y yp y etrazolium dye reduction assay d. Tetrazolium dye reduction assay The leukotoxicity assay was performed according to (8) using the tetrazolium dye reduction test. The formazan concentration representing PMN leukocyte viability was determined by measuring the absorbance values in an ELISA reader with dual wavelength (570 nm as test wavelength and 650 nm as reference). The leukotoxicity, expressed in percentage of cell death, and calculated as follows: 1- (absorbance of toxin treated cells / absorbance of control cells) ] x 100 The titer of leukotoxin was calculated as the reciprocal of the higher culture supernatant dilution causing ≥10% loss in viability of leukocytes. 111 Results 1. Bacterial isolates 1. Bacterial isolates A total of 110 bacterial isolates were recovered out of the 57 liver abscesses sampled in the current study. Out of these 110, only 45 isolates showed growing and staining characteristics compatible with F. necrophorum. Thus, the frequency rate of F. necrophorum induced liver abscesses in the current study is 78.9%. Investigation of the culture and staining characteristics, along with the physiological, biochemical and biological tests (Table 1) of these 45 isolates revealed identification of 2 groups of F. necrophorum isolates as follows: g p p Group 1: This group involved 34 bacterial isolates that showed characteristics compatible with those of F. necrophorum subsp. necrophorum. Group 2: This group involved 11 bacterial isolates that revealed characteristics compatible with those of F. necrophorum subsp. funduliforme. Discussion Forty five F. necrophorum induced liver abscesses out of the 57 cultured liver abscesses (with a frequency rate of 78.9%) have been reported in the current study. Such a higher frequency rate is often attributed to the mechanism by which liver abscess develops in view of the fact that the numbers of F. necrophorum bacteria in the rumen are often increased during the conditions of ruminal acidosis that follow sudden change to high-grain diets, because these bacteria use lactate rather than sugars as their major energy source for growth (6). This frequency rate (78.9%) is approximately compatible with that reported by (7) who stated that the incidence of F. necrophorum from cultured liver abscesses has ranged from 81 to 100% of abscesses. Both subspecies of F. necrophorum were recognized in the present study, however, the most frequently isolated one was F. necrophorum subsp. necrophorum (34 isolates recovered from 57 liver abscesses) compared to only 11 F. necrophorum subsp. funduliforme. This finding is not compatible with prevalence values of F. necrophorum induced liver abscesses in feedlot cattle reported by (18 and 19). In view of the variation in the type of diet on which the sampled cattle were nourished “cattle involved in the prevalence studies of the authors mentioned above (18 and 19) which are usually kept on high grain diet compared to those involved in the current study which are usually nourished on different types of diet”, this incompatibility in the prevalence values of subspecies F. necrophorum bacteria appeared to be realistic because the concentration of both subspecies of F. necrophorum bacteria in the rumen is markedly influenced by the type of diet (20). y y yp In this study, bovine polymorphonuclear neutrophils (PMN leukocytes) were used as target cells for leukotoxin of F. necrophorum organisms because there is general agreement that the leukotoxin of F. necrophorum is a soluble, proteinaceous and heat-labile exotoxin with specificity for ruminant’s neutrophils (6, 8, 21). The leukotoxin titer of F. necrophorum subsp. necrophorum isolates obtained in the present study ranged from 128 – 1024 (with a leukotoxin titer mean of 516±46), whereas that of F. necrophorum subsp. funduliforme isolates ranged from 0 – 128 (with a leukotoxin titer mean of 73±12). 2. Leukotoxicity titer y The leukotoxin titer values of the 34 F. necrophorum subsp. necrophorum isolates ranged from 128 to 1024 with a leukotoxin titer mean of 516±46. Fusobacterium necrophorum subsp. necrophorum isolates number 15, 16, 21 27, 33 and 34 showed the highest leukotoxin titer values (1024). On the other hand, the leukotoxin titer values of the 11 F. necrophorum subsp. fuduliforme isolates ranged from 0 to 128 with a leukotoxin titer mean of 73±12 (Table 2). Table 1: Identification of Fusobaterium necrophorum subspecies. Identification tests subsp. necrophorum subsp. funduliforme Gram’s stain Negative Negative Motility test Non motile Non motile Hemolysis test Wide beta hemolysis Narrow beta hemolysis Indole test + + Catalase test – – H2S production + + MR/ VP test – / – – / – Nitrate reduction test – – Gelatin liquefaction V V Fermentation of: Glucose W or – – Maltose – – Fructose – – Sucrose – – Lactose – – DNase test + – Phosphatase test + – Lipase test + – Agglutination of chicken erythrocytes + – V: variable reaction W: weak reaction Table 1: Identification of Fusobaterium necrophorum subspecies. Identification tests subsp. necrophorum subsp. funduliforme Gram’s stain Negative Negative Motility test Non motile Non motile Hemolysis test Wide beta hemolysis Narrow beta hemolysis Indole test + + Catalase test – – H2S production + + MR/ VP test – / – – / – Nitrate reduction test – – Gelatin liquefaction V V Fermentation of: Glucose W or – – Maltose – – Fructose – – Sucrose – – Lactose – – DNase test + – Phosphatase test + – Lipase test + – Agglutination of chicken erythrocytes + – V: variable reaction W: weak reaction Table 1: Identification of Fusobaterium necrophorum subspecies. 114 Proceeding of the Eleventh Veterinary Scientific Conference, 2012; 110 -114 Proceeding of the Eleventh Veterinary Scientific Conference, 2012; 110 -114 Table 2: Means of leukotoxin titers in culture supernatants of F. necrophorum subspecies necrophorum and F. necrophorum subspecies funduliforme isolates. Subspecies a Range of leukotoxin titer * Median of leukotoxin titer Mean of leukotoxin titer ± SE F. necrophorum subsp. necrophorum 128-1024 512 516±46 F. necrophorum subsp. funduliforme 0-128 64 73±12 * The range values mentioned in this table refer to the range of leukotoxin titer of 34 isolates of F. necrophorum subsp. necrophorum and 11 isolates of F. 2. Leukotoxicity titer necrophorum subspecies funduliforme investigated for their ability of leukotoxin production in the current study. 2: Means of leukotoxin titers in culture supernatants of F. necrophorum subspecies necrophorum and F. necrophorum subspecies funduliforme isolates. * The range values mentioned in this table refer to the range of leukotoxin titer of 34 isolates of F. necrophorum subsp. necrophorum and 11 isolates of F. necrophorum subspecies funduliforme investigated for their ability of leukotoxin production in the current study. * The range values mentioned in this table refer to the range of leukotoxin titer of 34 isolates of F. necrophorum subsp. necrophorum and 11 isolates of F. necrophorum subspecies funduliforme investigated for their ability of leukotoxin production in the current study. References 1. Scanlan, C. M., and Hathcock, T. L. (1983). Bovine rumenitis-liver abscess complex: a bacteriological review. Cornell Veternarian 73: 288-297. 2. Tan, Z. L., Nagaraja, T. G. and Chengappa, M. M. (1996). Fusobacterium necrophorum infections: virulence factors, pathogenic mechanism and control measures. Vet. Res. Commun. 20: 113–140. Proceeding of the Eleventh Veterinary Scientific Conference, 2012; 110 -114 3. Langworth, B. F. (1977). Fusobacterium necrophorum: Its Characteristics and Role as an Anima Pathogen. Bacteriol. Rev. 41: 373-390. worth, B. F. (1977). Fusobacterium necrophorum: Its Characteristics and Role as an Anim ogen. Bacteriol. Rev. 41: 373-390. 4. Shinjo, T.; Fujisawa, T. and Mitsuoka, T. (1991). Proposal of two subspecies of Fusobacterium necrophorum (Flu¨gge) Moore and Holdeman: Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flu¨gge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Halle´ 1898). Int. J. Syst. Bacteriol. 41: 395-397. 5. Clark, B. L.; Emery, D. L.; Stewart, D. J.; Dufty, J. H. and Anderson, D.A. (1986). Studies into immunization of cattle against interdigital necrobacillosis. Austral. Vet. J. 63: 107- l10. 6. Tan, Z. L.; Nagaraja, T. G.; Chengappa, M. M. and Smith, J. S. (1994). Biological and biochemical characterization of Fusobacterium necrophorum leukotoxin. Am. J. Vet. Res. 55: 515-521. 7. Nagaraja, T. G. and Chengappa, M. M. (1998). Liver abscesses in feedlot cattle. J. Anim 298. a, T. G. and Chengappa, M. M. (1998). Liver abscesses in feedlot cattle. J. Anim. Sci. 76: 287- 8. Tan, Z. L.; Nagaraja, T. G. and Chengappa, M. M. (1992). Factors affecting the leukotoxin activity of Fusobacterium necrophorum. Vet. Microbiol. 32: 15-28. 9. Casida, L. E. JR. (1965). Abundant microorganism in soil. Appl. Microbiol. 13: 327-334. 10. Cappuccino, J. G. and Sherman, N. (2008). Microbiology, A laboratory Manual, 8th ed., Pearson International Edition, New York. 11. Nagai, S.; Kanoe, M. and Toda, M. (1984). Purification and partial characterization of Fusobacterium necrophorum hemagglutinin. Zbl. Bakteriol. Hyg. [A] 258: 232-241. 12. Brown, R.; Collee, J. G. and Poxton, I. R. (1996). Bacteroides, Fusobacterium and other Gram- negative anaerobic rods; Anaerobic cocci; Identification of anaerobes. In: Collee, J. G.; Fraser, A. G.; Marmion, B. P.; Simmons, A. (eds.), Mackie & McCartney Practical Medical Microbiology, 14th ed., Churchill Livingstone, New York. 13. Collee, J. G.; Miles, R. S. and Watt, B. (1996). Tests for identification of bacteria. In: Collee, J. G.; Fraser, A. G.; Marmion, B. P.; Simmons, A. (eds.), Mackie & McCartney Practical Medical Microbiology, 14th ed., Churchill Livingstone, New York. 14. Alexander, S. K.; Strete, D.; Niles, M. J. (2004). Laboratory Exercises in Organismal and Molecular Microbiology, McGraw-Hill Higher Education, Inc., New York. 15. Saginala, S.; Nagaraja, T. G.; Lechtenberg, K. F.; Chengappa, M. M.; Kemp, K. E. and Hine, P. M. (1997). Discussion The difference in leukotoxin production may account for the difference in virulence between the two subspecies (22-25) and it explains why the subspecies necrophorum is encountered more frequently in bovine hepatic abscesses than the subspecies funduliforme (1, 7, 24). This finding is in agreement with those mentioned by several authors (6, 8, 19, 25) who reported that Fusobacterium necrophorum subsp. necrophorum produces more leukotoxin than F.necrophorum subsp. funduliforme. 114 Proceeding of the Eleventh Veterinary Scientific Conference, 2012; 110 -114 Effect of Fusobacterium necrophorum leukotoxoid vaccine on susceptibility to experimentally induced liver abscesses in cattle. J. Anim. Sci. 75: 1160-1166. 16. Reddy, P. G.; McVey, D. S.; Chengappa, M. M.; Blecha, F.; Minocha, H. C. and Baker, P. E. (1990). Bovine recombinant granulocyte-macrophage coloney stimulating factor enhances bovine neutrophil function in vitro. J. Am. Vet. Med. Assoc. 51: 1395-1399. 17. Weber, B.; Nickol, M. M.; Jagger, K. S. and Saelinger, C. B. (1982). Interaction of Pseudomonas exoproducts with phagocytic cell. Can. J. Microbiol. 28: 679-685. p p g y 18. Lechtenberg, K. F.; Nagaraja, T. G.; Leipold, H. W. and Chengappa, M. M. (1988). Bacteriologic and histologic studies of hepatic abscesses in cattle. Am. J. Vet. Res. 49: 58–62. 19. Tadepalli, S.; Stewart, G. C.; Nagaraja T. G. and Narayanan, S. K. (2008). Human Fusobacterium necrophorum strains have a leukotoxin gene and exhibit leukotoxic activity. J .Med. Microbiol. 57: 225-231. 20. Tan, Z. L.; Nagaraja, T. G. and Chengappa, M. M., (1994). Selective enumeration of Fusobacterium necrophorum from the bovine rumen. Appl. Env. Microbiol. 60: 1387-1389. 21. Kanoe, M.; Ishii, T.; Mizutani, K. and Blobel, H. (1986). Partial characterization of leukocidin from Fusobacterium necrophorum. Zbl. Bakteriol. Hyg. [A] 261: 170-176. 22. Shinjo, T.; Miyazato, S.; Kaneuchi, C. and Mitsuoka, T. (1981). Physiological and biochemical characteristics of Fusobacterium necrophorum biovar A and B strains and their deoxyribonucleic acid homology. Jpn. J. Vet. Sci. 43:233-241. gy 23. Berg, J. N., and Scanlan, C. M. (1982). Studies of Fusobacterium necrophorum from bovine hepatic abscesses: biotypes, quantitation, virulence, and antibiotic susceptibility. Am. J. Vet. Res. 43: 1580- 1586. 24. Smith, G. R. (1992). Pathogenicity of Fusobacterium necrophorum biovar B. Res. Vet. Sci. 52: 260- 261. 25. Tan, Z. L., Nagaraja, T. G. and Chengappa, M. M. (1994). Biochemical and biological characterization of ruminal Fusobacterium necrophorum. FEMS Microbiol. Lett. 120: 81-86. 112
https://openalex.org/W1995474415	https://zenodo.org/records/576519/files/ZK_article_2053.pdf	English	null	Revision of Khoikhoiinae (Hymenoptera, Braconidae)	ZooKeys	2,009	cc-by	13,913	Michael Sharkey1,†, Simon van Noort2,‡, James Whitfi eld3,§ Michael Sharkey1,†, Simon van Noort2,‡, James Whitfi eld3,§ 1 Department of Entomology, University of Kentucky, Lexington, Kentucky 40546-0091, USA 2 Natural History Division, South African Museum, Iziko Museums of Cape Town, PO Box 61, Cape Town 8000, South Africa 3 Department of Entomology, 320 Morrill Hall, University of Illinois at Urbana-Champaign, 505 S. Goodwin Ave, Urbana, IL 61801 USA † urn:lsid:zoobank.org:author:77B8EC3A-442C-4A7A-AF85-A31C27E257F2 ‡ urn:lsid:zoobank.org:author:7CCD166F-F1FA-43DA-B582-4E84EAF59AD1 § urn:lsid:zoobank.org:author:7A98AB5F-552D-4437-8F5D-C593CA713506 Corresponding author: James Whitfi eld (jwhitfi e@life.uiuc.edu) Academic editor: Norman Johnson \| Received 21 February 2009 \| Accepted 17 June 2009 \| Published 14 September 2009 urn:lsid:zoobank.org:pub:4AA0293D-DEC8-424C-9053-1CDD965CCC82 Citation: Sharkey M, van Noort S, Whitfi eld J (2009) Revision of Khoikhoiinae (Hymenoptera, Braconidae). In: Johnson N (Ed) Advances in the sys tematics of Hymenoptera. Festschrift in honour of Lubomír Masner. ZooKeys 20: 299–348. doi: 10.3897/zookeys.20.108 Abstracth Th e species of the two genera of Khoikhoiinae (Hymenoptera, Braconidae) are revised. Th irteen species are recognized, of which fi ve are new and eight were previously described: Khoikhoia anthelion Sharkey, sp. n., K. lission Mason, 1984, K. oligospilos Sharkey, sp. n., K. semiadusta Mason, 1983, K. solata Mason, 1983, K. townesi Mason, 1983, K. turneri Mason, 1984, Sania browni Sharkey, sp. n., S. capensis Mason, 1983, S. henryi Mason, 1983, S. marjoriae Mason, 1983, S. masneri Sharkey, sp. n., and S. masoni Sharkey, sp. n. All are from the Cape Region of South Africa, and all but one species are confi ned to the western Cape. A dichotomous key to species is presented; links to electronic interactive keys and to distribution maps are also included. Based on phylogenetic position and morphological characters, speculations on life history are made, and it is suggested that some species may be parasitoids of wood- or stem-boring Lepidoptera. Th e DELTA data matrix and images for the key are available at 10.3897/zookeys.20.108. app.1.ik; Intkey fi les are available at 10.3897/zookeys.20.108.app.2.ik; Lucid fi les in LIF and SDD for- mat are available at: doi:10.3897/zookeys.20.108.app.3.ik and doi:10.3897/zookeys.20.108.app.4.ik. Publishing of DELTA raw data will facilitate future workers to edit keys and to add newly discovered taxa. 299 Launched to accelerate biodiversity research A peer-reviewed open-access journal 299 Launched to accelerate biodiversity research A peer-reviewed open-access journal R ZooKeys 20: 299–348 (2009) doi: 10.3897/zookeys.20.108 www.pensoftonline.net/zookeys R ZooKeys 20: 299–348 (2009) doi: 10.3897/zookeys.20.108 www.pensoftonline.net/zookeys R ZooKeys 20: 299–348 (2009) doi: 10.3897/zookeys.20.108 www.pensoftonline.net/zookeys ikhoiinae (Hymenopte RESEARCH ARTICLE Copyright Michael Sharkey et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Keywords y South Africa, parasitoid wasps, Lepidoptera, Khoikhoia, Sania, taxonomy, systematics Copyright Michael Sharkey et al. This is an open access article distributed under the terms of the Creative Commons Attribution permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 300 Introduction Khoikhoiinae is a small subfamily of braconids in the microgastroid complex that is restricted to the Cape Region of South Africa. It was proposed by Mason (1983), who included two new genera, Khoikhoia and Sania, and six species, three of each genus. He also discussed its phylogenetic position within the microgastroid complex, and diff er- entiated it from the morphologically somewhat similar Cardiochilinae. Mason (1984) added two species that he discovered in the British Museum and added characters that further diff erentiated the two genera. Here we describe fi ve new species and re-describe Mason’s species. A fully illustrated dichotomous key is presented and links to on-line interactive and dichotomous keys are included. Mason proposed generic limits and synapomorphies, and described eight species based on only 11 specimens. We examined 76 specimens in the course of this study. Naturally some of Mason’s (1983, 1984) conclusions now seem to be erroneous in light of the new data. For example, in Mason’s (1983) key, he used several characters to distinguish the two genera that we have discovered to be present in both. We too are still working with a very limited set of specimens and our conclusions are tentative. Materials and methods All species descriptions and the dichotomous key were generated using DELTA soft- ware (http://delta-intkey.com). Data, species names, characters and character states were entered into Delta Editor (Dallwitz 1980; Dallwitz et al. 1999). Th e “tokey” fi le was edited to select and weight the characters used for the dichotomous key, and the modifi ed fi le was exported from DELTA to produce the key which was then lightly edited to produce the fi nal version (Dallwitz 1974; Dallwitz 1980; Dallwitz et al. 1993). Th e interactive key was produced in a similar manner using the DELTA fi le “toint” and the software IntKey (Dallwitz 1980; Dallwitz et al. 1993; Dallwitz et al. 1995). All source fi les and images used in this publication are available http:// sharkeylab.org/sharkeylab/Misc/datasets/DeltaFiles/KhoikhoiinaeDeltaFiles.zip and in Appendix 1 of the present paper (doi:10.3897/zookeys.20.108.app.1.ds). Th ese fi les are open to the public and future researchers are welcome to download them if they wish to modify, correct, or add to it for publication. All new species have been registered with Zoobank (Polaszek et al. 2005). Online interactive matrix and dichotomous keys were also produced using Lucid (www.lucidcentral.org), and are available on Waspweb at: http:// www.waspweb.org/Ichneumonoidea/Braconidae/Keys/index.htm. The key to Khoikhoiinae is available in three formats. Lucid Phoenix keys are dichotomous and a choice needs to be made at each key couplet to continue. Lucid matrix keys, on the other hand, use a different approach where relevant states from multiple character features can be selected independently until identification is achieved. For more information concerning Lucid keys visit www.lucidcentral.org <http:// Revision of Khoikhoiinae (Hymenoptera, Braconidae) 301 www.lucidcentral.org>. Files are provided as appendices in two formats enabling conversion of the Lucid matrix key to other platforms. 1. Lucid Interchange Format version 3 (LIF3) files are XML-based files that store all the Lucid3 key data, allowing exchange of the key with other key developers. 2. SDD files are XML-based files structured using the internationally agreed SDD (Structure of Descriptive Data) Schema. SDD files may be used to exchange Lucid keys with other SDD-compliant applications. Measurements were taken with a Microcode II stage measurement device manu- factured by Boeckeler Instruments. Images were taken with three diff erent instrument systems: 1. Automontage soft- ware using a 3CCD JVC digital camera attached to a Leica MZ16 stereo microscope. 2. EntoVision micro-imaging system. Materials and methods Th is system included a Leica M16 compound microscope with a JVC KY-75U 3-CCD digital video camera attached that fed im- age data to a notebook. Th e program Cartograph 5.6.0 was then used to merge an image series into a single in-focus image. Lighting was achieved using techniques summarized in Buffi ngton et al. (2005), Kerr et al. (2009) and Buffi ngton and Gates (2009). 3. SEM photographs of gold coated specimens and ESEM photographs of uncoated type series specimens were taken with a Philips XL30 ESEM-FEG fi eld- emission environmental scanning electron microscope at the Imaging Technology Group, Beckman Institute, University of Illinois, with digital images captured di- rectly onto computer. Species descriptions are for the holotype specimen. If the holotype was a male and female specimens were known, the female specimens was used to describe female spe- cifi c characters. Within the holotype description, variation in other specimens of both sexes is given in angled brackets, < >. Where there are clear diff erences in male and female specimens in non-sexual characters these are mentioned in a separate section below the holotype description. Kentucky, Lexington, Kentucky 40546 0091, USA. Natural History Museum (BMNH) Cromwell Road, London SW7 5BD, UK. Museum depositories: p American Entomological Institute (AEI) 3005 SW 56th Avenue, Gainesville, FL 32608-5047, USA. Canadian National Collection of Insects (CNCI) Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, K.W. Neatby Building, 960 Carling Ave. Ottawa, Ontario K1A 0C6, CANADA Illinois Natural History Survey (INHS) 1816 South Oak Street, MC 652, Cham- paign, IL 61820. Iziko South African Museum (SAMC) Natural History Division, South African Museum, Iziko Museums of Cape Town, PO Box 61, Cape Town 8000, South Africa. Hymenoptera Institute Collection (HIC) Department of Entomology, University of Kentucky, Lexington, Kentucky 40546–0091, USA.h y g y Th e Natural History Museum (BMNH) Cromwell Road, London SW7 5BD, UK. Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 302 Figure 1. Ovipositor morphology, showing variation within Khoikhoiinae A Sania marjoriae Mason 1983 B Sania masoni Sharkey, sp. n. C Sania browni Sharkey, sp. n. D Sania masneri Sharkey, sp. n. E Khoikhoia turneri Mason, 1984 F Khoikhoia townesi Mason, 1983. Figure 1. Ovipositor morphology, showing variation within Khoikhoiinae A Sania marjoriae Mason 1983 B Sania masoni Sharkey, sp. n. C Sania browni Sharkey, sp. n. D Sania masneri Sharkey, sp. n. E Khoikhoia turneri Mason, 1984 F Khoikhoia townesi Mason, 1983. Life history and evolution Host associations are unknown for all species of Khoikhoiinae; however, morphological and phylogenetic clues allow for some conjecture. All reliable host records for members of the microgastroid braconids are Lepidoptera, although occasional uncorroborated records from other insect orders have been published, and one reasonable record is from Trichoptera (Achterberg 2002). Phylogenetic studies of the microgastroids (re- Revision of Khoikhoiinae (Hymenoptera, Braconidae) 303 Figure 2. Scanning electron micrographs A Khoikhoia anthelion Sharkey, sp. n., female, mesosoma and fi rst two metasomal tergites, dorsal view B Sania browni Sharkey, sp. n., female, propodeum and fi rst three metasomal tergites, dorsal view C Sania marjoriae Mason, 1983, female, propodeum and laterotergites, lateral view D Sania masneri Sharkey, sp. n., male, genitalia E Sania masoni Sharkey, sp. n., male, meta- soma, lateral view, showing genitalia F Khoikhoia anthelion Sharkey, sp. n., female, ovipositor, lateral view. Figure 2. Scanning electron micrographs A Khoikhoia anthelion Sharkey, sp. n., female, mesosoma and fi rst two metasomal tergites, dorsal view B Sania browni Sharkey, sp. n., female, propodeum and fi rst three metasomal tergites, dorsal view C Sania marjoriae Mason, 1983, female, propodeum and laterotergites, lateral view D Sania masneri Sharkey, sp. n., male, genitalia E Sania masoni Sharkey, sp. n., male, meta- soma, lateral view, showing genitalia F Khoikhoia anthelion Sharkey, sp. n., female, ovipositor, lateral view. viewed in Murphy et al. 2008), although far from robust in support for some clades, clearly place Cheloninae as sister to the remaining subfamilies, so we can infer that members of Khoikhoiinae are parasitoids of Lepidoptera. All microgastroids are larval or egg-larval parasitoids (attacking the egg stage and developing in the larval stage). Since egg-larval parasitism occurs in all known chelonines and is also present in many microgastrines, it could possibly be part of the ground plan for the non-chelonine microgastroids (Ruberson and Whitfi eld 1996 ). Th e very thin, sharp ovipositors of khoikhoiines (Figs. 1, 2F) are consistent with this scenario. Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 304 Figure 3. Scanning electron micrographs A Sania marjoriae Mason, 1983, female, head lateral view B Sania masoni Sharkey, sp. n., female, head, lateral view C Khoikhoia anthelion Sharkey, sp. n., female, antennal scape (showing apical fl ange) and details of antennal scrobe, lateral view D Khoikhoia anthelion Sharkey, sp. Life history and evolution n., female, subalar region, lateral view E Sania marjoriae Mason, 1983, female, subalar region, lateral view F Sania masoni Sharkey, sp. n., female, subalar region, lateral view. Figure 3. Scanning electron micrographs A Sania marjoriae Mason, 1983, female, head lateral view B Sania masoni Sharkey, sp. n., female, head, lateral view C Khoikhoia anthelion Sharkey, sp. n., female, antennal scape (showing apical fl ange) and details of antennal scrobe, lateral view D Khoikhoia anthelion Sharkey, sp. n., female, subalar region, lateral view E Sania marjoriae Mason, 1983, female, subalar region, lateral view F Sania masoni Sharkey, sp. n., female, subalar region, lateral view. Many ichneumonoids that emerge from the host larvae after it has spun a pupal chamber have twisted mandibles that are thought to be eff ective in cutting through the silken threads of their host. Th is is the case with many members of Microgastrinae. Th e family Agathidinae consists exclusively of parasitoids of Lepidoptera larvae, and most members have twisted scissor-like mandibles. Th e one known exception to this is in the clade composed of the genera Crassomicrodus and Agathirsia, both of which are found primarily in semiarid regions of the southwest of the USA, Mexico and Central America (Pucci and Sharkey 2004). Although it is necessary to extract from only one Revision of Khoikhoiinae (Hymenoptera, Braconidae) 305 host record (Acontia cretata Noctuidae), these are parasitoids of caterpillars that do not spin cocoons, and cutting mandibles are not required to escape the host’s silken chamber. We suspect, therefore, that the hosts of khoikhoiines do not spin cocoons. p p Another odd morphological characteristic possessed by many species of Khoikhoiinae is a well-excavated antennal scrobe (Figs. 3A–B). Th is is a feature that, in the Braconidae at least, is restricted to taxa attacking wood-boring hosts. Th e antennal scrobes function to protect the antennae as the adult braconid emerges from woody substrate. Th ese braconids, e.g., Helcon, Capitonious, also have robust mandibles much like those of khoikhoiines. g p Perhaps the most informative characteristic of Khoikhoiinae is the fl ared scape. Th e scape of all species of Khoikhoia, and two species of Sania (S. marjoriae, and S. masneri), is fl ared apically and the apical surface is not a simple ridge, but rather it includes an area with considerable surface (Figs. 3A–C). Life history and evolution Th is is another characteristic that, within the Braconidae, is restricted to species attacking wood-boring hosts, e.g. many Braconinae including Cyclaulax and Hemibracon (see Figs. 37 and 41 in Quicke 1997). We do not believe that there has been any published conjecture on function, but we suggest that these structures are used to help prevent back-slippage when the adult wasp is escaping its host’s habitat through a wooden tunnel. While moving forward through the tunnel the scape would be directed posteriorly and housed within the antennal scrobe. Th e fl anged part of the scape would be directed posterodorsally. As the wasp moved forward in the tunnel the scape would be pushed against the scrobe but any backwards move- ment would cause the fl ange of the scape to lock into the dorsal surface of the tunnel. In essence the movement of the wasp forward in the tunnel is analogous to a ratchet. Four of the six species of Sania do not have a strongly fl ared scape and also lack a well-developed antennal scrobe. Also, the species of Sania have less robust mandibles compared with those of Khoikhoia. Th e four species of Sania that lack a fl ared scape (S. browni, S. capensis, S. henryi, and S. masoni) may not be attacking wood-boring hosts. p y g g In summary, we posit that some members of Khoikhoiinae attack wood-boring (or possibly stem-boring or woody infl orescence boring) Lepidoptera. Infl orescences and in- fructescences of Protea species are bored by the larvae of a range of coleopteran and lepidop- teran species (Coetzee and Giliomee 1987, Wright and Samways 1999, 2000). Th e most likely candidate would be a basal lepidopteran clade that is restricted, or mostly confi ned, to the Cape Region of South Africa, where all species of Khoikhoiinae have been collected. Another potential host candidate is a lepidopteran that feeds on roots, in which case the antennal and mandibular modifi cations of Khoikhoiinae might be used to tunnel through densely packed soil. One candidate host clade is the enigmatic Prototheoridae (Lepidoptera: Hepialoidea). Th is family is confi ned to southern Africa, and most diverse in the Cape region of South Africa (Davis 1996). Unfortunately knowledge of its life history is lacking. g g g Khoikhoiinae are associated with the Fynbos and Succulent Karoo biomes (Ru- therford et al. Life history and evolution 2006), which form the major component of the Cape Floristic region. Th ese biomes have been recognized as globally important biodiversity hotspots (My- ers et al. 2000; Latimer 2005), as well as a center of distribution and endemism for plants (Cowling et al. 1998), and numerous invertebrate taxa, including the fi gitid sub- family Pycnostigminae and the recently discovered insect order Mantophasmatodea, Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 306 both groups having their center of species richness in the Cape Region (Picker et al. 2002, Buffi ngton and van Noort 2007, Damgaard et al. 2008). It is plausible that the Khoikhoiinae may have diversifi ed with the evolution and radiation of the Cape Flo- ristic Region 3–5 million years ago (Goldblatt 1997; Linder et al. 1992; Linder 2003). Several of the new species described here were collected in signifi cant numbers in Malaise traps. For example, 21 specimens of Sania masoni were collected over a 20 day period from a single Malaise trap. Th is suggests that khoikhoiines may be locally com- mon, and is consistent with the idea some species or their hosts are gregarious. p g g Relationships among member subfamilies in the microgastroid complex are dis- cussed by Mason (1983) and Whitfi eld and Mason (1994) with respect to morpho- logical evidence, and by Murphy et al. (2008) with respect to molecular evidence from seven genes. Despite analyses of a relatively large amount of DNA sequence data (Mur- phy et al. 2008) and of a signifi cant data set of comparative morphological characters (Whitfi eld and Mason 1994), the exact placement of Khoikhoiinae among the non- cheloninae microgastroids is still uncertain, although its inclusion within this complex is highly supported. Our understanding is likely to improve considerably when the host biology and larval stages of khoikhoiines become known, and when a larger sample of the fauna can be included in molecular phylogenetic analysis (the study by Murphy et al. (2008) featured only one Khoikhoia and three Sania species). Th e recent study of bracovirus origins in the microgastroid complex by Bezier et al. (2009) attempted to confi rm the presence of bracovirus structural genes across the complex using PCR, but was unable to confi rm the presence of these genes in either Sania or Khoikhoia. Life history and evolution It is nevertheless likely, due to the phylogenetic position of khoikhoiines, that they do contain mutualistic bracoviruses, and that the negative PCR results were due to either mismatches in primer design or marginal specimen quality for molecular study. 4(3). Mesopleuron at least partly smooth with punctures ....................................6 4’ Mesopleuron entirely rugosopunctate or rugose ..........................................5 Key to genera and species of Khoikhoiinae Median tergite 1 partly smooth posterolaterally, rugose anteriorly ................. ...................................................................... Sania masoni Sharkey, sp. n. 12’ Median tergite 1 completely rugose .............................Sania henryi Mason 11(10). Median tergite 1 partly smooth posterolaterally, rugose anteriorly; posterola- teral margin straight. .....................................Sania masneri Sharkey, sp. n. 11’ Median tergite 1 completely rugose; posterolateral margin curved ................ .............................................................................. Sania marjoriae Mason 11 11' 12(10). Median tergite 1 partly smooth posterolaterally, rugose anteriorly ................. ...................................................................... Sania masoni Sharkey, sp. n. 12’ Median tergite 1 completely rugose .............................Sania henryi Mason 12' 12' 12 12 Key to genera and species of Khoikhoiinae An illustrated, interactive IntKey is available at http://sharkeylab.org/sharkeylab/shar- keyKeys.php. Interactive Lucid matrix and Lucid Phoenix keys are available at: http:// www.waspweb.org/Ichneumonoidea/Braconidae/Keys/index.htm 1. Clypeus with medial tooth. ..................................................Khoikhoia .....2 1’ Clypeus without medial tooth..................................................... Sania .....8 1 1' yp u u d 1 1' 1' Revision of Khoikhoiinae (Hymenoptera, Braconidae) 307 2(1). Several large punctures arranged in a longitudinal row on middle lobe of mesoscutum posteromedially...............Khoikhoia anthelion Sharkey, sp. n. 2’ Medial lobe of mesoscutum lacking longitudinal row of large punctures pos- teromedially ................................................................................................3 2 2' 2' 3(2). Notauli entirely crenulate. ..........................................................................4 3’ Notauli crenulations weak and partly eff aced ................................................ ......................................................... Khoikhoia oligospilos Sharkey, sp. n. 3(2). Notauli entirely crenulate. ..........................................................................4 3’ Notauli crenulations weak and partly eff aced ................................................ ......................................................... Khoikhoia oligospilos Sharkey, sp. n. 3 3' 3' 4(3). Mesopleuron at least partly smooth with punctures ....................................6 4’ Mesopleuron entirely rugosopunctate or rugose ..........................................5 4 4' Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 308 7(6) Median tergite 1 mostly smooth with weak microsculpture 6(4). Mesosoma entirely black except tegula somewhat paler ................................. .....................................................................Khoikhoia semiadusta Mason 6’ Mesosoma partly brown ..............................................................................7 6 6' 6' 5 5' 5(4). Middle lobe of scutum smooth with punctures anteromedially, otherwise ru- gosopunctate .......................................................Khoikhoia turneri Mason 5’ Middle lobe of scutum entirely rugosopunctate ............................................ ...........................................................................Khoikhoia townesi Mason 5 5' 6 6' 6' 7(6). Median tergite 1 mostly smooth with weak microsculpture .......................... ............................................................................. Khoikhoia solata Mason 7’ Median tergite 1 mostly rugose .............................Khoikhoia lission Mason 7 7' 7 7' Revision of Khoikhoiinae (Hymenoptera, Braconidae) Revision of Khoikhoiinae (Hymenoptera, Braconidae) 309 309 8(1). Discrimen smooth or with a few barely perceptible crenulae .......................9 8’ Discrimen crenulate ..................................................................................10 10 8' 8 8' 8 9(8). Metapleuron mostly rugose, lacking a large smooth lightly punctate area. ..... ................................................................................ Sania capensis Mason 9’ Metapleuron partly rugose but with a large smooth lightly punctate area. ..... ..................................................................... Sania browni Sharkey, sp. n. 9 9 9' 9 9' 10(8). Scape fl ared apicoanteriorly with expanded apical surface .........................11 10’ Scape not fl ared apicoanteriorly and lacking expanded apical surface ........12 9' Scape fl ared apicoanteriorly with expanded apical surface .........................11 Scape not fl ared apicoanteriorly and lacking expanded apical surface ........12 12 10 10 10' 10' 10 10' 10 Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 310 Taxonomy 11 11' 12' 12 12(10). Khoikhoiinae Mason, 1983 Description. Head. Number of fl agellomeres variable within species ranging from about 20–40, antenna often sexually dimorphic, female fl agellum compressed api- cally with fl agellomeres shorter and wider than males, sometimes fl agellomeres trans- verse in females, though usually slightly to 3× longer than wide; fl agellomeres some- times sexually dimorphic with fl agellum of females tapering strongly towards apex, males tapering slightly as in most other microgastroids; (this type of dimorphism is also found in a number of species of Cheloninae, e.g., Ascogaster abdominator (Dahlbom, 1833)); fl agellar placodes short, about 1/3 to 1/4 length of fl agellom- eres in males, not arranged into two rows; antennal scrobe present, though weak in most species of Sania; frons with elevated, coarsely sculptured, protuberance lat- erad antennal scrobe, eff ectively increasing functional depth of scrobe, protuberance Revision of Khoikhoiinae (Hymenoptera, Braconidae) 311 variously developed from weak to strong depending on species and generally more developed in members of Khoikhoia; malar suture distinct; mandible thick and long, not greatly twisted to function as scissors; temple large, presumably to hold large mandibular muscles; maxillary palpus 5-segmented, basal two segments fused; la- bial palpus 4-segmented; labrum setose, semicircular, usually exposed but capable of folding behind the clypeus, clypeus weakly concave (Sania) or with median and sometimes lateral teeth (Khoikhoia). Mesosoma. Propleuron lacking carina on posterolateral margin and lacking ventral fl ange overlapping ventral corner of pronotum; subalar region of meso- pleuron with a smooth glabrous area posteriorly, more pronounced in species of Khoikhoia; notauli impressed, crenulate and meeting posteriorly where they gener- ally extend to the transscutal articulation; transscutal articulation complete and depressed; postscutellar depression well developed, more so in Khoikhoia; propo- deum mostly rugose with median longitudinal carina; epicnemial carina absent; tarsal claws simple; hind tarsomeres lacking longitudinal ridge of setae; hind basi- tarsus and to a lesser degree some other hind tarsomeres laterally compressed; api- cal abscissa of Rs of forewing decurved and not tracheated; forewing 1a crossvein long and strong; forewing 2a crossvein absent; forewing 1cu-a far apical to M; forewing 2cu-a absent; forewing 2nd submarginal cell quadrate, tapering apically with r-m crossvein mostly unsclerotized; hindwing crossvein r long but not tubu- lar; hindwing r-m absent. Metasoma. Khoikhoia Mason, 1983 Type species: Khoikhoia townesi Mason, 1983 Type species: Khoikhoia townesi Mason, 1983 Diagnosis: Clypeus with median and sometimes lateral teeth; mandible large, face rugose to rugosopunctate; subalar area of mesopleuron with vertical carina weak or absent, and with posterior convex smooth glabrous area. Distribution: Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Khoikhoia g pp p p g Etymology: “Th e genus name is feminine, dedicated to the original inhabitants of the South African cape region, the Khoikhoi, who were known to the Dutch settlers as Hottentots because of their unique clicking speech.” (Mason 1983: p. 53). Khoikhoiinae Mason, 1983 Median tergite 1 with a median longitudinal groove; laterotergite 1 membranous (or absent according to Mason’s 1983 interpretation) and with micro- striae, spiracle on laterotergite 1; hypopygium size variable from more than half length of metasoma to approximately 1/5 length of metasoma; ovipositor length variable from barely exserted to almost as long as the metasoma; setae of ovipositor sheath sometimes restricted to apex where they can be very long, e.g., K. anthelion. g g Distribution. Restricted to the Western, Eastern and Northern Cape Provinces of South Africa, in localities varying from sea level to 1000 meters. Distribution map is available at http://sharkeylab.org/sharkeylab/Misc/generalmapper.php?table=khoikhoi inae&subfamily=Khoikhoiinae. Biodiversity. Including the new species proposed here, there are 13 species of Khoikhoiinae. Based on morphological evidence, most of these seem quite distinct, although K. turneri and K. townesi may constitute one species. Nonetheless, there are undoubtedly many more species. Of the seven species of Khoikhoia, all but two are represented by one specimen, and the two exceptions are known from two specimens. Two of these species were collected during intensive programs of Malaise trap sam- pling at two localities spanning a couple of years suggesting that in contrast to Sania, Khoikhoia species may be rare. Malaise traps seem eff ective in capturing specimens, and further intensive Malaise trap sampling targeting under-collected habitats in the Cape and perhaps other areas of southern Africa will undoubtedly result in the discov- ery of many more species. y y p Hosts. Unknown, probably larval Lepidoptera (See Life History section above). 312 Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) Male. Antenna highly sexually dimorphic with the female antennae shorter and tapering much more rapidly than those of male. Species Descriptions of Khoikhoiinae Khoikhoia anthelion Sharkey, sp. n. urn:lsid:zoobank.org:act:D613345A-984A-4DEF-89AA-C5A91BD6FA84 Khoikhoia anthelion Sharkey, sp. n. urn:lsid:zoobank.org:act:D613345A-984A-4DEF-89AA-C5A91BD6FA84 Holotype female. Body. Length. 6.8 mm. Color. Mostly black with laterotergite 1 pale and the following areas yellowish brown: gena, posterodorsal apex of pronotum, tegula, legs except coxae, trochanters, and some tarsomeres (Fig. 4A–C). Forewing mostly weakly infuscate but less so anterobasally and at midlength (Fig. 5E). <Male paratype very similar to holotype, with somewhat more extensive yellowish brown color on the pronotum.> Head. Number of fl agellomeres 29 <29–30>. Scape fl ared apicoanteriorly with expanded apical surface (Fig. 3C). Antennal scrobe shallow (Fig. 3C); rugose medially, smooth laterally except with few transverse striae immediately posterad antennal insertion (Fig. 3C). Gena with wide irregular punctures, lacking distinct rugose striae (Fig. 5A). Face entirely rugose (Fig. 4E). Area between antennal scrobe and inner orbit of eye with weak protuberance (Fig. 4E). Vertex with strong coarse punctures (Fig. 4F). Posterior orbit of eye with distinct crenulate margin (Fig. 5A). Mesosoma. Middle lobe of scutum entirely smooth with punctures (Fig. 4D). Notauli entirely crenulate (Fig. 4D). Mesopleuron entirely rugosopunctate or rugose (Figs. 3D, 4C). Sternaulus long, occupying most of length of mesopleuron, indicated by vertically elongated crenulae (Figs. 3D, 4C). Discrimen (median longitudinal ventral sulcus between mesopleura) crenulate. Metapleuron mostly rugose, lacking a large smooth lightly punctate area (Fig. 4C). Propodeum entirely rugose without smooth area posterolaterally (Fig. 5C). Metasoma. Median tergite 1 not distinctly narrowed posteriorly (Figs. 2A, 5C), completely rugose (Figs. 2A, 5C). Hypopygium less than 1/3 length of metasoma (Fig. 5D); extending past apical tergum (Fig. 5D). Ovipositor barely exserted, much shorter than metasoma (Figs. 2F, 5D). Length of setae of ovipositor sheath as much as 4x as long as width of sheath (Figs. 2F, 5D). g g Male. Antenna highly sexually dimorphic with the female antennae shorter and tapering much more rapidly than those of male. Revision of Khoikhoiinae (Hymenoptera, Braconidae) 313 Diagnosis. Mesopleuron entirely rugosopunctate or rugose (Fig. 4C); middle lobe of scutum entirely smooth with punctures (Fig. 4D). Material Examined. Holotype female: South Africa, Western Cape, Kogelberg Na- ture Reserve, MT, KO98-M42, Mesic Mtn. Fynbos, last burnt c. 1988, 34°16.481’S, 19°01.033’E, 199m, 16.IX-16.X.1999, S. van Noort. SAM-HYM-P0024705 (SAMC) Paratype. Male same data as holotype (HIC). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Khoikhoia&species=anthelion. Figure 4. Khoikhoia anthelion Sharkey, sp. Species Descriptions of Khoikhoiinae n., female, holotype A habitus, lateral view B habitus, dorsal view C mesosoma, lateral view D mesosoma, dorsal view E head, anterior view F head, dorsal view. Figure 4. Khoikhoia anthelion Sharkey, sp. n., female, holotype A habitus, lateral view B habitus, dorsal view C mesosoma, lateral view D mesosoma, dorsal view E head, anterior view F head, dorsal view. 314 Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) Etymology. Anthelion (Greek) is the diminutive of anthele, tuft or plume of a reed. Th e name refers to the unique tuft of setae on the ovipositor sheaths. Khoikhoia lission Mason, 1984 Holotype female. Body Length. 4.7 mm. Color. Mottled dark brown and yellowish brown except laterotergite 1 whitish, otherwise metasoma mostly pale brown, head and Figure 5. Khoikhoia anthelion Sharkey, sp. n., female, holotype A head, lateral view B head, anterior view C propodeum, metasomal tergites, dorsal view D ovipositor, lateral view E wings F data labels. Figure 5. Khoikhoia anthelion Sharkey, sp. n., female, holotype A head, lateral view B head, anterior view C propodeum, metasomal tergites, dorsal view D ovipositor, lateral view E wings F data labels. Revision of Khoikhoiinae (Hymenoptera, Braconidae) 315 mesothorax predominantly yellowish brown (Fig. 6). Forewing entirely infuscate (Fig. 7E). Head. Number of fl agellomeres 34. Scape fl ared apicoanteriorly with expanded apical surface. Antennal scrobe deep (Fig. 7B); entirely transversely rugosostriate. Gena transversely rugosostriate anteriorly, rugosopunctate at midlength, longitudinally ru- gosostriate posteriorly (Fig. 7B). Face entirely rugose (Fig. 7A). Area between antennal scrobe and inner orbit of eye with protuberance (Fig. 7A). Vertex with weak punctures (Fig. 7B). Posterior orbit of eye with distinct crenulate margin (Fig. 7B). Mesosoma. Middle lobe of scutum entirely smooth with punctures (Fig. 7C). Notauli entirely crenu- Figure 6. Khoikhoia lission Mason, 1984, female, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Figure 6. Khoikhoia lission Mason, 1984, female, holotype A habitus, dorsal view B habitus, lateral view Figure 6. Khoikhoia lission Mason, 1984, female, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Figure 6. Khoikhoia lission Mason, 1984, female, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 316 late (Fig. 7C). Mesopleuron at least partly smooth with punctures (Fig. 6C). Sternaulus weakly indicated by a slight depression, not diff erentially sculptured (Fig. 6C) <this is likely to be variable>. Discrimen (median longitudinal ventral sulcus between mesop- leura) crenulate. Metapleuron mostly rugose, lacking a large smooth lightly punctate area (Fig. 6C). Propodeum entirely rugose without smooth area posterolaterally (Fig. 7C). Metasoma. Median tergite 1 not distinctly narrowed posteriorly (Fig. 7D), completely rugose but with rugae weak posterolaterally (Fig. 7D). Diagnosis. Mesopleuron at least partly smooth with punctures (Fig. 6C); notauli entirely crenulate; mesosoma partly or entirely brown; median tergite 1 completely rugose (Fig. 7C). ; p y y ; g p y g ( g 7 ) Figure 7. Khoikhoia lission Mason, 1984, female, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels Figure 7. Khoikhoia lission Mason, 1984, female, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels Revision of Khoikhoiinae (Hymenoptera, Braconidae) 317 Male. Unknown. Material Examined. Holotype female: South Africa, Witzenberg Vall., S.W. Cape Prov., 914m, 19.I.1921, R.E. Turner (BMNH). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Khoikhoia&species=lission. Etymology. Mason (1984) did not include the etymology but the interpreta- tion seems rather straightforward. Lission (Greek) is the diminutive of lissos, meaning smooth or polished and referring to the mesosoma which is smooth relative to other members of the genus. smooth or polished and referring to the mesosoma which is smooth relative to other members of the genus. Figure 8. Khoikhoia oligospilos Sharkey, sp. n., male, holotype A habitus, dorsal view B habitus, lateral view C mesosoma, lateral view. Figure 8. Khoikhoia oligospilos Sharkey, sp. n., male, holotype A habitus, dorsal view B habitus, lateral view C mesosoma, lateral view. 318 Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) Khoikhoia oligospilos Sharkey, sp. n. Holotype male. Body. Length. 6.0 mm. Color. Mottled black and brown except laterotergite 1 whitish, otherwise metasoma mostly brown (Fig. 8). Forewing mostly weakly infuscate but less so anterobasally and at midlength (Fig. 8A). Head. Number of fl agellomeres 31. Scape fl ared apicoanteriorly with expanded apical surface (Fig. 9B). Antennal scrobe deep (Fig. 9E); entirely transversely rugosostriate, somewhat Figure 9. Khoikhoia oligospilos Sharkey, sp. n., male, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E head, dorsal view F data labels. Figure 9. Khoikhoia oligospilos Sharkey, sp. n., male, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E head, dorsal view F data labels. Revision of Khoikhoiinae (Hymenoptera, Braconidae) 319 smoother dorsomedially. Gena with wide irregular punctures, lacking distinct ru- gose striae (Fig. 9B). Face entirely rugose (Fig. 9A). Area between antennal scrobe and inner orbit of eye with protuberance (Fig. 9A). Vertex with weak punctures (Fig. 9E). Posterior orbit of eye with distinct crenulate margin (Fig. 9B). Mesosoma. Middle lobe of scutum entirely smooth with punctures (Fig. 9C). Notauli crenula- tions weak and partly eff aced (Fig. 9C). Mesopleuron at least partly smooth with punctures (Fig. 8C). Sternaulus absent (Fig. 8C). Discrimen (median longitudinal ventral sulcus between mesopleura) crenulate. Metapleuron mostly rugose, lacking a large smooth lightly punctate area, or partly rugose but with a large smooth lightly punctate area. Propodeum entirely rugose without smooth area posterolaterally (Fig. 9D). Metasoma. Median tergite 1 not distinctly narrowed posteriorly (Fig. 9D), partly smooth with punctures especially posteriorly, with weak rugae or microsculp- ture anteriorly (Fig. 9D). Female. Unknown. Diagnosis. Notaular crenulations weak and partly eff aced (Fig. 9C) Diagnosis. Notaular crenulations weak and partly eff aced (Fig. 9C). Material Examined. Holotype female: South Africa, Western Cape, Lang- berg Farm, 3 km 270° W Langebaanweg, MT, LW02-N2-M229, Sand Plain Fyn- bos, 32°58.461’S, 18°07.344’E, 51m, 29.X-5.XI.2003, S. van Noort. SAM-HYM- P0024985 (SAMC). Material Examined. Holotype female: South Africa, Western Cape, Lang- berg Farm, 3 km 270° W Langebaanweg, MT, LW02-N2-M229, Sand Plain Fyn- bos, 32°58.461’S, 18°07.344’E, 51m, 29.X-5.XI.2003, S. van Noort. SAM-HYM- P0024985 (SAMC). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Khoikhoia&species=oligospilos. Etymology. Oligo- is from the Greek, oligos meaning few, and spilos meaning spot. Th e name refers to the reduction of crenulae on the notauli. Diagnosis. Body of mesosoma entirely black except tegula brown (Fig. 10). Material Examined. Holotype male: South Africa: Jonkershoek, near Stellen- bosch, 33°57’33.74”S, 18°55’10.23”E, 227m, 24.XII.1970 , V. Whitehead (AEI). Etymology. “From the Latin meaning half tanned by the sun in reference to the wings, which are brown apically and hyaline basally.” (Mason 1983: p. 55). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab c/generalmapper.php?table=khoikhoiinae&genus=Khoikhoia&species=semiadusta Diagnosis. Body of mesosoma entirely black except tegula brown (Fig. 10). Material Examined. Holotype male: South Africa: Jonkershoek, near Stellen- bosch, 33°57’33.74”S, 18°55’10.23”E, 227m, 24.XII.1970 , V. Whitehead (AEI). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Khoikhoia&species=semiadusta. Etymology. “From the Latin meaning half tanned by the sun in reference to the wings, which are brown apically and hyaline basally.” (Mason 1983: p. 55). bosch, 33°57 33.74 S, 18°55 10.23 E, 227m, 24.XII.1970 , V. Whitehead (AEI). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Khoikhoia&species=semiadusta. Etymology. “From the Latin meaning half tanned by the sun in reference to the wings, which are brown apically and hyaline basally.” (Mason 1983: p. 55). Khoikhoia semiadusta Mason, 1983 Holotype male. Body. Length. 7.0 mm. Color. Mostly black with reddish brown infu- sions on temple, lateral frons, tegula, and parts of legs; laterotergite 1 whitish; lower gena and malar space yellowish white (Fig. 10). Forewing clear basally, infuscate in apical half (Fig. 11E). Head. Number of fl agellomeres 37. Scape fl ared apicoanteriorly with expanded apical surface (Fig. 11B). Antennal scrobe deep (Fig. 11B); entirely transversely rugosostriate. Gena entirely longitudinally rugosostriate, striae weaker posteriorly (Fig. 11B). Face entirely rugose (Fig. 11A). Area between antennal scrobe and inner orbit of eye with protuberance (Fig. 11A). Vertex with strong coarse punc- tures (Fig. 10A). Posterior orbit of eye with distinct crenulate margin (Fig. 11B). Meso- soma. Middle lobe of scutum entirely smooth with punctures (Fig. 11C). Notauli entirely crenulate (Fig. 11C). Mesopleuron at least partly smooth with punctures (Fig. 10C). Sternaulus weakly indicated at midlength with slightly deeper sculpture (Fig. 10C). Discrimen (median longitudinal ventral sulcus between mesopleura) crenulate. Metapleuron mostly rugose, lacking a large smooth lightly punctate area. Propodeum entirely rugose without smooth area posterolaterally (Fig. 11C). Metasoma. Median tergite 1 not distinctly narrowed posteriorly (Fig. 11D), completely rugose (Fig. 11D). Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 320 Figure 10. Khoikhoia semiadusta Mason, 1983, male, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Figure 10. Khoikhoia semiadusta Mason, 1983, male, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Figure 10. Khoikhoia semiadusta Mason, 1983, male, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Female. Unknown. Material Examined. Holotype male: South Africa: Jonkershoek, near Stellen- bosch, 33°57’33.74”S, 18°55’10.23”E, 227m, 24.XII.1970 , V. Whitehead (AEI). Material Examined. Holotype male: South Africa: Jonkershoek, near Stellen- bosch, 33°57’33.74”S, 18°55’10.23”E, 227m, 24.XII.1970 , V. Whitehead (AEI). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Khoikhoia&species=semiadusta. Etymology. “From the Latin meaning half tanned by the sun in reference to the wings, which are brown apically and hyaline basally.” (Mason 1983: p. 55). Etymology. “From the Latin meaning half tanned by the sun in reference to the wings, which are brown apically and hyaline basally.” (Mason 1983: p. 55). Revision of Khoikhoiinae (Hymenoptera, Braconidae) 321 Figure 11. Khoikhoia semiadusta Mason, 1983, male, holotype A head anterior view B head, lateral view. Khoikhoia semiadusta Mason, 1983 C mesosoma, dorsal view D metasomal tergites, dorsal view E wings F data labels. Figure 11. Khoikhoia semiadusta Mason, 1983, male, holotype A head anterior view B head, lateral view. C mesosoma, dorsal view D metasomal tergites, dorsal view E wings F data labels. Khoikhoia solata Mason, 1983 Holotype male. Body. Length. 7.2 mm. Color. Mottled black and brown, except laterotergite 1 whitish, otherwise metasoma mostly brown with some pale yellowish- brown areas on mesosoma and ventral gena (Fig. 12). Forewing entirely infuscate (Fig. 13E). Head. Number of fl agellomeres 32 <32–33>. Scape fl ared apicoanteri- orly with expanded apical surface (Fig. 13A, B). Antennal scrobe deep (Fig. 13B); entirely transversely rugosostriate (Fig. 13B). Gena entirely longitudinally rugosos- Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 322 triate, striae weaker posteriorly (Fig. 13A, B). Face entirely rugose (Fig. 13A). Area between antennal scrobe and inner orbit of eye with protuberance (Fig. 13A). Vertex with weak punctures (Fig. 12A). Posterior orbit of eye with distinct crenulate margin (Fig. 13B). Mesosoma. Middle lobe of scutum entirely smooth with punctures (Fig. 13C). Notauli entirely crenulate (Fig. 13C). Mesopleuron at least partly smooth with punctures (Fig. 12C). Sternaulus absent or diffi cult to discern due to dense sculpture of mesopleuron (Fig. 12C). Discrimen (median longitudinal ventral sulcus between mesopleura) crenulate. Metapleuron mostly rugose, lacking a large smooth lightly Figure 12. Khoikhoia solata Mason, 1983, male, holotype A habitus, dorsal view B habitus, lateral view. C head, mesosoma, lateral view. Figure 12. Khoikhoia solata Mason, 1983, male, holotype A habitus, dorsal view B habitus, lateral view. Figure 12. Khoikhoia solata Mason, 1983, male, holotype A habitus, dorsal view B habitus, lateral view. C head, mesosoma, lateral view. Figure 12. Khoikhoia solata Mason, 1983, male, holotype A habitus, dorsal view B habitus, lateral view. C head, mesosoma, lateral view. B Revision of Khoikhoiinae (Hymenoptera, Braconidae) 323 Revision of Khoikhoiinae (Hymenoptera, Braconidae) 323 punctate area (Fig. 12C). Propodeum entirely rugose without smooth area posterola- terally (Fig. 13C, D). Metasoma. Median tergite 1 not distinctly narrowed posteriorly (Fig. 13D), partly smooth with punctures especially posteriorly, with weak rugae or microsculpture anteriorly (Fig. 13D). Female. Unknown. Diagnosis. Tergite 1 partly smooth with punctures posteriorly (Fig. 13D); notauli entirely crenulate (Fig. 13C). Figure 13. Khoikhoia solata Mason, 1983, male, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Figure 13. Khoikhoia solata Mason, 1983, male, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Khoikhoia solata Mason, 1983 Michael Sharkey Simon van No 4 324 Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) Material Examined. Holotype male: South Africa: Jonkershoek, near Stellen- bosch, 33°57’33.74”S, 18°55’10.23”E, 227m, 18.XII.1970, V. Whitehead (AEI). Distribution Distribution map is available at http://sharkeylab org/sharkeylab/ Material Examined. Holotype male: South Africa: Jonkershoek, near Stellen- bosch, 33°57’33.74”S, 18°55’10.23”E, 227m, 18.XII.1970, V. Whitehead (AEI). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Khoikhoia&species=solata. Etymology. “From Latin meaning sunburned, an allusion to the color and to the fully infuscated wings.” (Mason 1983: p. 54). Figure 14. Khoikhoia townesi Mason, 1983, female, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Figure 14. Khoikhoia townesi Mason, 1983, female, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Revision of Khoikhoiinae (Hymenoptera, Braconidae) 325 Male. Unknown. Diagnosis. Middle lobe of scutum entirely rugosopunctate (Fig. 15C); mesopleuron entirely rugosopunctate (Fig. 14C). Khoikhoia townesi is very similar to K. turneri, and may be a senior synonym. Th e only appreciable diff erences are slight sculptural diff erences on the face and the middle lobe of the mesoscutum, and rather slight color diff erences (see fi gure associated with couplet 5 in the key). fi Material Examined. Holotype female: South Africa, Grahamstown, 33°18’37.50”S, 26°31’30.22”E, 545m, II.1972, Fred Gess (AEI). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Khoikhoia&species=townesi. h Th e sole specimen of this species, together with the single specimen of Sania henryi (Northern Cape Province) are the only representatives of Khoikhoiinae collected out- side of the Western Cape Province. Like its congeners, Khoikhoia townesi is probably still associated with Fynbos, as Grahamstown lies at the extreme eastern limits of this biome. Etymology. Although not specifi ed in Mason (1983), this is a patronym for Henry Townes. Khoikhoia townesi Mason, 1983 Holotype female. Body Length. 6.4 mm. Color. Mottled dark brown and yellowish brown except laterotergite 1 and margins of most metasomal sclerites whitish, head and mesothorax predominantly yellowish brown (Fig. 14). Forewing mostly weakly infuscate but less so anterobasally and at midlength (Fig. 15E), or clear basally, in- fuscate in apical half. Head. Number of fl agellomeres 35 <31–39>. Scape fl ared api- coanteriorly with expanded apical surface (Fig. 15B). Antennal scrobe deep (Fig. 15B); entirely transversely rugosostriate. Gena transversely rugosostriate anteriorly, longitu- Figure 15. Khoikhoia townesi Mason, 1983, female, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Figure 15. Khoikhoia townesi Mason, 1983, female, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 326 dinally rugosostriate posteriorly (Fig. 15A, B). Face entirely rugose (Fig. 15A). Area between antennal scrobe and inner orbit of eye with protuberance (Fig. 15A). Vertex with strong coarse punctures (Fig. 14A). Posterior orbit of eye with distinct crenulate margin (Fig. 15B). Mesosoma. Middle lobe of scutum entirely rugosopunctate (Fig. 15C). Notauli entirely crenulate (Fig. 15C). Mesopleuron entirely rugosopunctate or rugose (Fig. 14C). Sternaulus absent or diffi cult to discern due to dense sculpture of mesopleuron (Fig. 14C). Discrimen (median longitudinal ventral sulcus between mes- opleura) crenulate. Metapleuron mostly rugose, lacking a large smooth lightly punc- tate area (Fig. 14C). Propodeum entirely rugose without smooth area posterolaterally (Fig. 15C, D). Metasoma. Median tergite 1 not distinctly narrowed posteriorly (Fig. 15D); completely rugose (Fig. 15D). Hypopygium less than 1/3 length of metasoma (Fig. 1F, 14B); not extending past apical tergum (Fig. 1F, 14B). Ovipositor barely exserted, much shorter than metasoma (Fig. 1F, 14B). Setae of ovipositor sheath not longer than 2× width of ovipositor sheath (Fig. 1F). Male. Unknown. Khoikhoia turneri Mason, 1984 Holotype female. Body. Length. 6.8 mm. Color. Mottled black and brown except laterotergite 1 whitish, body, particularly metasoma, predominantly dark brown to black (Fig. 16). Forewing entirely infuscate (Fig. 17E). Head. Number of fl agellomer- es 34 <30–38>. Scape fl ared apicoanteriorly with expanded apical surface (Fig. 17B). Antennal scrobe deep (Fig. 17B); entirely transversely rugosostriate. Gena entirely longitudinally rugosostriate, striae weaker posteriorly (Fig. 17A). Face entirely rugose (Fig. 17A). Area between antennal scrobe and inner orbit of eye with protuberance (Fig. 17A). Vertex with strong coarse punctures (Fig. 16A). Posterior orbit of eye Revision of Khoikhoiinae (Hymenoptera, Braconidae) 327 with distinct crenulate margin (Fig. 17B). Mesosoma. Middle lobe of scutum smooth with punctures anteromedially, otherwise rugosopunctate (Fig. 17C). Notauli entire- ly crenulate (Fig. 17C). Mesopleuron entirely rugosopunctate or rugose (Fig. 16C). Sternaulus absent or diffi cult to discern due to dense sculpture of mesopleuron (Fig. 16C). Discrimen (median longitudinal ventral sulcus between mesopleura) crenulate. Metapleuron mostly rugose, lacking a large smooth lightly punctate area (Fig. 16C). Propodeum entirely rugose without smooth area posterolaterally (Fig. 17D). Meta- soma. Median tergite 1 not distinctly narrowed posteriorly (Fig. 17D); completely ) ( d g ud u u p u ) u Metapleuron mostly rugose, lacking a large smooth lightly punctate area (Fig. 16C). Propodeum entirely rugose without smooth area posterolaterally (Fig. 17D). Meta- soma. Median tergite 1 not distinctly narrowed posteriorly (Fig. 17D); completely Figure 16. K hoikhoia turneri Mason, 1984, female, holotype A habitus, dorsal view B habitus, lateral view. C head, mesosoma, lateral view. Figure 16. K hoikhoia turneri Mason, 1984, female, holotype A habitus, dorsal view B habitus, lateral view. C head, mesosoma, lateral view. Figure 16. K hoikhoia turneri Mason, 1984, female, holotype A habitus, dorsal view B habitus, lateral view. C head, mesosoma, lateral view. Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 328 rugose (Fig. 17D). Hypopygium less than 1/3 length of metasoma (Fig. 1E, 16B); not extending past apical tergum (Fig. 1E, 16B). Ovipositor barely exserted, much shorter than metasoma (Fig. 1E, 16B). Setae of ovipositor sheath not longer than 2× width of ovipositor sheath (Fig. 1E). Male. Unknown. Diagnosis. Middle lobe of scutum smooth with punctures anteromedially, oth- erwise rugosopunctate (Fig. 17C); mesopleuron entirely rugosopunctate (Fig. 16C). Diagnosis. Middle lobe of scutum smooth with punctures anteromedially, oth- erwise rugosopunctate (Fig. 17C); mesopleuron entirely rugosopunctate (Fig. 16C). Material Examined. Khoikhoia turneri Mason, 1984 Holotype female: South Africa, Ceres, Cape Province, 33°22’14.08”S, 19°18”22.10”E, 457m, I.1921, R.E.Turner (BMNH). Material Examined. Holotype female: South Africa, Ceres, Cape Province, 33°22’14.08”S, 19°18”22.10”E, 457m, I.1921, R.E.Turner (BMNH). , , , , ( ) Figure 17. Khoikhoia turneri Mason, 1984, female, holotype A head anterior view B head, lateral view. C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Figure 17. Khoikhoia turneri Mason, 1984, female, holotype A head anterior view B head, lateral view. C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Revision of Khoikhoiinae (Hymenoptera, Braconidae) 329 Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Khoikhoia&species=turneri. g pp p p g p Etymology. Although not specifi ed in Mason (1984), this is a patronym for R. Turner, the collector of the holotype. Sania Mason, 1983 Type species. Sania marjoriae Mason, 1983 Type species. Sania marjoriae Mason, 1983 Diagnosis. Clypeus weakly concave, lacking teeth; face smooth to weakly punctate; subalar area of mesopleuron with strong vertical carina, and with posterior fl at smooth to weakly convex glabrous area, not nearly as well developed as that of Khoikhoia. Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Sania. Etymology. “A feminine generic name, dedicated to the San people, a few of whom still follow their ancient hunting life in remote and arid parts of southern Africa.” (Mason 1983: p. 51). Sania browni Sharkey, sp. n. l d b k Sania browni Sharkey, sp. n. urn:lsid:zoobank.org:act:035CFE8B-7302-4940-A085-1A58E9E99762 y p urn:lsid:zoobank.org:act:035CFE8B-7302-4940-A085-1A58E9E99762 Holotype female. Body Length. 3.5 mm <3.4 – 4.4 mm>. Color. Mostly yellowish brown except as follows: head mostly black except ventrally; mesosoma including legs with most sclerites with some black infusions; metasoma mostly brownish black, with lateral areas of terga yellowish white, and pleural area of tergum 1 white; ovipositor sheath white basally, brown apically; <quite variable, from mostly black to mostly yel- lowish brown (Fig. 18); mesosoma always with extensive orangish brown; diff ering from most other species in that all specimens have extensive pale whitish yellow on all lateral terga (Fig. 18A)>. Forewing entirely infuscate (Fig. 19E). Head. Number of fl agellomeres, 23 <23–25>. Scape not fl ared apicoanteriorly and lacking expanded apical surface (Fig. 18C,F). Antennal scrobe fl at (Fig. 18F); entirely smooth with weak punctures. Clypeus mostly smooth with weak punctures (Fig. 18E); without median tooth (Fig. 18E, 19A). Gena mostly smooth with weak punctures (Fig. 18E,F). Face mostly punctate with some weak rugae especially dorsally (Fig. 18E). Area between antennal scrobe and inner orbit of eye without protuberance (Fig. 18E). Vertex with weak punctures (Fig. 18D). Posterior orbit of eye lacking distinct crenulate margin (Fig. 18C). Mesosoma. Middle lobe of scutum entirely smooth with punctures (Fig. 18D). Notauli entirely crenulate (Fig. 18D). Mesopleuron at least partly smooth with punctures (Fig. 18C, 19C). Sternaulus only present at midlength (Figs. 18C, 19C), or absent (Fig. 18C). Discrimen (median longitudinal ventral sulcus between mesop- leura) smooth or with a few barely perceptible crenulae (Fig. 19B). Metapleuron partly Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 330 rugose but with a large smooth lightly punctate area (Figs. 18C, 19C). Propodeum entirely rugose without smooth area posterolaterally (Figs. 18D, 19D). Metasoma. Median tergite 1 distinctly narrowed posteriorly (Figs. 2B,19D); mostly smooth with few rugae near midlength (Figs. 2B, 19D). Hypopygium less than 1/3 length of meta- soma (Figs. 1C, 18A); not extending past apical tergum (Fig. 1C). Ovipositor barely exserted, much shorter than metasoma (Figs. 1C, 18A). Length of setae of ovipositor sheath none longer than 2x width of ovipositor sheath (Fig. 1C). Male. Antenna not sexually dimorphic. Figure 18. Sania browni Sharkey, sp. n., female, paratype A habitus, lateral view B habitus, dorsal view C head, mesosoma, lateral view D head, mesosoma, dorsal view E head, anterior view F head, lateral view. Sania browni Sharkey, sp. n. l d b k Figure 18. Sania browni Sharkey, sp. n., female, paratype A habitus, lateral view B habitus, dorsal view C head, mesosoma, lateral view D head, mesosoma, dorsal view E head, anterior view F head, lateral view. 331 Revision of Khoikhoiinae (Hymenoptera, Braconidae) Diagnosis. Discrimen smooth or with a few barely perceptible crenulae (Fig. 19B); metapleuron partly rugose but with a large smooth lightly punctate area (Fig. 19C). Diagnosis. Discrimen smooth or with a few barely perceptible crenulae (Fig. 19B); metapleuron partly rugose but with a large smooth lightly punctate area (Fig. 19C). Material Examined. Holotype Female: South Africa, Western Cape, 15km N Citrusdal, Koedoeskop Farm, Malaise across hillside trail, 32°29’18”S, 18°57’30”E, 220m, 5–25.X.2004, ME Irwin, FD Parker. SA-34 (SAMC) Material Examined. Holotype Female: South Africa, Western Cape, 15km N Citrusdal, Koedoeskop Farm, Malaise across hillside trail, 32°29’18”S, 18°57’30”E, 220m, 5–25.X.2004, ME Irwin, FD Parker. SA-34 (SAMC) Paratypes. South Africa, Western Cape: 2 females, 3 males, same data as holo- type. 1 female, 9 males, 10 km S. Clanwilliam, Malaise on sandy hill, 32°13’39”S, 18°50’50”E, 140m, 5–25.X.2004, ME Irwin, FD Parker, M Hauser. 2 females, 1 male, Paratypes. South Africa, Western Cape: 2 females, 3 males, same data as holo- type. 1 female, 9 males, 10 km S. Clanwilliam, Malaise on sandy hill, 32°13’39”S, 18°50’50”E, 140m, 5–25.X.2004, ME Irwin, FD Parker, M Hauser. 2 females, 1 male, Figure 19. Sania browni Sharkey, sp. n., female, paratype A head ventral view B discrimen C mesosoma, lateral view D mesosoma & metasoma, dorsal view E wings F data labels. Figure 19. Sania browni Sharkey, sp. n., female, paratype A head ventral view B discrimen C mesosoma, lateral view D mesosoma & metasoma, dorsal view E wings F data labels. Figure 19. Sania browni Sharkey, sp. n., female, paratype A head ventral view B discrimen C mesosoma, lateral view D mesosoma & metasoma, dorsal view E wings F data labels. Michael Sharkey Simon van Noo 2 332 Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 14km NW Robertson@hwy R60, Malaise in dry wash, 33°46.65’S 19°45.69’E, 270 m, 7.X.2002, ME Irwin, FD Parker, RSA-15 (SAMC, AEI, HIC, CNCI, INHS). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Sania&species=browni. g g Note: Th is species is referred to as Sania sp. n. 3 in the molecular study by Murphy et al. (2008). Etymology. Sania browni Sharkey, sp. n. l d b k Named in honor of Brian Brown, a dipterist of outstanding repute and long time collaborator of the senior author. and long time collaborator of the senior author. Figure 20. Sania capensis Mason, 1983, male, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Figure 20. Sania capensis Mason, 1983, male, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Revision of Khoikhoiinae (Hymenoptera, Braconidae) 333 Sania capensis Mason, 1983 Holotype male: South Africa: Jonkershoek, near Stellen- bosch, 33°57’33.74”S, 18°55’10.23”E, 227m, 18.XII.1970, V. Whitehead (AEI) Paratypes. 2 males, South Africa: Jonkershoek, near Stellenbosch, 33°57’33.74”S, 18°55’10.23”E, 227m, 11.I.1971, V. Whitehead (AEI). Paratypes. 2 males, South Africa: Jonkershoek, near Stellenbosch, 33°57’33.74”S, 18°55’10.23”E, 227m, 11.I.1971, V. Whitehead (AEI). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Sania&species=capensis. Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Sania&species=capensis. g g Etymology. Not mentioned in Mason (1983), but obviously a reference to the Cape region of South Africa. Etymology. Not mentioned in Mason (1983), but obviously a reference to the Cape region of South Africa. Sania capensis Mason, 1983 Holotype male. Body Length. 4.0 mm. <4.0–4.1 mm> Color. Mottled black and brown except laterotergite 1 and margins of most metasomal sclerites whitish, left and right sides of the holotype vary considerably with the right side much lighter (Fig. 20). Forewing mostly weakly infuscate, but less so anterobasally and at midlength (Fig. 21E), or clear basally, infuscate in apical half. Head. Number of fl agellomeres 26 (23–29). Scape not fl ared apicoanteriorly and lacking expanded apical surface (Fig. 21B). An- Figure 21. Sania capensis Mason, 1983, male, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Figure 21. Sania capensis Mason, 1983, male, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 334 tennal scrobe fl at (Fig. 21A); smooth with weak punctures medially, weakly and ir- regularly transverse-striate laterally. Clypeus mostly smooth with weak punctures (Fig. 21A); without median tooth (Fig. 21A). Gena mostly smooth with weak punctures (Fig. 21A). Face weakly punctate throughout (Fig. 21A). Area between antennal scrobe and inner orbit of eye without protuberance (Fig. 21A). Vertex with weak punctures (Fig. 20A). Posterior orbit of eye lacking distinct crenulate margin (Figs. 20C, 21B). Mesosoma. Middle lobe of scutum entirely smooth with punctures (Fig. 21C). Notauli entirely crenulate (Fig. 21C). Mesopleuron at least partly smooth with punctures (Fig. 20C). Sternaulus only present at midlength (Fig. 20C). Discrimen (median longitudi- nal ventral sulcus between mesopleura) smooth or with a few barely perceptible crenu- lae. Metapleuron mostly rugose, lacking a large smooth lightly punctate area (Fig. 20C). Propodeum entirely rugose without smooth area posterolaterally (Fig. 21C). Metasoma. Median tergite 1 distinctly narrowed posteriorly (Fig. 21D); partly smooth with punc- tures especially posteriorly, with weak rugae or microsculpture anteriorly (Fig. 21D). Female. Unknown. Female. Unknown. Diagnosis. Discrimen smooth or with a few barely perceptible crenulae; metap- leuron mostly rugose, lacking a large smooth lightly punctate area (Fig. 20C). Diagnosis. Discrimen smooth or with a few barely perceptible crenulae; metap- leuron mostly rugose, lacking a large smooth lightly punctate area (Fig. 20C). Material Examined. Holotype male: South Africa: Jonkershoek, near Stellen- bosch, 33°57’33.74”S, 18°55’10.23”E, 227m, 18.XII.1970, V. Whitehead (AEI) Material Examined. Sania henryi Mason, 1983 Holotype male. Body Length. 4.4 mm. Color. Mottled black and brown except lateroter- gite 1 and margins of most metasomal sclerites whitish, otherwise metasoma mostly brown (Figs. 22A, B). Forewing entirely infuscate (Fig. 22). Head. Number of fl agellomeres 24 <20–28>. Scape not fl ared apicoanteriorly and lacking expanded apical surface (Fig. 23B). Antennal scrobe shallow (Fig. 23B); mostly smooth lacking punctures and microsetae, with very weak irregular sculpture laterally. Clypeus mostly smooth with weak punctures (Fig. 23A); without median tooth (Fig. 23A). Gena with weak rugose microsculpture (Fig. 23A, B). Face mostly punctate with some weak rugae especially dorsally (Fig. 23A). Area between antennal scrobe and inner orbit of eye without protuberance (Fig. 23A). Vertex with weak punctures (Fig. 22A). Posterior orbit of eye with distinct crenulate margin (Figs. 22C, 23B), or lacking distinct crenulate margin. Mesosoma. Middle lobe of scutum en- tirely smooth with punctures (Fig. 23C). Notauli entirely crenulate (Fig. 23C). Mesopleu- ron at least partly smooth with punctures (Fig. 22C). Sternaulus long, occupying most of length of mesopleuron (Fig. 22C). Discrimen (median longitudinal ventral sulcus between Revision of Khoikhoiinae (Hymenoptera, Braconidae) 335 mesopleura) crenulate. Metapleuron mostly rugose, lacking a large smooth lightly punctate area (Fig. 22C), or partly rugose but with a large smooth lightly punctate area (Fig. 22C). Propodeum entirely rugose without smooth area posterolaterally (Fig. 23D). Metasoma. Median tergite 1 distinctly narrowed posteriorly (Fig. 23D); completely rugose (Fig. 23D). Female. Unknown. Diagnosis. Scape not fl ared apicoanteriorly and lacking expanded apical surface (Fig. 23B); discrimen crenulate; mesosoma partly or entirely brown (Fig. 22C). Diagnosis. Scape not fl ared apicoanteriorly and lacking expanded apical surface (Fig. 23B); discrimen crenulate; mesosoma partly or entirely brown (Fig. 22C). Diagnosis. Scape not fl ared apicoanteriorly and lacking expanded apical surface (Fig. 23B); discrimen crenulate; mesosoma partly or entirely brown (Fig. 22C). Material Examined. Holotype male: South Africa, Garies, Cape, 30°33’35.77”S, 17°59’24.91”E, 223m, 23.IX.1970, H. & M. Townes (AEI). Material Examined. Holotype male: South Africa, Garies, Cape, 30°33’35.77”S, 17°59’24.91”E, 223m, 23.IX.1970, H. & M. Townes (AEI). Material Examined. Holotype male: South Africa, Garies, Cape, 30°33’35.77”S, 17°59’24.91”E, 223m, 23.IX.1970, H. & M. Townes (AEI). 17°59’24.91”E, 223m, 23.IX.1970, H. & M. Townes (AEI). Figure 22. Sania henryi Mason, 1983, male, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Figure 22. Sania marjoriae Mason, 1983 Sania marjoriae Mason, 1983 Holotype female. Body Length. 4.0 mm <3.4 – 4.0 mm.>. Color. Mostly dark brown to black; mottled black or dark brown and pale brown except laterotergite 1 and mar- gins of most metasomal sclerites whitish, left and right sides of the holotype vary con- siderably with the right side much lighter (Fig. 24), <the extent of pale brown color varies considerably but dark brown to black always predominates in the examined speci- mens>. Forewing mostly weakly infuscate but less so anterobasally and at midlength (Fig. 25E), or clear basally, infuscate in apical half. Head. Number of fl agellomeres 28 <24 – 28>. Scape fl ared apicoanteriorly with expanded apical surface (Figs. 3A, 25B). Antennal scrobe shallow (Figs. 3A, 25B), or fl at; mostly smooth lacking punctures and microsetae, with very weak irregular sculpture laterally. Clypeus mostly smooth with weak punctures (Fig. 25A); without median tooth (Fig. 25A). Gena with weak rugose microsculpture (Fig. 25A). Face mostly punctate with some weak rugae especially dor- sally (Fig. 25A). Area between antennal scrobe and inner orbit of eye without protu- berance (Fig. 25A). Vertex with weak punctures (Fig. 24A). Posterior orbit of eye with distinct crenulate margin, or lacking distinct crenulate margin (Fig. 25B). Mesosoma. Middle lobe of scutum entirely smooth with punctures (Fig. 25C). Notauli entirely crenulate (Fig. 25C). Mesopleuron at least partly smooth with punctures (Figs. 3E, 24C). Sternaulus long, occupying most of length of mesopleuron (Figs. 3E, 24C). Dis- crimen (median longitudinal ventral sulcus between mesopleura) crenulate. Metapleu- ron mostly rugose, lacking a large smooth lightly punctate area (Fig. 24C). Propodeum mostly rugose with smooth area posterolaterally (Fig. 25C). Metasoma. Median tergite 1 distinctly narrowed posteriorly (Figs. 2C, 25D) completely rugose (Figs. 2C, 25D). Hypopygium subequal to half length of metasoma (Figs. 1A, 24B); extending past api- cal tergum (Figs. 1A, 24B). Ovipositor more than half as long as metasoma (Figs. 1A, 24B). Setae of ovipositor sheath 1 to 2× as long as sheath width (Figs. 1A, 24B). g g Male. Antenna slightly sexually dimorphic with the female antennae shorter and tapering slightly more rapidly than those of male. Diagnosis. Scape fl ared apicoanteriorly with expanded apical surface; median tergite 1 distinctly narrowed posteriorly and completely rugose (Fig. 25D). Material Examined. Holotype female: South Africa: Jonkershoek, near Stellen- bosch, 33°57’33.74”S, 18°55’10.23”E, 227m, 18.XII.1970, V. Whitehead (AEI) Other specimens. Note. Th is species is referred to as Sania sp. n. 4 in the molecular study by Murphy et al. (2008). Sania henryi Mason, 1983 Sania henryi Mason, 1983, male, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 336 Figure 23. Sania henryi Mason, 1983, male, holotype A head anterior view B head, lateral view C meso- soma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Figure 23. Sania henryi Mason, 1983, male, holotype A head anterior view B head, lateral view C meso- soma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Sania&species=henryi. Together with the single specimen of Khoikhoia townesi (Eastern Cape Province) these are the only two representatives of Khoikhoiinae collected outside of the Western Cape Province. Sania henryi is the only Khoikhoiinae so far recorded as being associated with the Succulent Karoo biome, a biodiversity hotspot (Myers et al. 2000). We expect that further undiscovered species will be collected in this habitat. p Etymology. Not mentioned in Mason (1983) but clearly a patronym for Henry Townes. Revision of Khoikhoiinae (Hymenoptera, Braconidae) 337 Sania masneri Sharkey, sp. n. Sania masneri Sharkey, sp. n. urn:lsid:zoobank.org:act:49E6B48F-F40B-479C-9704-8EDFAE402EB9 Sania marjoriae Mason, 1983 5 females, 9 males, South Africa: Western Cape, 10 km S. Clanwilliam, Malaise on sandy hill, 32°13’39”S, 18°50’50”E, 140m, 5–25.X.2004, ME Irwin, FD Parker, M Hauser (SAMC, HIC, INHS). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Sania&species=marjoriae. Etymology. “Dedicated to Marjorie Townes, whose quiet labors over many years have contributed so greatly to the production of the ‘Townes and Townes’ team.” (Mason 1983: p. 51). Note. Th is species is referred to as Sania sp. n. 4 in the molecular study by Murphy et al. (2008). Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 338 y J fi y Figure 24. Sania marjoriae Mason, 1983, female, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Figure 24. Sania marjoriae Mason, 1983, female, holotype A habitus, dorsal view B habitus, lateral view Figure 24. Sania marjoriae Mason, 1983, female, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Sania masneri Sharkey, sp. n. Sania masneri Sharkey, sp. n. urn:lsid:zoobank.org:act:49E6B48F-F40B-479C-9704-8EDFAE402EB9 urn:lsid:zoobank.org:act:49E6B48F-F40B-479C-9704-8EDFAE402EB9 Holotype female. Body Length. 5.2 mm <male 4.2. mm>. Color. Mottled black and brown except laterotergite 1 and margins of most metasomal sclerites whitish, otherwise metasoma mostly brown (Fig. 26). Forewing clear basally, infuscate in apical half (Fig. 27E). Head. Number of fl agellomeres 28 <male also 28>. Scape fl ared apicoanteriorly with expanded apical surface (Fig. 27B). Antennal scrobe shallow (Fig. 27B); smooth medially, transversely striate laterally. Clypeus mostly smooth with weak punctures (Fig. Revision of Khoikhoiinae (Hymenoptera, Braconidae) 339 Figure 25. Sania marjoriae Mason, 1983, female, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Figure 25. Sania marjoriae Mason, 1983, female, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. C 27A); without median tooth (Fig. 27A). Gena mostly smooth with weak punctures (Fig. 27A). Face mostly punctate with some weak rugae especially dorsally (Fig. 27A). Area between antennal scrobe and inner orbit of eye with protuberance, or without pro- tuberance. Vertex with weak punctures (Fig. 26A). Posterior orbit of eye with distinct crenulate margin (Fig. 27B), or lacking distinct crenulate margin. Mesosoma. Middle lobe of scutum entirely smooth with punctures (Fig. 27C). Notauli entirely crenulate (Fig. 27C). Mesopleuron at least partly smooth with punctures (Fig. 26C). Sternaulus long occupying most of length of mesopleuron, or only present at midlength, or absent or diffi cult to discern due to dense sculpture of mesopleuron (Fig. 26C). Discrimen Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 340 (median longitudinal ventral sulcus between mesopleura) crenulate. Metapleuron most- ly rugose, lacking a large smooth lightly punctate area (Fig. 26C). Propodeum entirely rugose without smooth area posterolaterally (Fig. 27C). Metasoma. Median tergite 1 distinctly narrowed posteriorly (Fig. 27C); partly smooth with punctures especially pos- teriorly, with weak rugae or microsculpture anteriorly (Fig. 27C). Hypopygium sub- equal to half length of metasoma (Figs. 1D, 26B); extending past apical tergum (Figs. 1D, 26B). Ovipositor more than half as long as metasoma (Figs. 1D, 26B). Setae of ovipositor sheath as much as 3 or 4× as long as width of sheath (Figs. 1D, 26B). p g g Figure 26. Sania masneri Sharkey, sp. Sania masneri Sharkey, sp. n. urn:lsid:zoobank.org:act:49E6B48F-F40B-479C-9704-8EDFAE402EB9 n., female, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Figure 26. Sania masneri Sharkey, sp. n., female, holotype A habitus, dorsal view B habitus, lateral view Figure 26. Sania masneri Sharkey, sp. n., female, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Revision of Khoikhoiinae (Hymenoptera, Braconidae) 341 Male. Antenna highly sexually dimorphic with the female antennae shorter and tapering much more rapidly than those of male. Genitalia large (Fig. 2D). Diagnosis. Scape fl ared apicoanteriorly with expanded apical surface (Fig. 27B); median tergite 1 distinctly narrowed posteriorly and smooth with punctures postero- laterally (Fig. 27D). Material Examined. Holotype female: South Africa, Western Cape, 10 km S. Clanwilliam, Malaise on sandy hill, 32°13’39”S, 18°5:0’50”E, 140m, 5–25.X.2004, ME Irwin, FD Parker, M Hauser (SAMC) Material Examined. Holotype female: South Africa, Western Cape, 10 km S. Clanwilliam, Malaise on sandy hill, 32°13’39”S, 18°5:0’50”E, 140m, 5–25.X.2004, ME Irwin, FD Parker, M Hauser (SAMC) Paratypes. 2 males, same data as holotype (SAMC, HIC). Figure 27. Sania masneri Sharkey, sp. n., female, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Figure 27. Sania masneri Sharkey, sp. n., female, holotype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. 342 Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Sania&species=masneri. Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Sania&species=masneri. g pp p p g p Etymology. Dedicated to Lubomír Masner for his dedication to systematics and for the inspiration that he has given to so many of us. Note. Th is species is referred to as Sania sp. n. 1 in the molecular study by Murphy et al. (2008). Figure 28. Sania masoni Sharkey, sp. n., female, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Figure 28. Sania masoni Sharkey, sp. n., female, holotype A habitus, dorsal view B habitus, lateral view C head, mesosoma, lateral view. Revision of Khoikhoiinae (Hymenoptera, Braconidae) 343 Sania masoni Sharkey, sp. n. y p urn:lsid:zoobank.org:act:DDC57A2D-20B9-4003-9B29-468B1100B7A4 urn:lsid:zoobank.org:act:DDC57A2D-20B9-4003-9B29-468B1100B7A4 Holotype female. Body Length. 4.8 mm <4.7–5.2>. Color. All black except: mandible and parts of all segments of fore- and midlegs reddish or yellowish brown, hind tibia red- dish brown basally, laterotergite and sternum of metasomal tergum 1 whitish (Fig. 28) <pale color, reddish or yellowish brown, varies from as in the holotype to completely ab- sent, although laterotergite and sternum of metasomal tergum 1 always whitish>. Forew- ing mostly weakly infuscate but less so anterobasally and at midlength, or clear basally, Figure 29. Sania masoni Sharkey, sp. n., female, paratype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Figure 29. Sania masoni Sharkey, sp. n., female, paratype A head anterior view B head, lateral view C mesosoma dorsal view D propodeum metasomal tergites dorsal view E wings F data labels Figure 29. Sania masoni Sharkey, sp. n., female, paratype A head anterior view B head, lateral view C mesosoma, dorsal view D propodeum, metasomal tergites, dorsal view E wings F data labels. Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) 344 infuscate in apical half (Fig. 29E). Head. Number of fl agellomeres 24 <22–24>. Scape not fl ared apicoanteriorly and lacking expanded apical surface (Figs. 3B, 29A, B). Anten- nal scrobe fl at (Figs. 3B, 29B); variable, from mostly transverse-striate, to mostly smooth with punctures. Clypeus mostly smooth with weak punctures (Fig. 29A), without median tooth (Fig. 29A). Gena mostly smooth with weak punctures (Fig. 29A). Face mostly punc- tate with some weak rugae especially dorsally (Fig. 29A). Area between antennal scrobe and inner orbit of eye with protuberance (Fig. 29A), or without protuberance. Vertex with weak punctures (Fig. 28A). Posterior orbit of eye lacking distinct crenulate margin (Fig. 29B). Mesosoma. Middle lobe of scutum entirely smooth with punctures (Fig. 29C). Notauli entirely crenulate (Fig. 29C). Mesopleuron at least partly smooth with punctures (Figs. 3F, 28C). Sternaulus only present at midlength, or absent (Figs. 3F, 28C). Discri- men (median longitudinal ventral sulcus between mesopleura) crenulate. Metapleuron mostly rugose, lacking a large smooth lightly punctate area (Fig. 28C). Propodeum en- tirely rugose without smooth area posterolaterally (Fig. 29C). Metasoma. Median tergite 1 not distinctly narrowed posteriorly (Fig. 29D); partly smooth with punctures especially posteriorly, with weak rugae or microsculpture anteriorly (Fig. 29D). y p urn:lsid:zoobank.org:act:DDC57A2D-20B9-4003-9B29-468B1100B7A4 Hypopygium less than 1/3 length of metasoma (Figs. 1B, 28B); not extending past apical tergum (Figs. 1B, 28B). Ovipositor barely exserted, much shorter than metasoma (Figs. 1B, 28B). Setae of ovipositor sheath not longer than 2× width of ovipositor sheath (Figs. 1B, 28B). Male. Antenna slightly sexually dimorphic with the female antennae shorter and tapering slightly more rapidly than those of male. Metasoma (Fig. 2E).l Diagnosis. Scape not fl ared apicoanteriorly and lacking expanded apical surface (Figs. 3B, 29A, B); discrimen crenulate; mesosoma black (Fig. 28A, C). Material Examined. Holotype female: South Africa, Western Cape, 10 km S. Clanwilliam, Malaise on sandy hill, 32°13’39”S, 18°50’50”E, 140m, 5–25.X.2004, ME Irwin, FD Parker, M Hauser (SAMC) Paratypes. 3 females, 18 males, South Africa, Western Cape, 15km N Citrus- dal, Koedoeskop Farm, Malaise across hillside trail, 32°29’18”S, 18°57’30”E, 220m, 5–25.X.2004, ME Irwin, FD Parker, SA-34 (SAMC, AEI, HIC, CNCI, INHS). Paratypes. 3 females, 18 males, South Africa, Western Cape, 15km N Citrus- dal, Koedoeskop Farm, Malaise across hillside trail, 32°29’18”S, 18°57’30”E, 220m, 5–25.X.2004, ME Irwin, FD Parker, SA-34 (SAMC, AEI, HIC, CNCI, INHS). Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Sania&species=masoni. Distribution. Distribution map is available at http://sharkeylab.org/sharkeylab/ Misc/generalmapper.php?table=khoikhoiinae&genus=Sania&species=masoni. Etymology. Dedicated to the late W.R.M. (Bill) Mason for his many contribu- tions to Hymenopterology including the discovery of the subfamily Khoikhoiinae. Etymology. Dedicated to the late W.R.M. (Bill) Mason for his many contribu- tions to Hymenopterology including the discovery of the subfamily Khoikhoiinae. Acknowledgements Th is article is dedicated to Lubomír Masner. Each of the three authors has been touched by his enthusiasm and each of us has benefi ted from his extensive and profound knowl- edge of Hymenoptera. We all wish him many more productive and happy years. We hope that including his name alongside those of his mentors and friends, Henry and Marjorie Townes, and Bill Mason in the subfamily Khoikhoiinae will please him. Sup- port for MS was provided by NSF EF-0337220 and by a South African National Re- search Foundation grant GUN 61497 (SvN) and an Italian/South African Scientifi c Revision of Khoikhoiinae (Hymenoptera, Braconidae) 345 and Technological Co-operation grant GUN 2068865 awarded to SvN and Prof. M. Olmi (University of Tuscia).Th anks to David Wahl, Gavin Broad and Andy Bennett for providing specimens, to David Wagner for insights into possible lepidopteran hosts, and to Scott Robinson and Alex Wild for help and direction with the ESEM images. References 346 Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) Latimer AM, Silander JA, Cowling RM (2005) Neutral ecological theory reveals isolation and rapid speciation in a biodiversity hot spot. Science 309: 1722–1725. p p y p Linder HP (2003) The radiation of the Cape flora, southern Africa. Biological Reviews 78: 597–638. Linder HP, Meadows, ME, Cowling RM (1992) History of the Cape fl ora. In: Cowling RM (Ed) Th e Ecology of Fynbos, Nutrients, Fire and Diversity. Oxford University Press, Ox- ford, UK, 113–134. Mason WRM (1983) A new South African subfamily related to Cardiochilinae (Hymenop- tera: Braconidae). Contributions to the American Entomological Institute 20: 49–62. Mason WRM (1984) Two new species of Khoikhoiinae (Hym.: Braconidae). Oriental Insects 18: 285–288. Murphy N, Banks JC, Whitfi eld JB, Austin AD (2008) Phylogeny of the parasitic microgas- troid subfamilies (Hymenoptera: Braconidae) based on sequence data from seven genes, with an improved time estimate of the origin of the lineage. Molecular Phylogenetics and Evolution 47: 378–395. Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403: 853–858. Picker MD, Colville JF, van Noort S (2002) Mantophasmatodea now in South Africa. Science 297: 1475. Polaszek A, Agosti D, Alonso-Zarazaga M, Beccaloni G, de Place Bjørn P, Bouchet P, Brothers DJ, Earl of Cranbrook, Evenhuis NL, Godfray HCJ, Johnson NF, Krell FT, Lipscomb D, Lyal CHC, Mace GM, Mawatari SF, Miller SE, Minelli A, Morris S, Ng PKL, Patterson DJ, Pyle RL, Robinson N, Rogo L, Taverne J, Th ompson FC, van Tol J, Wheeler QD, Wilson EO (2005) A universal register for animal names. Nature 437: 477. Pucci T, Sharkey M (2004) A revision of Agathirsia Westwood (Hymenoptera: Braconidae: Agathid- inae) with notes on mouthpart morphology. Journal of Hymenoptera Research 13(1): 64–107. Quicke DLJ (1997) Braconinae. In: Wharton RA, Marsh PM, Sharkey MJ (Eds) Manual of the New World genera of the family Braconidae (Hymenoptera). International Society of Hymenopterists Special Publication No. 1, 149–176. Ruberson JR, Whitfi eld JB (1996) Facultative egg-larval parasitism of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) by Cotesia marginiventris (Hymenoptera: Bra- conidae). Florida Entomologist 79: Rutherford MC, Mucina L, Powrie LW (2006) Biomes and bioregions of Southern Africa. In: Mucina L, Rutherford MC (Eds) Th e vegetation of South Africa, Lesotho and Swaziland. SANBI, Pretoria, 30–51. References Achterberg C van (2002) Apanteles (Choeras) gielisi sp. n. (Hymenoptera: Braconidae: Micro- gastrinae) from the Netherlands and the fi rst report of Trichoptera as host of Braconidae. Zoologische Mededelingen 76: 53–60. Bezier A, Annheim M, Herbiniere J, Wetterwald C, Gyapay G, Bernard-Samain S, Wincker P, Roditi I, Heller M, Belghazi M, Pfi ster-Wilhelm R, Periquet G, Dupuy C, Huguet E, Volkoff A-N, Lanzrein B, Drezen J-M (2009). Polydnaviruses of braconid wasps derive from an ancestral nudivirus. Science 323: 926–930. Buffi ngton ML, Burks R, McNeil L (2005) Advanced techniques for imaging microhymenop- tera. American Entomologist 51: 50–54 Buffi ngton ML, Gates M (2009) Advanced imaging techniques II: using a compound micro- scope for photographing point-mount specimens. American Entomologist 54: 222–224. Buffi ngton ML, van Noort S (2007) A world revision of the Pycnostigminae (Cynipoidea: Figitidae) with descriptions of seven new species. Zootaxa 1392: 1–30. Coetzee JH, Giliomee JH (1987) Borers and other inhabitants of the infructescence of Protea repens (L). (Proteaceae) in the Western Cape. Phytophylactica 19:1–6. Cowling RM, Rundel PW, Desmet PG, Esler KJ (1998) Extraordinarily high regional-scale plant diversity in southern African arid lands: subcontinental and global comparisons. Di- versity and Distributions 4: 27–36. Dallwitz MJ (1974) A fl exible computer program for generating identifi cation keys. Systematic Zoology 23: 50–7. Dallwitz MJ (1980) A general system for coding taxonomic descriptions. Taxon 29: 41–6. Dallwitz MJ, Paine TA, Zurcher EJ (1993 onwards) User’s guide to the DELTA System: a general system for processing taxonomic descriptions. 4th edition. http://delta-intkey.com. Dallwitz MJ, Paine TA, Zurcher EJ (1995 onwards) User’s guide to Intkey: a program for inter- active identifi cation and information retrieval. http://delta-intkey.com. i Dallwitz MJ, Paine TA, Zurcher EJ (1999 onwards) User’s guide to the DELTA Editor. http:// delta-intkey.com. Damgaard J, Klass K-D, Picker MD, Buder G (2008) Phylogeny of the Heelwalkers (Insecta: Mantophasmatodea) based on mtDNA sequences, with evidence for additional taxa in South Africa. Molecular Phylogenetics and Evolution 47: 443–462. Davis DR (1996). A revision of the southern African family Prototheoridae (Lepidoptera: Hep- ialoidea). Entomologica Scandinavica 27: 393–439. Goldblatt P (1997) Floristic diversity in the Cape Flora of South Africa. Biodiversity and Con- servation 6: 359–377. Kerr P, Fisher E, Buffi ngton ML (2009) Dome lighting for insect imaging under a microscope. American Entomologist, 54: 198–200. Appendix 1. DELTA data matrix, images, and other fi les to the key of the Khoikhoiinae (Hymenop- tera, Braconidae). doi:10.3897/zookeys.20.108.app.1.ik. Copyright notice: Th is dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). Th e Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Citation: Sharkey M, van Noort S, Whitfi eld J (2009) DELTA data matrix and images to the key of the of Khoikhoiinae (Hymenoptera, Braconidae). doi:10.3897/zookeys.20.108.app.1.ik. Dataset published in: Zookeys 20: 299–348. doi:10.3897/zookeys.20.108. Citation: Sharkey M, van Noort S, Whitfi eld J (2009) DELTA data matrix and images to the key of the of Khoikhoiinae (Hymenoptera, Braconidae). doi:10.3897/zookeys.20.108.app.1.ik. Dataset published in: Zookeys 20: 299–348. doi:10.3897/zookeys.20.108. References Whitfi eld JB, Mason WRM (1994) Mendesellinae, a new subfamily of braconid wasps (Hy- menoptera, Braconidae) with a review of relationships within the microgastroid assem- blage. Systematic Entomology 19: 61–76. Wright M.G, Samways MJ (1999) Plant characteristics determine insect borer assemblages on Protea species in the Cape fynbos, and importance for conservation management. Biodiver- sity and Conservation 8:1089–1100. Wright MG, Samways MJ (2000) Biogeography and species richness of endophagous insects associated with Proteaceae in South Africa. African Journal of Ecology 38: 16–22. Revision of Khoikhoiinae (Hymenoptera, Braconidae) 347 Citation: Sharkey M, van Noort S, Whitfi eld J (2009) Interactive key, in IntKey format, to the species of Khoikhoiinae (Hymenoptera, Braconidae). doi:10.3897/zookeys.20.108.app.2.ik. Dataset published in: Zookeys 20: 299–348. doi:10.3897/zookeys.20.108. Citation: Sharkey M, van Noort S, Whitfi eld J (2009) DELTA data matrix and images to the key of the of Khoikhoiinae (Hymenoptera, Braconidae). doi:10.3897/zookeys.20.108.app.1.ik. Dataset published in: Zookeys 20: 299–348. doi:10.3897/zookeys.20.108. Appendix 2. Interactive key, in IntKey format, to the species of Khoikhoiinae (Hymenoptera, Bra- conidae). doi:10.3897/zookeys.20.108.app.2.ik. Note: To run the identifi cation key, you will need Windows 95/NT or a later ver- sion. You also need to download Intkey software and reboot your computer, if it is not already installed. Th e software package, Intkey, can be downloaded from http:// delta-intkey.com/www/programs.htm. Once Intkey is installed you need only click on the .ink fi le (below) and the key will open. Click on any character on the left to begin. More details on how to use Intkey effi ciently are found at http://fl orabase.calm.wa.gov. au/help/keys/intkey_tutorial.pdf. Copyright notice: Th is dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). Th e Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. 348 Michael Sharkey, Simon van Noort & James Whitfi eld / ZooKeys 20: 299–348 (2009) Appendix 3. Appendix 3. Lucid Interchange Format version 3 (LIF3) fi le. Th is is an XML-based fi le that sto res all the Lucid3 key data, allowing exchange of the key with other key developers doi:10.3897/zookeys.20.108.app.3.ik.h Lucid Interchange Format version 3 (LIF3) fi le. Th is is an XML-based fi le that sto- res all the Lucid3 key data, allowing exchange of the key with other key developers. doi:10.3897/zookeys.20.108.app.3.ik. Copyright notice: Th is dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). Th e Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Citation: Sharkey M, van Noort S, Whitfi eld J (2009) Lucid Interchange Format version 3 (LIF3) fi le to the species of Khoikhoiinae (Hymenoptera, Braconidae). doi:10.3897/zookeys.20.108.app.3.ik. Dataset published in: Zookeys 20: 299–348. doi:10.3897/zookeys.20.108 Citation: Sharkey M, van Noort S, Whitfi eld J (2009) Lucid Interchange Format version 3 (LIF3) fi le to the species of Khoikhoiinae (Hymenoptera, Braconidae). doi:10.3897/zookeys.20.108.app.3.ik. Dataset published in: Zookeys 20: 299–348. doi:10.3897/zookeys.20.108 Citation: Sharkey M, van Noort S, Whitfi eld J (2009) Lucid SDD fi le to the species of Khoikhoiinae (Hymenopte- ra, Braconidae). doi:10.3897/zookeys.20.108.app.4.ik. Dataset published in: Zookeys 20: 299–348. doi:10.3897/zoo- keys.20.108 Citation: Sharkey M, van Noort S, Whitfi eld J (2009) Lucid Interchange Format version 3 (LIF3) fi le to the species of Khoikhoiinae (Hymenoptera, Braconidae). doi:10.3897/zookeys.20.108.app.3.ik. Dataset published in: Zookeys 20: 299–348. doi:10.3897/zookeys.20.108 Appendix 4. Lucid SDD fi le. Th is is a XML-based fi le structured using the internationally agreed SDD (Structure of Descriptive Data) Schema. Th is SDD fi le may be used to exchange the Lu- cid key with other SDD-compliant applications. doi:10.3897/zookeys.20.108.app.4.ik. Copyright notice: Th is dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). Th e Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Citation: Sharkey M, van Noort S, Whitfi eld J (2009) Lucid SDD fi le to the species of Khoikhoiinae (Hymenopte- ra, Braconidae). doi:10.3897/zookeys.20.108.app.4.ik. Dataset published in: Zookeys 20: 299–348. doi:10.3897/zoo- keys.20.108
https://openalex.org/W2799796067	https://link.springer.com/content/pdf/10.1007/s13201-018-0711-0.pdf	English	null	Groundwater quality assessment of the quaternary unconsolidated sedimentary basin near the Pi river using fuzzy evaluation technique	Applied water science	2,018	cc-by	10,604	Abstract The present study was carried out to assess the groundwater quality for drinking purposes in the Quaternary Unconsoli- dated Sedimentary Basin of the North Chengdu Plain, China. Six groups of water samples (S1, S2, S3, S4, S5, and S6) are selected in the study area. These samples were analyzed for 19 different physicochemical water quality parameters to assess groundwater quality. The physicochemical parameters of groundwater were compared with China’s Quality Standards for Groundwater (GB/T14848-93). Interpretation of physicochemical data revealed that groundwater in the basin was slightly alkaline. Total hardness and total dissolved solid values show that the investigated water is classified as very hard and fresh water, respectively. The sustainability of groundwater for drinking purposes was assessed based on the fuzzy mathematics evaluation (FME) method. The results of the assessment were classified into five groups based on their relative suitability for portable use (grade I = most suitable to grade V = least suitable), according to (GB/T 14848-93). The assessment results reveal that the quality of groundwater in most of the wells was class I, II and III and suitable for drinking purposes, but well (S2) has been found to be in class V, which is classified as very poor and cannot be used for drinking. Also, the FME method was compared with the comprehensive evaluation method. The FME method was found to be more comprehensive and reasonable to assess groundwater quality. This study can provide an important frame of reference for decision making on improving groundwater quality in the study area and nearby surrounding. Keywords Groundwater quality · Groundwater pollution · Fuzzy mathematics · Physicochemical parameters Groundwater quality · Groundwater pollution · Fuzzy mathematics · Physicochemical parameters Groundwater quality assessment of the quaternary unconsolidated sedimentary basin near the Pi river using fuzzy evaluation technique Adam Khalifa Mohamed1,2 · Dan Liu1 · Mohamed A. A. Mohamed2 · Kai Song1 Received: 18 July 2016 / Accepted: 11 April 2018 / Published online: 20 April 2018 © The Author(s) 2018 1 Faculty of Geoscience and Environmental Engineering, Southwest Jiaotong University, Chengdu, China 2 Faculty of Water and Environmental Engineering, Sudan University of Science and Technology, Khartoum, Sudan Applied Water Science (2018) 8:65 https://doi.org/10.1007/s13201-018-0711-0 Applied Water Science (2018) 8:65 https://doi.org/10.1007/s13201-018-0711-0 ORIGINAL ARTICLE ORIGINAL ARTICLE Description of study area The study area is located between longitudes 103°54′02″ to 104°16′54″ and latitudes 30°40′40″ to 30°57′58″ in the North Chengdu Plain and bounded by many rivers, which are tributaries of the Minjiang river and is divided into many villages with the total population of over 140,000. The pri- mary source of drinking water and agricultural irrigation in the study area is groundwater from the Unconsolidated Quaternary sediments, which provides water to residents. It has a sub-tropical humid monsoon climate with four seasons. The study area is located between longitudes 103°54′02″ to 104°16′54″ and latitudes 30°40′40″ to 30°57′58″ in the North Chengdu Plain and bounded by many rivers, which are tributaries of the Minjiang river and is divided into many villages with the total population of over 140,000. The pri- mary source of drinking water and agricultural irrigation in the study area is groundwater from the Unconsolidated Quaternary sediments, which provides water to residents. It has a sub-tropical humid monsoon climate with four seasons. Compared to other areas in the same climatic zone, features such as low temperature, less sunshine, and rainy weather are more frequent. The mean annual temperature is 10.4 °C, the average temperature of coldest months and the hottest month is 4.6 and 24.4 °C, respectively. The region is dominated by NW wind, with a maximum wind speed of 17 m/s and aver- age wind speed of 1.3 m/s. No typhoons are observed. The annual maximum relative humidity is 80%, and the mini- mum relative humidity is 75%. The annual maximum of absolute moisture content is 15.2, and the minimum is 14.3. Rainfall is the main recharge source of groundwater. The average annual precipitation is 1134.8 mm. Moreover, the longest continuous period of rainfall is 28 days. The Long- qiao water plant has been established on the right bank of the Pi river at a distance of 38 m. The daily production capacity of the Longqiao water plant is about 10,000–12,000 m3/d, and the daily water supply is 8000–10,000 m3/d. However, the groundwater is exposed to the risk of contamination from different sources. The main pollution sources of groundwater in the study area comes from the domestic sewage water, and industrial wastewater discharged routinely into the Pi river, which is estimated at 8.4 × 103 m3/d and agricultural pollution from using chemical fertilizers and pesticide. Materials and methods At present, many methods are available at home and abroad, and proven to be powerful in water quality assess- ment, such as the principal component analysis (Gangopad- hyay et al. 2001), neural network model (Wu et al. 2007), Bayesian discrimination method (Chen et al. 2009), entropy method (Chun-rong and Jun 2011), water pollution index method (Liu et al. 2013), grey clustering method (Zhang 2014), statistical analysis method (Liu et al. 2015) and oth- ers. However, these methods cannot directly reflect pol- lution characteristics, and the linear relationship between those variables may have some effects on the results (Li et al. 2018). In the same vein, the methods above have their own merits, but they are less feasible and challenging to popularize in the regional groundwater pollution assessment due to the complex and changeable environmental problems. Furthermore, in all environmental quality assessments, there is uncertainty about environmental risk, because of incon- sistency and peculiarities of each groundwater pollutant. Introduction may threaten human health and plant growth (Zhu et al. 2014, Hosseini-Moghari et al. 2015). Most water contami- nation originates from the disposal of solid wastes from dif- ferent human activities, such as agriculture, construction and industry, and the disposal of domestic and industrial waste- water is discharged into rivers through the sewer systems (Li et al. 2015, Zhang et al. 2015, Kumar et al. 2016). These systems may often leak wastewater into shallow aquifers, directly or indirectly and groundwater supplies from nearby wells are affected if they are exposed to these wastewater pollutants (Li et al. 2016, Qishlaqi et al. 2017, Pinto 2015). Groundwater is very important in day to day life for people and society (Shigut et al. 2017). It has not only been used for supplying potable water to both urban and rural areas but also essential for irrigation, economic development, and protection of environmental and ecological balance (Cheng and Fanhai 2012, Srinivas et al. 2015, Kumar et al. 2015, Al- Ahmadi 2013). Recently, providing good quality water for drinking is considered a fundamental requirement for public health protection. Consequently, the poor quality of water When groundwater is polluted, its quality cannot be restored by stopping the contaminants from the sources. Shallow, unconsolidated aquifers are particularly vulner- able to contamination, which may persist in groundwater for many years or decades (Li et al. 2016, Liu et al. 2010). Meanwhile, it becomes necessary to monitor the groundwa- ter quality regularly and devise ways to protect it (Kaur et al. 2014, Shigut et al. 2017). In the study area, the shallow aqui- fer in the Quaternary Unconsolidated Sedimentary Basin, * Adam Khalifa Mohamed adamkh124@yahoo.com Dan Liu liudan‑swju@163.com 1 Faculty of Geoscience and Environmental Engineering, Southwest Jiaotong University, Chengdu, China 2 Faculty of Water and Environmental Engineering, Sudan University of Science and Technology, Khartoum, Sudan * Adam Khalifa Mohamed adamkh124@yahoo.com Dan Liu liudan‑swju@163.com 2 Faculty of Water and Environmental Engineering, Sudan University of Science and Technology, Khartoum, Sudan Vol.:(0123456789) 1 3 65 Page 2 of 12 65 Page 2 of 12 Applied Water Science (2018) 8:65 65 the technique characteristics of the evaluation methods used. Therefore, this study will be an essential reference for future studies. It will also be useful for the local decision makers in regional groundwater management and protection. Introduction the largest source of drinking water in the North Chengdu plain, China, is, however, constantly impacted by agricul- ture, industry, mining, and other human activities. This has challenged the water resource managers and forced them to pay attention to the evaluation of groundwater contamina- tion in the study area based on the recognized assessment methods. 1 3 Sample collection and analysis analyzed. pH and total dissolved solids (TDS) were meas- ured in situ using a portable pH and TDS meters because the parameters are likely to change during transport. Water sampling methods were according to (Kent and Payne 1988). The samples were analyzed for 19 various physicochemical parameters, include hydrogen ion concentration (pH), total hardness (TH), total dissolved solids (TDS), potassium (K+), sodium (Na2+), calcium (Ca2+), magnesium (Mg2+) sulfates ( SO2− 4 ), chlorides (Cl−), bicarbonates ( HCO− 3 ), potassium permanganate index (CODMn), nitrate ( NO− 3 ), nitrite ( NO− 2 ), ammonia ( NH+ 4 ), iron (Fe), manganese (Mn), arsenic (As), chromium (Cr6+), and lead (Pb). These parameters were used as index indicators to evaluate the groundwater contamina- tion risk in the study area. This selection was based on their importance to the water quality and the potential impact on human health and their pollution potential on groundwater resource in particular (Zhang et al. 2017). Detection methods for each parameter were as follows: the concentrations of K+ and Na+ were measured using flame photometer. TH, Ca2+, Mg2+, Cl−, and HCO− 3 were analyzed by titrimetric methods. The concentrations of CODMn, NH+ 4 , NO− 3 , NO− 2 , and SO2− 4 were determined using spectrophotometer technique. And finally, the Fe, Mn, As, Cr6+, and Pb concentrations were then measured using atomic absorption spectrometry. In this study, groundwater samples were obtained from six monitoring wells (S1, S2, S3, S4, S5, and S6) from the shal- low aquifer in the Quaternary Unconsolidated Sedimentary Basin near the Pi river, China. These six monitoring wells are located between the Pi river and Longqiao drinking water supply plant. The locations of groundwater samples are displayed in (Fig. 1). The sampling wells (S1, S2, and S3) are inside the wall of Longqiao water plant, while the sampling wells (S4, S5, and S6) outside the Longqiao water plant wall. Well (S2) is near the septic tank of the plant workers, and the wells (S1 and S3) are near the manage- ment offices and workers residences, respectively. Whereas, the well (S4) is near the Pi river bridge and the sampling wells (S5 and S6) are in the middle of farms existing in the area. These wells are used as a group in the Longqiao water plant to provide supply drinking water to residents in the Longqiao town and its surrounding area. Description of study area It is believed that most of the wastewater is infiltrated into the shallow aquifer in the Quaternary Unconsolidated Sedimen- tary Basin in the area, because of its shallow depth of the groundwater level (2.0–10 m) and relatively high hydraulic conductivity (k = 10–50 m/day). So, it is necessary to evalu- ate the quality of drinking water in this region since it is closely linked to people’s health. To overcome the shortcomings associated with the above methods and respond to the call of water resource manag- ers, fuzzy mathematics evaluation (FME) method was used. FME method has a large range of applications which could help in identifying and overcoming any uncertainty regard- ing the risk of groundwater contamination using member- ship functions (Mujumdar and Sasikumar 2002, Ma et al. 2010, Zhang et al. 2012, Kamrani et al. 2016). It has also been proven effective to deal with complex and changeable environmental problems (Singh et al. 2017), and also con- trolling the effect of monitoring errors on assessment results (Ghasemi et al. 2014).i This study is considered as the first of its kind to assess groundwater quality in this region and nearby surrounding. To show the advantage of FME, it was compared with Com- prehensive Evaluation Method (CEM), which is the most common method of assessing the quality of groundwater for drinking in China, and recommended by the quality standard for groundwater of China (GB/T 14848-93) (Wu and Sun 2016, Su et al. 2017). This method provides a holistic view of groundwater quality status and appropriateness for drink- ing purposes by considering various water quality param- eters based on simple mathematical-numerical tools. There- fore, the aims of this study are: (1) evaluating groundwater quality condition and its suitability for drinking purposes in the Quaternary Unconsolidated Sedimentary Basin, (2) iden- tifying the main pollutants which influence the groundwater quality and (3) compare the evaluation results to learn about 1 3 Applied Water Science (2018) 8:65 Page 3 of 12 65 Fig. 1 Location of study area and groundwater samples Determining the evaluation factors Depending on the circumstances of environment, the evalu- ation factors set U = (u1, u2,…,un) = (pH, TH, TDS, SO2− 4 , 1 3 Fig. 2 Flow chart of the fuzzy mathematics method 1 3 Fig. Sample collection and analysis It was observed that the location of wells is exposed to the risk of ground- water pollution resulting from the domestic sewage water and industrial wastewater discharged into the Pi river. Also, unsuitable use of agricultural chemicals, unhealthy open def- ecation and many more potentially infiltrate into the shallow aquifer. This built the foundation for selecting the location for research samples to evaluate because they have relative importance as source of drinking water in the region. The samples were collected after 10 min of pumping and stored in clean 500-mL glass bottles that were thoroughly washed with detergent and rinsed with deionized water. The samples were sent to the laboratory of Environmental Engineering Center of Sichuan Geological Engineering Investigation and kept in a refrigerator at a temperature below 4 °C until In this study, the groundwater suitability for drinking and domestic purposes was evaluated by complying the values of various groundwater quality parameters according to China’s Quality Standards for Groundwater (GB/T14848-93), since it is the only way to assess groundwater quality in China by specifying the classification of groundwater quality, ground- water quality monitoring, evaluation methods and ground- water quality protection (Chinese 1993). It also corresponds to the methods applied to assess the quality of groundwater 1 3 study area mples Applied Water Science (2018) 8:65 65 Page 4 of 12 65 Page 4 of 12 65 in this paper by dividing the groundwater quality into five categories: excellent, good, moderate, poor and very poor. in this paper by dividing the groundwater quality into five categories: excellent, good, moderate, poor and very poor. Cl−, CODMn, NO− 3 , NO− 2 , NH+ 4 , Fe, Mn, As, Cr6+, and Pb), which represent the overall situation of groundwater quality of the Quaternary Unconsolidated Sedimentary Basin. The element u (i =1, 2,…n) is measured value of pollutants that affect the quality of groundwater. Principle for fuzzy mathematics evaluation method Fuzzy mathematics was proposed by Zadeh in 1965 (Mahapatra et al. 2011, Zhang et al. 2009) as a new way to represent vagueness in everyday life. This method is proven to be capable of dealing with complex systems under uncer- tain and imprecise conditions (Gharibi et al. 2012, Singh et al. 2017). Risk assessment of groundwater contamination could be a challenge because it often involves many ground- water quality parameters. Fuzzy mathematics can simplify this risk assessment process (Zhang et al. 2012). It takes the effectual measurement of pollutant concentration compared with its evaluation criteria. Through accepted linear func- tion, it calculates each pollution element of membership on the level of groundwater contamination. Afterwards, fuzzy matrix can be set, and the weight of each single element value can be obtained by calculation, which constitutes the weight factors matrix. Last, the membership matrix and the weight factors matrix are multiplied, and the evaluation results can be derived (Agoubi et al. 2016, Feng et al. 2012). Figure 2 shows the steps of creating the fuzzy mathematics model. Determining the evaluation standard This paper adopts the Chinese national standards of ground- water quality (GB/T 14848-93) as the evaluation standard, which was drafted by the Ministry of Geology and Min- eral Resources of the People’s Republic of China. Based on groundwater quality in China and human health requirement as well as the objective of water protection, the standard (GB/T 14848-93) classifies groundwater quality into five grades (I, II, III, IV, and V). The quality evaluation grades of water V =(v1, v2,…vm) are represented by vi (i =1, 2,…m), and it is the standard classification value of groundwater for any contamination, which includes five levels: excellent (grade I), good (grade II), moderate (grade III), poor (grade IV), and very poor (grade V). Groundwater with grades I and II is of excellent and good quality and is suitable for all uses. Grade III is moderate-quality water, which is generally suitable for drinking, irrigation, and industrial production. Grade IV is poor quality water, which is fit only for irriga- tion and industrial production and may be used for drinking after proper treatment. Grade V groundwater is very poor quality water that cannot be used for any purpose (Chinese 1993). The classification of these grades about each evalua- tion factor is given in Table 1. Determining the evaluation factors 2 Flow chart of the fuzzy mathematics method Table 1 Classification of groundwater quality based on the Chinese national standard (GB/T 14848-93) Parameters Grades I II III IV V pH ≤ 6.5 ≤ 7.0 ≤ 7.5 ≤ 8.0 > 8.5 TH (mg/L) ≤ 150 ≤ 300 ≤ 450 ≤ 550 > 550 TDS (mg/L) ≤ 300 ≤ 500 ≤ 1000 ≤ 2000 > 2000 SO4 (mg/L) ≤ 50 ≤ 150 ≤ 250 ≤ 350 > 350 Cl (mg/L) ≤ 50 ≤ 150 ≤ 250 ≤ 350 > 350 CODMn (mg/L) ≤ 1.0 ≤ 2.0 ≤ 3.0 ≤ 10 > 10 NO3 (mg/L) ≤ 2.0 ≤ 5.0 ≤ 20 ≤ 30 > 30 NO2 (mg/L) ≤ 0.001 ≤ 0.01 ≤ 0.02 ≤ 0.1 > 0.1 NH4 (mg/L) ≤ 0.02 ≤ 0.02 ≤ 0.2 ≤ 0.5 > 0.5 Fe (mg/L) ≤ 0.1 ≤ 0.2 ≤ 0.3 ≤ 1.5 > 1.5 Mn (mg/L) ≤ 0.05 ≤ 0.05 ≤ 0.1 ≤ 1.0 > 1.0 As (mg/L) ≤ 0.005 ≤ 0.01 ≤ 0.05 ≤ 0.05 > 0.05 Cr6+ (mg/L) ≤ 0.005 ≤ 0.01 ≤ 0.05 ≤ 0.1 > 0.1 Pb (mg/L) ≤ 0.005 ≤ 0.01 ≤ 0.05 ≤ 0.1 > 0.1 Classification Excellent Good Moderate Poor Very poor Table 1 Classification of groundwater quality based on the Chinese national standard (GB/T 14848-93) Parameters Grades I II III IV V pH ≤ 6.5 ≤ 7.0 ≤ 7.5 ≤ 8.0 > 8.5 TH (mg/L) ≤ 150 ≤ 300 ≤ 450 ≤ 550 > 550 TDS (mg/L) ≤ 300 ≤ 500 ≤ 1000 ≤ 2000 > 2000 SO4 (mg/L) ≤ 50 ≤ 150 ≤ 250 ≤ 350 > 350 Cl (mg/L) ≤ 50 ≤ 150 ≤ 250 ≤ 350 > 350 CODMn (mg/L) ≤ 1.0 ≤ 2.0 ≤ 3.0 ≤ 10 > 10 NO3 (mg/L) ≤ 2.0 ≤ 5.0 ≤ 20 ≤ 30 > 30 NO2 (mg/L) ≤ 0.001 ≤ 0.01 ≤ 0.02 ≤ 0.1 > 0.1 NH4 (mg/L) ≤ 0.02 ≤ 0.02 ≤ 0.2 ≤ 0.5 > 0.5 Fe (mg/L) ≤ 0.1 ≤ 0.2 ≤ 0.3 ≤ 1.5 > 1.5 Mn (mg/L) ≤ 0.05 ≤ 0.05 ≤ 0.1 ≤ 1.0 > 1.0 As (mg/L) ≤ 0.005 ≤ 0.01 ≤ 0.05 ≤ 0.05 > 0.05 Cr6+ (mg/L) ≤ 0.005 ≤ 0.01 ≤ 0.05 ≤ 0.1 > 0.1 Pb (mg/L) ≤ 0.005 ≤ 0.01 ≤ 0.05 ≤ 0.1 > 0.1 Classification Excellent Good Moderate Poor Very poor Table 1 Classification of groundwater quality based on the Chinese national standard (GB/T 14848-93) Fig. Determining the evaluation factors 2 Flow chart of the fuzzy mathematics method Applied Water Science (2018) 8:65 Page 5 of 12 65 Determining the weights of factors It can be rep- resented mathematically for any of the fourteen ground- water quality parameters with respect to five classification grades (I, II, III, IV, and V) as follows (Lermontov et al. 2009):when j = 1, where Ci is the measured values of index i, Si is the standard value for index i, n is the grading number of water quality standard, and αij is the jth sample value under the ith level of classification factor. To make the fuzzy compositional operation, the weight of each single factor must be normal- ized as follows: (3) Wi = Ci Si × 1 n∑ i=1 Ci Si , (3) (6) 𝜇A(x) = ⎧ ⎪ ⎨ ⎪⎩ 1 x ≤𝛼ij 𝛼ij+1−x 𝛼ij+1−𝛼ij 𝛼ij < x < 𝛼ij+1 0 x > 𝛼ij+1 , (6) where Wi represents the normalized weight of the evaluated index i. when j = 2, 3, 4 when j = 2, 3, 4 Based on the above equation, the weight set of the single factor can be determined A = { w 1, w2, … wi}. when j = 5 (7) 𝜇A(x) = ⎧ ⎪ ⎪ ⎨ ⎪ ⎪⎩ 0 x < 𝛼ij−1 𝛼ij+1−x 𝛼ij+1−𝛼ij 𝛼ij ≤x < 𝛼ij+1 x−𝛼ij−1 𝛼ij−𝛼ij−1 𝛼ij−1 ≤x < 𝛼ij 0 x < 𝛼ij+1 , (8) 𝜇A(x) = ⎧ ⎪ ⎨ ⎪⎩ 0 x < 𝛼ij−1 x−𝛼ij−1 𝛼ij−𝛼ij−1 𝛼 ij−1 < x < 𝛼ij 1 x ≥𝛼ij , (7) 𝜇A(x) = ⎧ ⎪ ⎪ ⎨ ⎪ ⎪⎩ 0 x < 𝛼ij−1 𝛼ij+1−x 𝛼ij+1−𝛼ij 𝛼ij ≤x < 𝛼ij+1 x−𝛼ij−1 𝛼ij−𝛼ij−1 𝛼ij−1 ≤x < 𝛼ij 0 x < 𝛼ij+1 , Determining the weights of factors where each element of X is mapped to a value between 0 and where each element of X is mapped to a value between 0 and 1. This value is referred to as membership value or degree of membership, and it is used to determine the degree of membership of each rating factor. Hence, the fuzzy set A is defined by its MF: pp 1. This value is referred to as membership value or degree of membership, and it is used to determine the degree of membership of each rating factor. Hence, the fuzzy set A is defined by its MF: The weights of factors are important elements in the math- ematical model of FME technique, which reverses the posi- tion and role of each factor in the measures of comprehen- sive decision making, and the result of the comprehensive evaluation is directly affected by it. The equations for weight is as follows (Zhang 2014): (5) 휇A(x) = {(휇A(x)), x ∈X, 휇A(x) ∈[0, 1] } . 휇A(x) = {(휇A(x)), x ∈X, 휇A(x) ∈[0, 1] } . (5) The membership function sets are represented through triangular, trapezoidal, Gaussian, Pseudo exponential, Sig- moidal and other shapes of fuzzy numbers (Miao et al. 2014, Srinivas et al. 2017). Generally, water quality parameter’s impact is represented by certain range of values, and the firing level of the conclusion is computed as the product of dismissal levels from the antecedent (Agoubi et al. 2016). In this study, the triangular membership function is used to normalize the crisp inputs because of its simplicity and computational efficiency and provide an environment more conductive to human-in-the-loop knowledge acquisition (Mahapatra et al. 2011, Caniani et al. 2015). It can be rep- resented mathematically for any of the fourteen ground- water quality parameters with respect to five classification grades (I, II, III, IV, and V) as follows (Lermontov et al. 2009):when j = 1, (1) Wi = Ci Si , (2) Si = 1 n n ∑ j=1 훼ij, (1) (2) In this study, the triangular membership function is used to normalize the crisp inputs because of its simplicity and computational efficiency and provide an environment more conductive to human-in-the-loop knowledge acquisition (Mahapatra et al. 2011, Caniani et al. 2015). Fuzzy evaluation of groundwater quality The fuzzy evaluation matrix B for groundwater quality evaluation is obtained by the compositional process of the weight A and the fuzzy evaluation matrix R of each single grade for I, II, III, IV, and V respectively. The principle of FME method can be described by the mathematical Eq. (10), with the main purpose of weighting evaluation factors (Hao et al. 2012). Groundwater quality parameters The concentration values of groundwater quality parameters obtained from the six wells in the study area were statistically analyzed. It should be noted that for each well, four samples were taken at different periods and there was no apparent change in the analysis results of these samples and, therefore, it was taken as the average. The results represented as maxi- mum, minimum, mean and standard deviation for the main groundwater quality parameters are shown in Table 4. Their levels were compared with the acceptable limits recommended by the national quality standards for groundwater of China (GB/T 14848-93) to see if they were suitable for drinking. (10) B = A × R = { w1, w2, … wi} × ⎡ ⎢ ⎢ ⎢ ⎢⎣ r11 r12 ⋯ r1m r21 r22 ⋯ r2m ⋮ ⋮ ⋮ rn1 rn2 ⋯ rnm ⎤ ⎥ ⎥ ⎥ ⎥⎦ (10) B = A × R = { w1, w2, … wi} × ⎡ ⎢ ⎢ ⎢ ⎢⎣ r11 r12 ⋯ r1m r21 r22 ⋯ r2m ⋮ ⋮ ⋮ rn1 rn2 ⋯ rnm ⎤ ⎥ ⎥ ⎥ ⎥⎦ From the principle of maximum membership, the grade of groundwater quality is determined. Determination of the membership and relation matrix R (7) A fuzzy set is completely characterized by its membership function (MF). The (MF) has been used to assess ground- water quality according to the standards. The level of mem- bership belongs to a type of fuzzy information which over- comes the differences among water index grade standards in different places (Zhang 2014). The FME begins with the concept of a fuzzy set. The fuzzy set describes the relation- ship between an uncertain quantity (x) and a membership function (μ). The elements of fuzzy set membership may be described as a number in the interval [0, 1] (Nasr et al. 2012). The greater the value of membership, the higher the membership qualifications. When the value of membership is 1, it is subordinated completely, and when the value of membership is 0, it is subordinated incompletely. The mem- bership degree of the fuzzy set is defined over a domain X which takes the form: (8) 𝜇A(x) = ⎧ ⎪ ⎨ ⎪⎩ 0 x < 𝛼ij−1 x−𝛼ij−1 𝛼ij−𝛼ij−1 𝛼 ij−1 < x < 𝛼ij 1 x ≥𝛼ij , (8) 훼ij, 훼 ij+1 and 훼ij−1 are the different levels of groundwater qual- ity standards and x is the real measured concentration of each factor.i The membership function can be described of five twin grades and the fuzzy relationship matrix R is formed as follows: 휇A ∶X →[0, 1], (4) 1 3 65 Page 6 of 12 Applied Water Science (2018) 8:65 65 Page 6 of 12 65 Page where n denotes the number of indices selected for the assessment, F is the value of CEM for a given sample, and Fi represents the assigned value for the ith index by the quality standard for groundwater (GB/T 14848-93). F is the average value of each individual component score of Fi, and (Fi)max is the maximum value of the individual component evalu- ation score Fi. Once F is determined, groundwater quality classification can be obtained as per the criteria in Table 3 (Wu and Sun 2016). (9) R = ⎡ ⎢ ⎢ ⎢⎣ r11 r12 ⋯r1m r21 r22 ⋯r2m ⋮ ⋮ ⋮ rn1 rn2 ⋯rnm ⎤ ⎥ ⎥ ⎥⎦ . (9) Comprehensive evaluation method The values of these concentrations for all the groundwater samples collected in this study are within the permissible limit of (GB/T 14848-93) for ground- water quality except sample S2 of NH+ 4 which exceeds the acceptable limit of 0.2 mg/L according to the national quality standards for groundwater (GB/T 14848-93). Finally, the con- centrations of Fe, Mn, As, Cr6+, and Pb in groundwater were 0.086–0.23 mg/L with the mean 0.59 mg/L, 0.01–0.21 mg/L with a mean of 0.07 mg/L, 0.0002–0.0002 mg/L with the mean 0.0002 mg/L, 0.002–0.004 mg/L with a mean 0.003 mg/L and 0.005–0.01 mg/L with an average of 0.008 mg/L, respectively. Among them, Fe and Mn are found to exceed the permissi- ble limits of drinking water as set by (GB/T 14848-93) of 0.3 and 0.1 mg/L, respectively, and remaining concentrations fall within the permissible limits of (GB/T 14848-93) for all groundwater samples. For Fe concentration, two samples 15.96 to 20.92, 0.23 to 0.29, 1.17 to 3.96, 0.003 to 0.005 and 0.020 to 0.68 mg/L, respectively, with a cumulative average for each concentration of 60.22, 17.61, 0.25, 2.40, 0.004 and 0.14 mg/L, respectively. The values of these concentrations for all the groundwater samples collected in this study are within the permissible limit of (GB/T 14848-93) for ground- water quality except sample S2 of NH+ 4 which exceeds the acceptable limit of 0.2 mg/L according to the national quality standards for groundwater (GB/T 14848-93). Finally, the con- centrations of Fe, Mn, As, Cr6+, and Pb in groundwater were 0.086–0.23 mg/L with the mean 0.59 mg/L, 0.01–0.21 mg/L with a mean of 0.07 mg/L, 0.0002–0.0002 mg/L with the mean 0.0002 mg/L, 0.002–0.004 mg/L with a mean 0.003 mg/L and 0.005–0.01 mg/L with an average of 0.008 mg/L, respectively. Among them, Fe and Mn are found to exceed the permissi- ble limits of drinking water as set by (GB/T 14848-93) of 0.3 and 0.1 mg/L, respectively, and remaining concentrations fall within the permissible limits of (GB/T 14848-93) for all groundwater samples. For Fe concentration, two samples exceed the permissible limit represented in sites (S2, S4) and for Mn concentration, one sample exceeds the permis- sible limit at the site (S2). According to the mean values, the dominance of cations is Ca2+ > Mg2+ > Na+ > K+ and anions is HCO− 3 > SO2− 4 > Cl−, which makes the predominant type of groundwater to be Ca–Mg-HCO3 type. Comprehensive evaluation method As shown in Table 5, the pH of the groundwater is within the range of 6.7–7.5 with the mean of 7.27, indicating that the groundwater in the study area is slightly alkaline for most of the groundwater samples. All samples fall within limits (6.5 and 8.5) of quality standard for groundwater of China (GB/T 14848-93). The average concentrations of water K+, Ca2+, Mg2+, and HCO− 3 were 2.018, 92.34, 17.23, and 299.0 mg/L, respectively, and no limits set for these param- eters have been stated of quality standard for groundwater of China (GB/T 14848-93). The Na+ concentration was deter- mined between 6.00 and 12.00 mg/L with an average of 9.417 mg/L. All samples of groundwater are found below the permissible limit of (GB/T 14848-93). The concentrations of TH and TDS range from 260.20 to 355.30 mg/L and 331.80 to 415.40 mg/L, with an average of both 301.50 and 368.97, respectively. This indicates that the quality of all ground- water in the study area is classified as very hard to TH and fresh water to TDS, respectively, according to (Todd 1980, Sawyer and McCarty 1967). In this study, the concentra- tions of (TH and TDS) are found below the allowable limit of (GB/T 14848-93) for all groundwater samples. At the same time, the concentrations of SO2− 4 , Cl−, CODMn, NO− 3 , NO− 2 , and NH+ 4 in groundwater range from 52.83 to 68.20, The comprehensive evaluation method (CEM) is based on the classification of individual component to determine the quality category required as in Table 2 to determine the individual component evaluation score of Fi, and press the comprehensive evaluation to determine the value of F. The evaluation is carried out using the quality standard for groundwater of China (GB/T 14848-93) as classified in the FME technique. (Chinese 1993). As per standard, CEM can be computed as follows (Su et al. Comprehensive evaluation method As mentioned above, the groundwater quality indicators exceeding the standard ratio in the study area were mainly Fe, Mn, and NH4 for sampling points S2 and S4 (Fig. 3). All units of parameters are in mg/L except pH, St.dev standard deviation Comprehensive evaluation method 2017): (11) F = √ F2 + F2max 2 (i = 1, 2, 3, … n), (12) F = 1 n ∑n i=1 Fi (i = 1, 2, 3, … n), (11) (12) Table 2 Individual component classification Page 7 of 12 65 Applied Water Science (2018) 8:65 ge 7 of 12 65 65 Table 4 Descriptive statistics of groundwater quality variables for six sampling wells in the study area Note: Standard refers to the quality standard for groundwater developed by China (GB/T14848-93) (Chi- nese 1993) All units of parameters are in mg/L except pH, St.dev standard deviation Parameters Unit Min Max Mean St.dev Standard (Class III) pH – 6.70 7.50 7.27 0.29 6.5–8.5 TH mg/L 260.20 355.30 301.50 42.11 ≤ 450 TDS mg/L 331.80 415.40 368.97 36.67 ≤ 1000 K+ mg/L 1.50 2.30 2.018 0.34 – Na+ mg/L 6.00 12.00 9.417 2.25 ≤ 200 Ca2+ mg/L 77.15 111.20 92.34 15.22 – Mg2+ mg/L 15.81 18.85 17.23 1.13 – SO4 2− mg/L 52.83 68.20 60.22 6.644 ≤ 250 Cl− mg/L 15.96 20.92 17.61 2.304 ≤ 250 HCO3 − mg/L 250.20 347.80 299.00 40.06 – CODMn mg/L 0.23 0.29 0.25 0.02 3 NO3 − mg/L 1.17 3.96 2.40 0.92 ≤ 20 NO2 − mg/L 0.003 0.005 0.004 0.0006 ≤ 0.07 NH4 + mg/L 0.020 0.68 0.14 0.26 ≤ 0.2 Fe mg/L 0.086 2.23 0.59 0.82 ≤ 0.3 Mn mg/L 0.01 0.21 0.07 0.08 ≤ 0.1 As mg/L 0.0002 0.0002 0.0002 0.00 ≤ 0.05 Cr6+ mg/L 0.002 0.004 0.003 0.001 ≤ 0.01 Pb mg/L 0.005 0.01 0.008 0.002 ≤ 0.05 Table 5 The weight value of groundwater quality parameters in well No. S2 Weight Factors PH TH TDS SO4 Cl CODMn NO3 NO2 NH4 Fe Mn As Cr6+ Pb Wi 0.974 0.732 0.297 0.068 0.071 0.039 0.064 0.089 2.363 2.720 0.389 0.005 0.043 0.079 Wi 0.123 0.092 0.037 0.009 0.009 0.005 0.008 0.011 0.298 0.343 0.049 0.001 0.005 0.010 Table 5 The weight value of groundwater quality parameters in well No. S2 15.96 to 20.92, 0.23 to 0.29, 1.17 to 3.96, 0.003 to 0.005 and 0.020 to 0.68 mg/L, respectively, with a cumulative average for each concentration of 60.22, 17.61, 0.25, 2.40, 0.004 and 0.14 mg/L, respectively. Note: Standard refers to the quality standard for groundwater developed by China (GB/T14848-93) (Chi- nese 1993) Weight of each evaluation factors According to Eqs. (1) (2) and (3), the corresponding weight values of all wells in the study area were obtained, using the (GB/T14848-93) standard (Table 1) and data from Table 4 for the fourteen selected indicators. The weight values for well No. S2 are shown in Table 5, as an example. The membership and relation matrix of different evaluation factors on various grades S2 Well Parameters I II III IV V S2 pH 0 0.20 0.80 0 0 TH 0 0.998 0.002 0 0 TDS 0.63 0.37 0 0 0 SO4 1 0 0 0 0 Cl 1 0 0 0 0 CODMn 1 0 0 0 0 NO3 1 0 0 0 0 NO2 0.56 0.44 0 0 0 NH4 0 0 0 0.1 0.9 Fe 0 0 0 0 1 Mn 0 0 0.88 0.12 0 As 1 0 0 0 0 Cr6+ 1 0 0 0 0 Pb 1 0 0 0 0 Based on Eqs. (6) (7) and (8), the degree of membership of each indicator to the groundwater quality grade is calculated. Each indicator is computed to have five levels of membership, and the fourteen selected indicators can get fourteen sets of numerical values. Accordingly, the corresponding fuzzy rela- tionship matrix R is achieved from the selected indicators of all wells in the study area which was computed. The membership for well No. S2 is shown in Table 6, as an example. Based on Eqs. (6) (7) and (8), the degree of membership of each indicator to the groundwater quality grade is calculated. Fig. 3 Groundwater quality indicators exceeding the standard ratio in the study area Fuzzy mathematics evaluation results of study area According to the above-mentioned relevant principles of the FME technique for the assessment of groundwater quality of the study area, the steps for evaluation results can be listed as follows: 1 3 Applied Water Science (2018) 8:65 65 Page 8 of 12 65 Page 8 of 12 S1 S2 S3 S4 S5 S6 0.0 0.5 1.0 1.5 2.0 2.5 Concentration mg/L Well name NH 4 + Fe Mn S1 S2 S3 S4 S5 S6 0.0 0.5 1.0 1.5 2.0 2.5 Concentration mg/L Well name NH 4 + Fe Mn Fig. 3 Groundwater quality indicators exceeding the standard ratio in the study area The membership and relation matrix of different evaluation factors on various grades Evaluation factors In this study, a FME technique was used to assess the groundwater quality of the quaternary unconsolidated sedimentary basin near the Pi river according to quality evaluation parameters and five classifications of groundwa- ter quality based on the Chinese national standard (GB/T 14848-93) (Table 1). The groundwater quality was assessed for six wells (S1, S2, S3, S4, S5, and S6) as selected in the study area. Of the 19 groundwater parameters analyzed, 14 parameters (pH, TH, TDS, SO4, Cl, CODMn, NO3, NO2, NH4, Mn, Fe, As, Cr6+ and Pb) were selected due to the fact the rest did not have the five-level division as required in the China’s standard for groundwater quality evaluation (GB/T 14848-93). This selection is also based on the fact that it is periodically monitored by the Local Environmental Protection Department for their vital importance to the water quality and potential influence on human health.f factors on various grades Based on Eqs. (6) (7) and (8), the degree of membership of each indicator to the groundwater quality grade is calculated. Each indicator is computed to have five levels of membership, and the fourteen selected indicators can get fourteen sets of numerical values. Accordingly, the corresponding fuzzy rela- tionship matrix R is achieved from the selected indicators of all wells in the study area which was computed. The membership for well No. S2 is shown in Table 6, as an example. This membership can be expressed by fuzzy relationship matrix, as follows: R = ⎡ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢⎣ 0 0.20 0.80 0 0 0 0.998 0.002 0 0 0.63 0.37 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0.56 0.44 0 0 0 0 0 0 0.1 0.9 0 0 0 0 1 0 0 0.88 0.12 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 ⎤ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥⎦ . S1 S2 S3 S4 S5 S6 0.0 0.5 Well name Fig. 3 Groundwater quality indicators exceeding the standard ratio in the study area Table 6 Membership of well No. B = (0.076, 0.136, 0.141, 0.036, 0.611). According to the principle of maximum membership to determine the groundwater quality level, 0.611 is the maxi- mum of all five values. Therefore, well No. S2 was found to belong to grade V, which is classified as very poor and their water quality cannot be used for any purpose. The degrada- tion in groundwater quality at this well is mainly due to high concentrations of NH4 and Fe (see Table 6). Similarly, the groundwater quality grades of the other wells in the study area were obtained. The results are shown in Table 7. Fig. 4 Comprehensive assessment of fuzzy technique for assessing groundwater quality From Table 7 and Fig. 4, the results of fuzzy evaluation matrix B for the other wells showed that the groundwater quality of well No. S1 is (0.579, 0.421, 0, 0, 0), and it found to belong to grade I, which is classified as excellent and their water quality is considered to be suitable for vari- ous purposes. The groundwater quality of wells No. S3 is (0.379, 0.477, 0.144, 0, 0), and categorized as being grade II, which is classified as good, and was also deemed to be suitable for all uses. Finally, the groundwater quality of wells No. S4, S5 and S6 are (0.264, 0.239, 0.466, 0.032), (0.214, 0.318, 0.468, 0, 0), (0.207, 0.319, 0.473, 0, 0), respectively, which fall under grade III and is classified as moderate, and their water quality is generally suitable for drinking, irriga- tion, and industrial production. From the six groundwater samples, grade I of groundwater occupies 16.67%, grade II 16.67% and grade III 50% that means about 83.33% of the groundwater samples could be used as drinking water source. However, S2 is grade V (16.67%), which cannot be used for drinking. This suggests that the groundwater quality in the study area, in general, is not bad. Through analysis of the water indicators, the elevated groundwater quality indi- cators were mainly NH4, Fe, and Mn which have resulted in the evaluation of well No. S2 as class V which is very poor. According to our field investigation in the study, the very poor groundwater quality of well No. S2 could be attributed to several reasons: 1. In the Basin of Minjiang as Pi river is a tributary of Minjiang river, sediment has a certain amount of Fe and Mn under the natural environmental conditions (Zeng Jichuan 2009, Li et al. Evaluation factors In this study, a FME technique was used to assess the groundwater quality of the quaternary unconsolidated sedimentary basin near the Pi river according to quality evaluation parameters and five classifications of groundwa- ter quality based on the Chinese national standard (GB/T 14848-93) (Table 1). The groundwater quality was assessed for six wells (S1, S2, S3, S4, S5, and S6) as selected in the study area. Of the 19 groundwater parameters analyzed, 14 parameters (pH, TH, TDS, SO4, Cl, CODMn, NO3, NO2, NH4, Mn, Fe, As, Cr6+ and Pb) were selected due to the fact the rest did not have the five-level division as required in the China’s standard for groundwater quality evaluation (GB/T 14848-93). This selection is also based on the fact that it is periodically monitored by the Local Environmental Protection Department for their vital importance to the water quality and potential influence on human health. This membership can be expressed by fuzzy relationship matrix, as follows: 1 3 Table 6 Membership of well No. S2 Well Parameters I II III IV V S2 pH 0 0.20 0.80 0 0 TH 0 0.998 0.002 0 0 TDS 0.63 0.37 0 0 0 SO4 1 0 0 0 0 Cl 1 0 0 0 0 CODMn 1 0 0 0 0 NO3 1 0 0 0 0 NO2 0.56 0.44 0 0 0 NH4 0 0 0 0.1 0.9 Fe 0 0 0 0 1 Mn 0 0 0.88 0.12 0 As 1 0 0 0 0 Cr6+ 1 0 0 0 0 Pb 1 0 0 0 0 1 3 Page 9 of 12 65 Applied Water Science (2018) 8:65 Page 9 of 12 65 Fuzzy evaluation of groundwater quality S1 S2 S3 S4 S5 S6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Grade value Well name Excellent Good Moderate Poor Very poor Final result Fig. 4 Comprehensive assessment of fuzzy technique for assessing groundwater quality Based on Eq. (10), the fuzzy evaluation matrix B for ground- water quality evaluation was obtained by the compositional process of the weight matrix A and the fuzzy evaluation matrix R. For example, the fuzzy evaluation matrix B of the well No. S2 is as follows: B = (0.076, 0.136, 0.141, 0.036, 0.611). Comparison between fuzzy mathematics evaluation technique and comprehensive evaluation method 1 3 The present study analyzed groundwater quality status of six wells in the Quaternary Unconsolidated Sedimentary Basin using FME technique and CEM. Based on the results of the assessment obtained from both methods, the holistic picture of groundwater quality within the study area was satisfactory and consistent with the actual situation of the study area under the prevailing conditions. The indices for both methods indicated that the groundwater quality in the study area was suitable for drinking at ratio 83.34 and 66.67%, respectively. The FME showed only the well No. 2 to be unsuitable for drinking water (see Table 7), while the CEM showed wells No. 2 and 4 to be unsuitable for drinking water (see Table 8). This is due to the technical difference of both methods. For instance, the FME approach uses membership degree to describe the limit between different pollution degrees for assessing groundwater quality. The FME technique takes into account the impression of each assessment factor on the evaluation result and deter- mines the major pollutants according to the weights of evalua- tion factors. This reflects how close the actual concentration of t s l p 4 w r t s m f r r w t r i i Table 8 Groundwater quality classification based on comprehensive evaluation method Well name S1 F 0.82 Grade II The present study analyzed groundwater quality status of six wells in the Quaternary Unconsolidated Sedimentary Basin using FME technique and CEM. Based on the results of the assessment obtained from both methods, the holistic picture of groundwater quality within the study area was satisfactory and consistent with the actual situation of the study area under the prevailing conditions. The indices for both methods indicated that the groundwater quality in the study area was suitable for drinking at ratio 83.34 and 66.67%, respectively. The FME showed only the well No. 2 to be unsuitable for drinking water (see Table 7), while the CEM showed wells No. 2 and 4 to be unsuitable for drinking water (see Table 8). This is due to the technical difference of both methods. For instance, the FME approach uses membership degree to describe the limit between different pollution degrees for assessing groundwater quality. The FME technique takes into account the impression of each assessment factor on the evaluation result and deter- mines the major pollutants according to the weights of evalua- tion factors. Groundwater quality evaluation based on comprehensive evaluation method S1 S2 S3 S4 S5 S6 0 1 2 3 4 5 6 7 8 F value Well name Fig. 5 Results of groundwater quality evaluation based on compre- hensive evaluation method In the study area, groundwater is a vital source of drinking water for residents. The CEM has been used to assess the qual- ity of groundwater of the Quaternary Unconsolidated Sedi- mentary Basin near the Pi river and to demonstrate the advan- tage of this study. In this method, the same physicochemical water quality parameters that were chosen in the FME method (14 parameters) have been used for six wells (S1, S2, S3, S4, S5, and S6). The results are shown in Table 8. As shown in Table 8 and Fig. 5, F values of all analyzed samples in the study area differ from 0.82 to 7.29, ranging from good quality to very poor quality. The results of the assessment showed that four groundwater samples (66.67% of all samples) are classified as good quality water (grade II) which is suitable for various purposes. Two groundwater samples (33.33% of all collected samples) are classified as poor and very poor quality water (grade IV and V), respectively, which are classified as unsuitable for drinking. The common contaminants in these samples are NH4, Fe, and Mn, which are mainly from the natu- ral environment, industrial and agricultural activities. Accord- ing to the results of the CEM, four groundwater samples (S1, S3, S5, and S6) could be used as drinking water source, while (S2 and S4) cannot be used as drinking water source. Fig. 5 Results of groundwater quality evaluation based on compre- hensive evaluation method boundary water quality index of water quality is, hence making the evaluation more comprehensive and reasonable. Whereas, the CEM of water highlights the largest factor of pollution as the index classification is based on the binary logic. Therefore, they cannot describe the continuity of environmental quality, and cannot objectively reflect the influence of the index value near the water quality grade limit for quality evaluation and classification. i Despite the FME and CEM generated almost similar results to the holistic picture of groundwater quality in the study area, the fuzzy indicator is recommended as the more useful indica- tor for the following advantages: B = (0.076, 0.136, 0.141, 0.036, 0.611). 2006). Thus, the over-limit ratio of Fe and Mn is considered natural environment. 2. The sample No. S2 was taken from the water plant, which is closest to the populated area, and the infiltra- tion of the plant domestic wastewater may lead to the increase of NH4 content. In essence, the groundwater quality of the well S2 is mainly influenced by human activities. 3. Excessive discharge of industrial waste water and domestic sewage is the main cause of groundwater pol- lution in the study area. Considering the aforementioned reasons, well No.2 is deemed contaminated and, there- fore, should be monitored periodically and protected from the causes of pollution to avoid being consumed by local residents. 1 3 Table 7 The fuzzy evaluation of groundwater quality Well name I II III IV V Results S1 0.579 0.421 0 0 0 I S2 0.076 0.136 0.141 0.036 0.611 V S3 0.379 0.477 0.144 0 0 II S4 0.264 0.239 0.466 0.032 0 III S5 0.214 0.318 0.468 0 0 III S6 0.207 0.319 0.473 0 0 III Applied Water Science (2018) 8:65 65 Page 10 of 12 65 Page 10 of 12 References Agoubi B, Souid F, Kharroubi A, Abdallaoui A (2016) Assessment of hot groundwater in an arid area in Tunisia using geochemical and fuzzy logic approaches. Environ Earth Sci 75:1497 Al-Ahmadi ME (2013) Hydrochemical characterization of groundwater in wadi Sayyah, Western Saudi Arabia. Appl Water Sci 3:721–732i 3 4 The FME technique results show that five of the sam- pled groundwater (S1, S3, S4, S5, and S6) are suitable of drinking water directly while the well (S2) is unsuitable for drinking unless treated. The common contaminants in these samples are NH4, Fe, and Mn, which are mainly from the natural environment, industrial and agricultural activities. Thus, authorities should give more attention to the pollution of NH4, Fe, and Mn to prevent deterioration of good water quality by taking some effective measures that are required to enhance the drinking water quality by delineating an effective water quality management plan. This method may play an important role in decision-making for the drinking water quality assessment because it proved effective in solv- ing problems of fuzzy boundaries. So, it is more objective and scientific and reliable in practice. Caniani D, Lioi D, Mancini I, Masi S (2015) Hierarchical classifica- tion of groundwater pollution risk of contaminated sites using fuzzy logic: a case study in the Basilicata region (Italy). Water 7:2013–2036 Chen H, Li X, Liu A (2009) Studies of water source determination method of mine water inrush based on Bayes’ multi-group step- wise discriminant analysis theory. Rock Soil Mech 30:3655–3659 Cheng Y, Fanhai M (2012) GW vulnerability assessment based on entropy and fuzzy method. Proc Inst Civ Eng 165:277 Chinese S. (1993) Quality Standard for Groundwater (GB/T 14848– 93). AQSIQ (General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China) (in Chinese) Chun-rong J, Jun Z (2011) Based on fuzzy weight matter element to evaluate the water quality of Jialing River in Nanchong, China. Proc Environ Sci 11:631–636 Feng L, Daohan W, Sijing F, Suzhen Y (2012) Study on the application of fuzzy mathematics in assessing the water quality of Qinghe Reservoir. In: Computer science and electronics engineering (ICCSEE), 2012 international conference on, pp 672–675. IEEE i The FME method was compared with CEM in this study. Conclusions Groundwater pollution is a vague concept because there are often no clear-cut boundaries that separate a “polluted” from an “unpolluted” sample. It is, therefore, necessary to develop a new method based on a fuzzy technique to give solutions that are robust and have a high level of confidence. Acknowledgements This research was supported by the Fundamental Research Funds for the Central Universities (2682015CX020). i In this study, six groundwater samples were collected, analyzed and assessed for drinking water quality in the Quaternary Unconsolidated Sedimentary Basin near the Pi river. The pH value of the groundwater was slightly alka- line for most of the groundwater samples to basic in nature. The groundwater is classified as very hard and fresh water based on TH and TDS, respectively. Groundwater quality parameters were compared with the acceptable limits recom- mended by the national quality standards for groundwater of China (GB/T 14848-93). From all groundwater parameters analyzed, NH4, Fe, and Mn were above the permissible lim- its of (GB/T 14848-93). The sequence of the abundance of major ions is found in the order of Ca2+ > Mg2+ > Na+ > K+ and anions is HCO− 3 > SO2− 4 > Cl−.i Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creativeco mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Comparison between fuzzy mathematics evaluation technique and comprehensive evaluation method This reflects how close the actual concentration of 1. Ability to describe a wide variety of non-linear relation- ships. 2. They tend to be simple since they are based on a set of local simple models. 3. Fuzzy mathematics can deal with and process missing data without compromising the final result. 4. Avoiding artificial precision as well as generating results which are more consistent with the ecological complexity of real-world issues. 5. Combining both qualitative and quanti- tative information to express the ecological status of the case study, which is a unique capability of fuzzy approach. In general, the results revealed that the knowledge-based models such as FME method were practical and flexible tools for incorporating the experts’ attitudes and modeling the cur- rent uncertainties associated with water resources and envi- ronmental perplexities. However, in the FME technique, the weight of evaluating indicators is determined by the moni- toring data compared to groundwater quality standard. As a result, when an abnormal value appears at some evaluating indicator, the condition of overestimating the weight of these indicators would lead to unrealistic evaluation results which Well name S1 S2 S3 S4 S5 S6 F 0.82 7.29 2.21 4.3 2.35 2.27 Grade II V II IV II II Table 8 Groundwater quality classification based on comprehensive evaluation method Table 8 Groundwater quality classification based on comprehensive evaluation method 1 3 Page 11 of 12 Applied Water Science (2018) 8:65 65 conditions accurately and precisely, specifically for the North Chengdu Plain, China. may not be in line with the actual situation of the studied area (Zou et al. 2006). may not be in line with the actual situation of the studied area (Zou et al. 2006). The degree of groundwater pollution risk has a direct con- nection to the water discharge and environmental vulnerabil- ity of the region. 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https://openalex.org/W3108957328	https://hal.archives-ouvertes.fr/hal-03009790/document	Latin	null	A Partial Metric Semantics of Higher-Order Types and Approximate Program Transformations	HAL (Le Centre pour la Communication Scientifique Directe)	2,021	cc-by	15,742	Funding Guillaume Geoﬀroy: ERC CoG 818616 “DIAPASoN”, ANR 16CE250011 “REPAS” 25 Paolo Pistone: ERC CoG 818616 “DIAPASoN”, ANR 16CE250011 “REPAS” 26 Funding Guillaume Geoﬀroy: ERC CoG 818616 “DIAPASoN”, ANR 16CE250011 “REPAS” 25 Paolo Pistone: ERC CoG 818616 “DIAPASoN”, ANR 16CE250011 “REPAS” 26 Funding Guillaume Geoﬀroy: ERC CoG 818616 “DIAPASoN”, ANR 16CE250011 “REPAS” 25 Paolo Pistone: ERC CoG 818616 “DIAPASoN”, ANR 16CE250011 “REPAS” 26 A Partial Metric Semantics of Higher-Order Types 1 and Approximate Program Transformations 2 Guillaume Geoﬀroy 3 Università di Bologna, Dipartimento Informatica, Scienza e Ingegneria, Italy 4 guillaume.geoﬀroy@unibo.it 5 Paolo Pistone 6 Università di Bologna, Dipartimento Informatica, Scienza e Ingegneria, Italy 7 paolo.pistone2@unibo.it 8 Paolo Pistone 6 Università di Bologna, Dipartimento Informatica, Scienza e Ingegneria, Italy 7 paolo.pistone2@unibo.it 8 © G. Geoﬀroy and P. Pistone; licensed under Creative Commons License CC-BY 42nd Conference on Very Important Topics (CVIT 2016). Editors: John Q. Open and Joan R. Access; Article No. 23; pp. 23:1–23:18 Leibniz International Proceedings in Informatics Schloss Dagstuhl – Leibniz-Zentrum für Informatik, Dagstuhl Publishing, Germany Leibniz International Proceedings in Informatics Abstract 9 Program semantics is traditionally concerned with program equivalence. However, in ﬁelds like 10 approximate, incremental and probabilistic computation, it is often useful to describe to which 11 extent two programs behave in a similar, although non equivalent way. This has motivated the 12 study of program (pseudo)metrics, which have found widespread applications, e.g. in diﬀerential 13 privacy. In this paper we show that the standard metric on real numbers can be lifted to higher-order 14 types in a novel way, yielding a metric semantics of the simply typed lambda-calculus in which 15 types are interpreted as quantale-valued partial metric spaces. Using such metrics we deﬁne a class 16 of higher-order denotational models, called diameter space models, that provide a quantitative 17 semantics of approximate program transformations. Noticeably, the distances between objects of 18 higher-types are elements of functional, thus non-numerical, quantales. This allows us to model 19 contextual reasoning about arbitrary functions, thus deviating from classic metric semantics. 20 2012 ACM Subject Classiﬁcation Theory of computation →Denotational semantics 21 Keywords and phrases Simply typed λ-calculus, program metrics, approximate program transfor- 22 mations, partial metric spaces 23 Digital Object Identiﬁer 10.4230/LIPIcs.CVIT.2016.23 24 Digital Object Identiﬁer 10.4230/LIPIcs.CVIT.2016.23 24 Funding Guillaume Geoﬀroy: ERC CoG 818616 “DIAPASoN”, ANR 16CE250011 “REPAS” 25 Paolo Pistone: ERC CoG 818616 “DIAPASoN”, ANR 16CE250011 “REPAS” 26 A Partial Metric Semantics of Higher-Order Programs 23:2 evaluated in C. Similar cases of contextual reasoning can be found in many areas of computer 45 science: for example in techniques from numerical analysis (e.g. the Gauss-Newton method), 46 in which a computationally intensive function is replaced by its Taylor’s expansion around 47 some given point, or in approximate computing techniques like loop perforation [38], in which 48 a compiler can be asked to skip a certain number of iterations of a loop in a program. 49 The Problem of Coupling Program Metrics with Higher-Order Types While several 50 frameworks for contextual reasoning have been developed in recent years [35, 20, 5, 44, 32], 51 these approaches suggest that describing program similarity for a fully higher-order language 52 in terms of program metrics still constitutes a major challenge. 53 In particular, when considering higher-order languages with a type Real for real numbers, 54 it is not clear how to lift the standard metric on Real to higher-order types, e.g. to Real →Real, 55 so that the distances between higher-order programs are measured in a contextual way. 56 A standard solution is to take the sup-distance, that is, to let, for f, g : Real →Real, 57 d(f, g) = sup{d(f(r), g(r)) \| r ∈Real}. This solution works well in models in which programs 58 are interpreted as non-expansive or Lipschitz-continuous maps [25, 5]. However such models 59 are not cartesian-closed1, so they do not account for the simply-typed lambda-calculus 60 in its full generality, but only for linear or sub-exponential variations of it (such as Fuzz 61 [35, 20, 5]). Also, it has been shown [13] that in a probabilistic setting the non-linearity of 62 higher-order programs has the eﬀect of trivialising metrics, that is, of forcing distances to be 63 either 0 or 1, hence collapsing program distances onto usual notions of program equivalence. 64 Most importantly, even if one restricts to a sub-exponential language, the sup-distance is 65 inadequate to account for contextual transformations as the replacement of λx. sin(x) by 66 λx.x around 0, as the sup-distance between these two programs is inﬁnite (see Fig. 3). 67 On the other side of the coin, other approaches like [44, 32] are fully contextual and 68 higher-order, but provide, at best, only weak approximations of a standard notion of metric. 1 In fact, cartesian closed categories of metric spaces and non-expansive functions do exist [19, 12], but, to our knowledge, none of these categories contains the real numbers with the standard metric. 1 Introduction 27 In program semantics one is usually interested in capturing notions of behavioral equivalence 28 between programs. However, in several ﬁelds like approximate [34], incremental [10, 2] and 29 probabilistic [13] computation, it is often more useful to be able to describe to which extent 30 two programs behave in a similar, although non equivalent way, so that one can measure the 31 change in the result produced by replacing one program by the other one. 32 This idea has motivated much literature on program (pseudo)metrics [4, 41, 5, 19, 6, 13, 11, 33 14, 21], that is, on semantics in which types are endowed with a notion of distance measuring 34 the diﬀerences in their behaviors. This approach has found widespread applications, for 35 example in diﬀerential privacy [35, 3, 7], where one is interested in measuring the sensitivity of 36 a program, i.e. its capacity to amplify changes in its inputs, and in the study of probabilistic 37 processes [16, 43, 11, 42]. 38 Recent literature [44, 32] has highlighted the importance of contextuality to reason about 39 program similarity: many common situations require to measure the error produced by a 40 transformation of the form C[t] ⇝C[u], which replaces a program t by u within a context 41 C[ ], as a function of the mismatch between t and u and of the sensitivity of the context C[ ] 42 itself. For instance, the error produced by replacing the program λx. sin(x) by the identity 43 function λx.x in a given context C will be highly sensitive to how close to 0 these functions are 44 A Partial Metric Semantics of Higher-Order Programs Figure 1 Diﬀerential logical relations do not yield partial metrics. x x −ε x + ε f(x) g(x) f g δ2 δ1 (a) In diﬀerential logical relations the dis- tance between two functions f, g : R →R, computed at (x, ε) is the maximum between δ1 = max{d(f(x), g(y)); y ∈[x−ε, x+ε]} and δ2 = max{d(g(x), f(y)); y ∈[x −ε, x + ε]}. x x −ε x + ε f(x) g(x) h(x) f g h d(f, g) d(g, h) δ = d(g, g) d(f, h) (b) The distance arising from diﬀerential log- ical relations is not a partial metric: the ex- ample above shows that d(f, h) > d(f, g) + d(g, h) −d(g, g) (with all distances computed at (x, ε)). x x −ε x + ε f(x) g(x) h(x) f g h d(f, g) d(g, h) δ = d(g, g) d(f, h) (b) The distance arising from diﬀerential log- ical relations is not a partial metric: the ex- ample above shows that d(f, h) > d(f, g) + d(g, h) −d(g, g) (with all distances computed at (x, ε)). x x −ε x + ε f(x) g(x) h(x) f g h d(f, g) d(g, h) δ = d(g, g) d(f, h) x x −ε x + ε f(x) g(x) f g δ2 δ1 (a) In diﬀerential logical relations the dis- tance between two functions f, g : R →R, computed at (x, ε) is the maximum between δ1 = max{d(f(x), g(y)); y ∈[x−ε, x+ε]} and δ2 = max{d(g(x), f(y)); y ∈[x −ε, x + ε]}. (b) The distance arising from diﬀerential log- ical relations is not a partial metric: the ex- ample above shows that d(f, h) > d(f, g) + d(g, h) −d(g, g) (with all distances computed at (x, ε)). Figure 1 Diﬀerential logical relations do not yield partial metrics. Figure 1 Diﬀerential logical relations do not yield partial metrics. Figure 1 Diﬀerential logical relations do not yield partial metrics. approximate values, LAM is a quantale, and δA : JAK →LAM is a function, called diameter, 88 which provides a quantitative measure of approximate values. The map δA generalizes some 89 properties of the diameter function of the standard metric on real numbers. A Partial Metric Semantics of Higher-Order Programs In particular, just 90 like the distance between two real numbers can be described as the diameter of the smallest 91 interval containing them, the map δA yields a generalized partial metric dA : \|A\| × \|A\| →LAM 92 in which the distance between two exact values of A is measured as the diameter of the 93 smallest approximate value containing them, i.e. dA(x, y) = δA(x ∨y). 94 Measuring Distances between Programs of Functional Type A primary source of inspira- 95 tion for our approach is the recent work by Dal Lago, Gavazzo and Yoshimizu on diﬀerential 96 logical relations [32]. This is a semantical framework for higher-order languages in which 97 a type is interpreted as a set X endowed with a kind of metric structure expressed by a 98 ternary relation ρ ⊆X × Q × X, where Q is an arbitrary quantale. To our knowledge, this 99 is the ﬁrst place were the idea of varying the quantales in which distances are measured is 100 introduced as a key ingredient to obtain a cartesian closed category. 101 However, although such a relation ρ induces a distance function dρ(x, y) = sup{ε \| 102 ρ(x, ε, y)}, this function is not a (partial) metric. We can show this fact with a simple 103 example: in this model the distance between two programs f, g : Real →Real is taken 104 in the quantale of functions from R × R∞ + to R∞ + : intuitively, d(f, g) associates a closed 105 interval [x −ε, x + ε] (corresponding to the pair (x, ε)) with the smallest distance δ such that 106 [f(x) −δ, f(x) + δ] and [g(x) −δ, g(x) + δ] both contain the images of [x −ε, x + ε] through 107 g and f respectively (see Fig. 1a). Then, as shown in Fig. 1b, by letting δ = d(g, g)(x, ε), 108 we have that d(g, g) sends the interval I = [x −ε, x + ε] onto the interval [g(x) −δ, g(x) + δ], 109 which has diameter 2δ, while the image of I has diameter δ, making the triangular law of 110 partial metrics fail. 111 By contrast, in our model, the distance between two programs f, g : Real →Real lives in 112 the quantale of monotone maps from approximate values of Real (i.e. closed intervals) to 113 positive reals. A Partial Metric Semantics of Higher-Order Programs 69 Nonetheless, these approaches introduce the idea, which we retain here, that program 70 diﬀerences must be taken as being themselves some kind of programs, relating errors in input 71 with errors in output, and that accordingly, programs should be split in two diﬀerent classes: 72 exact programs, computing mappings from well-deﬁned inputs to well-deﬁned outputs, and 73 approximate programs, mapping errors in the input to errors in the output. 74 Diameter Spaces In this paper we introduce a new semantic framework to reason about 75 program similarity and approximate program transformations based on a class of higher-order 76 denotational models that we call diameter space models. Compared to existing higher-order 77 frameworks, the main novelty of these models is that program similarities are measured by 78 associating each simple type with a generalized partial metric space, yielding a lifting of the 79 standard metric on Real to higher-order types. 80 Generalized partial metric spaces are a well-investigated class of metric spaces that has 81 been widely applied in program semantics [8, 9, 33, 37, 36, 26, 23]. Such spaces generalize 82 standard metric spaces in that distances need not be real numbers, but can be functions or 83 any other type of object that lives in a suitable quantale [25], and self-distances d(x, x) need 84 not be 0 (which leads to a stronger triangular inequality: d(x, y) + d(z, z) ≤d(x, z) + d(z, y)). 85 In our models a higher-order type A is interpreted as a 4-tuple (\|A\|, JAK, LAM, δA) called 86 a diameter space, where \|A\| is a set of exact values, JAK ⊂P(\|A\|) is a complete lattice o 87 23:3 G. Geoﬀroy and P. Pistone G. Geoﬀroy and P. Pistone x x −ε x + ε f(x) g(x) f g δ2 δ1 (a) In diﬀerential logical relations the dis- tance between two functions f, g : R →R, computed at (x, ε) is the maximum between δ1 = max{d(f(x), g(y)); y ∈[x−ε, x+ε]} and δ2 = max{d(g(x), f(y)); y ∈[x −ε, x + ε]}. x x −ε x + ε f(x) g(x) h(x) f g h d(f, g) d(g, h) δ = d(g, g) d(f, h) (b) The distance arising from diﬀerential log- ical relations is not a partial metric: the ex- ample above shows that d(f, h) > d(f, g) + d(g, h) −d(g, g) (with all distances computed at (x, ε)). A Partial Metric Semantics of Higher-Order Programs sin(x) and λx.x around 0 by the fact that their distance, applied to a small interval [−ε, ε] 124 around 0, is very close to the self-distance of λx. sin(x) on the same interval (as illustrated 125 in Fig. 3). Moreover, the triangular inequality of partial metrics can be used to infer new 126 bounds from previously established ones in a compositional way. 127 Diameter Space over a Cartesian Closed Category Our approach was devised primarily to 128 account for transformations in higher-order languages designed for real analysis computation 129 (like e.g. Real PCF [18]). However, diameter spaces can be constructed starting from any 130 higher-order programming language with a reasonable denotational semantics. In fact, for any 131 cartesian closed category C, we can construct a cartesian lax-closed category Diam(C), whose 132 morphisms can be seen as approximate versions of the morphisms of C. The “lax” preservation 133 of the cartesian closed structure reﬂects the fact that, by composing approximations in a 134 higher-order setting, also their error rates compose (typically, approximating non β-normal 135 λ-terms will lead to higher error-rates than approximating their β-normal forms). 136 The generality of our construction shows in particular that our partial metric semantics 137 requires no restrictions (e.g. Lipschitz-continuity) on morphisms, and adapts well to the 138 model one starts with: for instance, the category Diam(Set) contains a partial metric on 139 the set of all set-theoretic functions from R to R, while the categories Diam(Eﬀ) (where Eﬀ 140 is the eﬀective topos [27]) and Diam(Scott) show that our approach scales well to a more 141 computability-minded setting. 142 A Partial Metric Semantics of Higher-Order Programs 23:4 A Partial Metric Semantics of Higher-Order Programs x −ε x + ε f g h d(f, g) d(g, h) d(g, g) d(f, h) Figure 2 Our new metric is a partial met- ric: in the example above it can be seen that d(f, h) ≤d(f, g) + d(g, h) −d(g, g) (with all dis- tances computed in the interval [x −ε, x + ε]). −ε +ε sin(x) x δ δ′ Figure 3 The self-distances δ, δ′ of sin(x) and x in a small interval [−ε, ε] of 0 are very close. −ε +ε sin(x) x δ δ′ Figure 3 The self-distances δ, δ′ of sin(x) and x in a small interval [−ε, ε] of 0 are very close. x −ε x + ε f g h d(f, g) d(g, h) d(g, g) d(f, h) Figure 3 The self-distances δ, δ′ of sin(x) and x in a small interval [−ε, ε] of 0 are very close. Figure 2 Our new metric is a partial met- ric: in the example above it can be seen that d(f, h) ≤d(f, g) + d(g, h) −d(g, g) (with all dis- tances computed in the interval [x −ε, x + ε]). longer depict the “center” of the interval [x −ε, x + ε], and that the triangular inequality 117 works because in summing d(f, g) and d(g, h) the self-distance d(g, g) is counted twice. 118 Note that the distance of f from itself, which needs not be (constantly) 0, provides a 119 measure of the sensitivity of f, since it associates each interval a with the size of the interval 120 f(a) spanned by f on a (a similar feature is present in diﬀerential logical relations). 121 Note that the distance of f from itself, which needs not be (constantly) 0, provides a 119 measure of the sensitivity of f, since it associates each interval a with the size of the interval 120 f(a) spanned by f on a (a similar feature is present in diﬀerential logical relations). 121 The use of partial metrics with functional distances yields a rich and expressive framework 122 b l f F h l f The use of partial metrics with functional distances yields a rich and expressive framework 122 to reason about contextual transformations. For instance, we can express the closeness of 123 λx. A Partial Metric Semantics of Higher-Order Programs More precisely, this distance is the function that maps a closed interval a to 114 the diameter of the smallest interval containing both f(a) and g(a). This notion of distance 115 does satisfy all the axioms of a partial metric, as illustrated in Fig. 2. Observe that we no 116 CVIT 2016 2 Generalized Partial Metric Spaces 143 Partial metric spaces were introduced in the early nineties as a variant of metric spaces in 144 which self-distances can be non-zero. Such spaces have attracted much attention in program 145 semantics [8, 9, 33, 37, 36, 26, 23], due to their compatibility with standard constructions 146 from both domain theory (since their topology is T0) and usual metric topology (e.g. Cauchy 147 sequences, completeness, Banach-ﬁxed point theorem) [8, 33]. Generalized partial metric 148 23:5 G. Geoﬀroy and P. Pistone A generalized partial metric space (in short, GPMS) is the data of a set X, 169 a commutative integral quantale Q and a function d : X × X →Q such that: 170 for all x, y ∈X, d(x, x) ≤d(x, y), 171 for all x, y ∈X, d(x, x) ≤d(x, y), 171 for all x, y ∈X, if d(x, x) = d(x, y) = d(y, y), then x = y, 172 for all x, y ∈X, if d(x, x) = d(x, y) = d(y, y), then x = y, 172 for all x, y ∈X, d(x, y) = d(y, x), 173 for all x, y ∈X, d(x, y) = d(y, x), 173 for all x, y, z ∈X, d(x, z) + d(y, y) ≤d(x, y) + d(y, z). 174 for all x, y, z ∈X, d(x, z) + d(y, y) ≤d(x, y) + d(y, z). 174 for all x, y, z ∈X, d(x, z) + d(y, y) ≤d(x, y) + d(y, z). 174 For every metric space (X, d), the structure (X, ([0, ∞], +, ≤), d) is a GPMS. As is 175 well-known [8], any real-valued GPMS (X, [0, ∞], d) induces a metric d∗by letting 176 For every metric space (X, d), the structure (X, ([0, ∞], +, ≤), d) is a GPMS. As is 175 well-known [8], any real-valued GPMS (X, [0, ∞], d) induces a metric d∗by letting 176 (⋆) d∗(x, y) = 2d(x, y) −d(x, x) −d(y, y) (⋆) 177 d∗(x, y) = 2d(x, y) −d(x, x) −d(y, y) 177 For a more telling and somewhat archetypal example, take any set X and consider the set 178 X≤ω of all sequences of elements of X indexed by an ordinal less than or equal to ω. For all 179 such sequences s, t, let d(s, t) = 2−n ∈[0, ∞], where n is the length of the largest common 180 preﬁx to s and t: one can check that (X≤ω, [0, ∞], d) is indeed a generalized partial metric 181 space. In fact, if we interpret the preﬁxes of a sequence as pieces of partial information, 182 then we have d(s, s) = d(s, t) if and only if t is a reﬁnement of s (i.e. if it contains more 183 information), and d(s, s) = 0 if and only if s is total (i.e. if it cannot be reﬁned). G. Geoﬀroy and P. Pistone spaces, i.e. partial metric spaces whose metric takes values over an arbitrary quantale [25], 149 are well-investigated too [29, 28]. 150 In this paper we will only be concerned with partial metrics taking values over a commu- 151 tative integral quantale [25], of which we recall the deﬁnition below. 152 ▶Deﬁnition 1. A commutative integral quantale is a triple (Q, +, ≤) where: 153 (Q, ≤) is a complete lattice, 154 (Q, +) is a commutative monoid, 155 + commutes with arbitrary infs, 156 + commutes with arbitrary infs, 156 the least element of Q is neutral for +. 157 the least element of Q is neutral for +. 157 For readability, we have we have reversed the ordering with respect to the conventional 158 deﬁnition, so that for example, ([0, ∞], +, ≤) is a commutative integral quantale whose least 159 element is 0 (as opposed to “([0, ∞], +, ≥) is a commutative integral quantale whose largest 160 element is 0”, which is what we would get with the usual deﬁnition). It is straightforward to 161 check that for all commutative integral quantales Q, R, the product monoid Q × R equipped 162 with the product ordering is also a commutative integral quantale. In addition, for all posets 163 X, the set of monotone functions from X to Q, equipped with the pointwise monoid operation 164 and the pointwise ordering, is also a commutative integral quantale. Another example of 165 commutative integral quantale is given by the lattice of ideals of any commutative ring, with 166 the product of ideals as the monoid operation. 167 We recall now the deﬁnition of a generalized partial metric space: 168 ▶Deﬁnition 2. A generalized partial metric space (in short, GPMS) is the data of a set X, 169 a commutative integral quantale Q and a function d : X × X →Q such that: 170 ▶Deﬁnition 2. 3 Approximate Programs for the Simply-Typed λ-Calculus over Real 1 To illustrate our construction, we start from a relatively concrete example: we consider a 192 simply-typed lambda calculus with a base type Real and primitives for real numbers, and we 193 follow the plan outlined in the introduction, which yields for each simple type a notion of 194 approximate value, approximate function, diameter and distance between programs. Most 195 deﬁnitions are straightforward and intuitive: the interesting, not immediately obvious point 196 is that our construction does yield a partial metric on each type. 197 Simple types are deﬁned as follows: Real is a simple type; if A and B are simple 198 types, then A →B and A × B are simple types. For all n > 0, we ﬁx a set Fn of 199 functions from Rn to R. We consider the usual Curry-style simply-typed λ-calculus over 200 the types deﬁned above (the left and right projection are denoted by πL : A × B →A and 201 πR : A × B →B respectively, and the constructor for pairs by ⟨−, −⟩), enriched with the 202 following constants: for all r ∈R, a constant r : Real; for all n > 0 and all f ∈Fn, a constant 203 f : Real →. . . →Real →Real. We call this calculus STλC(Fn), and its terms are simply 204 called terms. We write t[x1 := u1, . . . , xn := un] to denote the simultaneous substitution 205 of u1, . . . , un for x1, . . . , xn in t. For all types A, we denote by ΛA the set of closed terms 206 of type A. The relation of β-reduction is enriched with the following rule, extended to all 207 contexts: for all n > 0, f ∈Fn, and r1, . . . , rn ∈R, fr1 . . . rn →β s, where s = f(r1, . . . , rn). 208 By standard arguments [1], this calculus has the properties of subject reduction, conﬂuence 209 and strong normalisation. 210 ▶Remark 3. The class of real-valued functions which can be computed in STλC(Fn) depends 211 on the choice we make for Fn. G. Geoﬀroy and P. Pistone 184 For a more telling and somewhat archetypal example, take any set X and consider the set 178 X≤ω of all sequences of elements of X indexed by an ordinal less than or equal to ω. For all 179 such sequences s, t, let d(s, t) = 2−n ∈[0, ∞], where n is the length of the largest common 180 preﬁx to s and t: one can check that (X≤ω, [0, ∞], d) is indeed a generalized partial metric 181 space. In fact, if we interpret the preﬁxes of a sequence as pieces of partial information, 182 then we have d(s, s) = d(s, t) if and only if t is a reﬁnement of s (i.e. if it contains more 183 information), and d(s, s) = 0 if and only if s is total (i.e. if it cannot be reﬁned). 184 One can check that for all partial metric spaces (X, Q, dX) and (Y, R, dY ), (X × 185 Y, Q × R, dX×Y ) is a generalized partial metric space, where dX×Y ((x1, y1), (x2, y2)) = 186 (dX(x1, x2), dY (y1, y2)). However, in general, it is not clear how one should deﬁne a partial 187 metric on a function space. In Section 3.2 we introduce a construction to obtain partial 188 metric spaces on function spaces by generalizing some properties of the standard diameter 189 function on sets of real numbers. 190 CVIT 2016 A Partial Metric Semantics of Higher-Order Programs 23:6 3 Approximate Programs for the Simply-Typed λ-Calculus over Real 1 With suitable choices (see for instance [40, 17, 18]) one can 212 obtain that all programs of type Real →Real compute continuous functions2, that all such 213 programs are integrable over closed intervals, or that all such programs are continuously 214 diﬀerentiable. 215 ▶Remark 3. The class of real-valued functions which can be computed in STλC(Fn) depends 211 on the choice we make for Fn. With suitable choices (see for instance [40, 17, 18]) one can 212 obtain that all programs of type Real →Real compute continuous functions2, that all such 213 programs are integrable over closed intervals, or that all such programs are continuously 214 diﬀerentiable. 215 In addition to the usual notion of β-equivalence between terms of STλC(Fn), we will 216 exploit also a stronger equivalence: given two closed terms t, u of type A, we say that t and u 217 are observationally equivalent and write t ≈A u if for all terms C such that x : A ⊢C : Real 218 is derivable, C[x := t] is β-equivalent to C[x := u] (which amounts to saying that they both 219 β-reduce to the same real number). It is clear that observational equivalence is a congruence 220 and that two β-equivalent terms are always observationally equivalent. 221 2 Note that for this to be possible, Fn cannot contain the identity function over Real. JRealK = {{t ∈ΛReal \| ∃r ∈I, t →∗ β r} \| I ⊆R is a compact interval or ∅or R}, 230 JA × BK = {a × b \| a ∈JAK, b ∈JBK}, where a × b = {t ∈ΛA×B \| πLt ∈a and πRt ∈b}, 231 JA →BK = {{t ∈ΛA→B \| ∀u ∈ΛA, tu ∈I(u)} \| I : ΛA →JBK}. 232 3.1 Approximate Values and Approximate Programs 222 The ﬁrst step of our construction for STλC(Fn) is to associate to each simple type A a set 223 JAK whose elements are certain sets of programs of type A that we call approximate values of 224 type A. A closed term t ∈ΛA represents a program with return type A and no parameters, 225 so an approximate value can be thought of as a speciﬁcation of a program with return type 226 A and no parameters up to a certain degree of error or approximation. 227 For each simple type A, the set of approximate values JAK ⊆P(ΛA) is deﬁned inductively 228 as follows: 229 JRealK = {{t ∈ΛReal \| ∃r ∈I, t →∗ β r} \| I ⊆R is a compact interval or ∅or R}, 230 23:7 G. Geoﬀroy and P. Pistone sin(x) + 1 cos(x) −1 sin(x + 1) (a) λx. sin(x + 1) is in [λx. sin(x) + 1, λx. cos(x) + 1]Real→Real. −1 1 −1 1 ε δ u[x] t[x] • • • • • r (b) ε = (∂(u) ◦∂(t))([−1, 1]) is bigger than δ = ∂(u ◦t)([−1, 1]) = [r, r]. Figure 4 Examples of functional approximate values and of approximate programs. sin(x) + 1 cos(x) −1 sin(x + 1) (a) λx. sin(x + 1) is in [λx. sin(x) + 1, λx. cos(x) + 1]Real→Real. −1 1 −1 1 ε δ u[x] t[x] • • • • • r (b) ε = (∂(u) ◦∂(t))([−1, 1]) is bigger than δ = ∂(u ◦t)([−1, 1]) = [r, r]. (b) ε = (∂(u) ◦∂(t))([−1, 1]) is bigger than δ = ∂(u ◦t)([−1, 1]) = [r, r]. (a) λx. sin(x + 1) is in [λx. sin(x) + 1, λx. cos(x) + 1]Real→Real. (a) λx. sin(x + 1) is in [λx. sin(x) + 1, λx. cos(x) + 1]Real→Real. Figure 4 Examples of functional approximate values and of approximate programs. The approximate values of type Real are sets of closed programs of type Real which 233 essentially coincide with the compact intervals of R, plus the empty set and R itself. An 234 approximate value in JA × BK is a “rectangle” a × b, with a ∈JAK and b ∈JBK, while an 235 approximate value in JA →BK is uniquely determined by a function I from closed terms 236 u ∈ΛA to approximate values I(u) ∈JBK. 3.1 Approximate Values and Approximate Programs 222 237 For example, any two terms t, u ∈ΛReal with normal forms q, r ∈R induce an approximate 238 value [t, u]Real = {v ∈ΛReal \| v →∗ β s ∧(q ≤s ≤r ∨q ≥s ≥r)} of type Real. Similarly, any 239 two terms t, u ∈ΛReal→Real induce an approximate value [t, u]Real→Real = {v ∈ΛReal→Real \| 240 ∀r ∈ΛReal vr ∈[tr, ur]Real}. For instance, if t = λx. sin(x) + 1 and u = λx. cos(x) −1, then 241 [t, u]Real→Real contains all closed terms corresponding to maps oscillating between sin(x) + 1 242 and cos(x) + 1 (e.g. the program λx. sin(x + 1), as illustrated in Fig. 4a). 243 For all A, the set JAK is a a subset of P(ΛA) closed under arbitrary intersections. We 244 deduce that JAK has arbitrary meets (given by intersections) and arbitrary joins W i∈I ai = 245 T{a ∈JAK \| ∀i ∈I ai ⊆a}, and thus JAK is a complete lattice. In particular, for all t ∈ΛA, 246 there is a least element of JAK that contains t, which will be denoted by t. One can check 247 that t = u if and only if t ≈A u. 248 Monotone functions from approximate values to approximate values represent approximate 249 programs. They behave like a model of the simply-typed λ-calculus in a weak sense, namely: 250 for all monotone functions ⃗α 7→c[⃗α] : JA1K × . . . × JAnK →JB →CK and ⃗α 7→b[⃗α] : 251 JA1K × . . . × JAnK →JBK, we can deﬁne a monotone function ⃗α 7→(c[⃗α] b[⃗α]) = sup{vu \| 252 v ∈c[⃗α], u ∈b[⃗α]} : JA1K × . . . × JAnK →JCK, 253 programs. They behave like a model of the simply typed λ calculus in a weak sense, namely: 250 for all monotone functions ⃗α 7→c[⃗α] : JA1K × . . . × JAnK →JB →CK and ⃗α 7→b[⃗α] : 251 JA1K × . . . × JAnK →JBK, we can deﬁne a monotone function ⃗α 7→(c[⃗α] b[⃗α]) = sup{vu \| 252 v ∈c[⃗α], u ∈b[⃗α]} : JA1K × . . . × JAnK →JCK, 253 p g y p y yp , y for all monotone functions ⃗α 7→c[⃗α] : JA1K × . . . × JAnK →JB →CK and ⃗α 7→b[⃗α] : 251 JA1K × . . . Beyond theoretical aspects (which will be made clearer in Section 5) Proposition 4 is also 267 j̸ i j and these two constructions are weakly compatible with β-reduction and η-expansion: 257 Proof. Without loss of generality, we can assume n = 0. Let v ∈λβ. c[β] and u ∈b. By 264 deﬁnition, tu ∈c[u], so tu ⊆c[u] ⊆c[b]. Therefore, (λβ. c[β]) b ⊆b. Let v ∈d. For all 265 u ∈ΛB, by deﬁnition, vu ∈du. Therefore, v ∈λβ. d β. ◀ 266 A Partial Metric Semantics of Higher-Order Programs 23:8 important in practice because it implies that if we compute an approximation of a program 268 from approximations of its parts and then simplify the resulting approximate program using 269 β-reduction and η-expansion, what we obtain is still a valid approximation of the original 270 program. 271 important in practice because it implies that if we compute an approximation of a program 268 from approximations of its parts and then simplify the resulting approximate program using 269 β-reduction and η-expansion, what we obtain is still a valid approximation of the original 270 program. 271 We can deﬁne a weak embedding from terms into approximate programs, by mapping 272 each term to its tightest approximation: for all terms t such that α1 : A1, . . . , αn : An ⊢t : B, 273 we deﬁne a monotone function ∂(t) : JA1K × · · · × JAnK →JBK by ∂(t)(a1, . . . , an) = 274 sup{tu1 . . . un \| u1 ∈a1, . . . , un ∈an}. 275 ▶Remark 5. The map ∂is constant on classes of observational equivalence, and one can 276 check that it is is weakly compatible with the constructions of the λ-calculus, in particular: 277 ∂(αi)(a1, . . . , an) = ai, 278 ∂(tu)(a1, . . . , an) ⊆∂(t)(a1, . . . , an) ∂(u)(a1, . . . , an), 279 ∂(λβ )( ) λβ ∂( )(β ) ∂(λβ.t)(a1, . . . , an) ⊆λβ. ∂(t)(β, a1, . . . , an). 280 This map ∂(t) can be taken as a measure of the sensitivity of t, as it maps an interva 281 This map ∂(t) can be taken as a measure of the sensitivity of t, as it maps an interval 281 a, that is a quantiﬁably uncertain input, to a quantiﬁably uncertain output ∂(t)(a). For 282 instance, if we take the term t[x] = sin(x) + 1 above, then ∂(t) : JRealK →JRealK sends the 283 interval [−π, π]Real into [0, 2]Real. 284 ▶Remark 6. When composing two maps ∂(t) and ∂(u), we might obtain a worse approxima- 285 tion than by computing ∂(t[u/x]) directly. For instance, let t[x] and u[x] be, respectively, 286 the discontinuous and Gaussian functions illustrated in Fig. 4b. A Partial Metric Semantics of Higher-Order Programs If a is the interval [−1, +1], 287 then ∂(t)(a) = [−1, 1], and since u[x := −1] = u[x := 1] ≃β r for some 0 < r < 1, we deduce 288 that ∂(u)(∂(t)(a)) = [−1, 1] ⊋[r, r] = ∂(u[t/x])(a). 289 3.1 Approximate Values and Approximate Programs 222 × JAnK →JBK, we can deﬁne a monotone function ⃗α 7→(c[⃗α] b[⃗α]) = sup{vu \| 252 v ∈c[⃗α], u ∈b[⃗α]} : JA1K × . . . × JAnK →JCK, 253 for all monotone functions ⃗α 7→c[⃗α] : JA1K × . . . × JAnK →JCK and all i ≤n, we can 254 deﬁne a monotone function (αj)j̸=i 7→(λαi. c[⃗α]) = {v ∈ΛAi→C \| ∀ti ∈ΛAi, vti ∈ 255 c[α1, . . . , ti, . . . , αn]} : Q j̸=iJAjK →JAi →CK, 256 for all monotone functions ⃗α 7→c[⃗α] : JA1K × . . . × JAnK →JCK and all i ≤n, we can 254 deﬁne a monotone function (αj)j̸=i 7→(λαi. c[⃗α]) = {v ∈ΛAi→C \| ∀ti ∈ΛAi, vti ∈ 255 c[α1, . . . , ti, . . . , αn]} : Q j̸=iJAjK →JAi →CK, 256 [ , , , , ]} Q j̸=iJ jK J K, and these two constructions are weakly compatible with β-reduction and η-expansion: 257 j̸ i j and these two constructions are weakly compatible with β-reduction and η-expansion: 257 ▶Proposition 4. For all monotone functions (⃗α, β) 7→c[⃗α, β] : JA1K×. . .×JAnK×JBK →JCK 258 and ⃗α 7→b[⃗α] : JA1K × . . . × JAnK →JBK, (⃗α 7→(λβ. c[⃗α, β]) b[⃗α]) ≤(⃗α 7→c[⃗α, b[⃗α]]), and 259 for all monotone functions ⃗α 7→d[⃗α] : JA1K × . . . × JAnK →JB →CK, (⃗α 7→λβ. d[⃗α] β) ≥ 260 (⃗α 7→d[⃗α]), where functions are ordered by pointwise inclusion. In other words, on approxi- 261 mate programs, β-reduction and η-expansion discard information, and conversely β-expansion 262 and η-reduction recover some information. 263 Proof. Without loss of generality, we can assume n = 0. Let v ∈λβ. c[β] and u ∈b. By 264 deﬁnition, tu ∈c[u], so tu ⊆c[u] ⊆c[b]. Therefore, (λβ. c[β]) b ⊆b. Let v ∈d. For all 265 u ∈ΛB, by deﬁnition, vu ∈du. Therefore, v ∈λβ. d β. ◀ 266 Proof. Without loss of generality, we can assume n = 0. Let v ∈λβ. c[β] and u ∈b. By 264 deﬁnition, tu ∈c[u], so tu ⊆c[u] ⊆c[b]. Therefore, (λβ. c[β]) b ⊆b. Let v ∈d. For all 265 u ∈ΛB, by deﬁnition, vu ∈du. Therefore, v ∈λβ. d β. ◀ 266 Beyond theoretical aspects (which will be made clearer in Section 5) Proposition 4 is also 267 CVIT 2016 3.2 A Partial Metric on Each Type 290 So far, we have associated each type A of STλC(Fn) with a complete lattice JAK ⊆P(ΛA) 291 of approximate values of type A, and each typed program t : A →B with an approximate 292 program ∂(t) (in fact, a monotone function) from approximate values of type A to approximate 293 values of type B. We will now exploit this structure to deﬁne, for each type A of STλC(Fn), 294 a generalized partial metric on the closed (exact) programs of type A. 295 The ﬁrst step is to deﬁne, for every simple type A, a commutative integral quantale 296 (LAM, ≤A, +A) of distances of type A: 297 The ﬁrst step is to deﬁne, for every simple type A, a commutative integral quantale 296 (LAM, ≤A, +A) of distances of type A: 297 (LAM, ≤A, +A) of distances of type A: 297 (LAM, ≤A, +A) of distances of type A: 297 (LRealM, ≤Real, +Real) = ([0, ∞], ≤, +), 298 (LRealM, ≤Real, +Real) = ([0, ∞], ≤, +), 298 LA × BM = LAM × LBM, 299 LA →BM = Poset(JAK, LBM). 300 LA →BM = Poset(JAK, LBM). 300 where, for two posets Q, R, Poset(Q, R) denotes the set of monotone functions from Q to R. 301 Observe that the quantale LA →BM is a set of functions over the approximate values of A. 302 where, for two posets Q, R, Poset(Q, R) denotes the set of monotone functions from Q to R. 301 Observe that the quantale LA →BM is a set of functions over the approximate values of A Observe that the quantale LA →BM is a set of functions over the approximate values of A. 302 For all simple types A, we now deﬁne a distance function dA : ΛA × ΛA →LAM: 303 ( ) \| \| For all simple types A, we now deﬁne a distance function dA : ΛA × ΛA →LAM: 303 L M dReal(t, u) = \|r −s\|, where r, s are the unique elements of R such that t →∗ β r and u 304 dA×B(t, u) = (dA(πLt, πLu), dB(πRt, πRu)), 305 dA×B(t, u) = (dA(πLt, πLu), dB(πRt, πRu)), 305 dA→B(t, u) = a 7→sup {dB(rv, sw) \| r, s ∈{t, u}, v, w ∈a}. 306 dA→B(t, u) = a 7→sup {dB(rv, sw) \| r, s ∈{t, u}, v, w ∈a}. G. Geoﬀroy and P. Pistone G. Geoﬀroy and P. Pistone a b δ(a ∪b) δ(a ∩b) δ(a) δ(b) Figure 5 The diameter function is modular over intersecting real intervals: diam(a∪b)+diam(a∩ b) = diam(a) + diam(b) for all a, b ∈[R] such that a ∩b ̸= ∅. This property is at the heart of our generalization of diameters. Observe that this property fails when a ∩b is empty. a b δ(a ∪b) δ(a ∩b) δ(a) δ(b) Figure 5 The diameter function is modular over intersecting real intervals: diam(a∪b)+diam(a∩ b) = diam(a) + diam(b) for all a, b ∈[R] such that a ∩b ̸= ∅. This property is at the heart of our generalization of diameters. Observe that this property fails when a ∩b is empty. by δA(a) = sup{dA(t, u) \| t, u ∈a}. The key to our objective will be to prove that δA is sub- 313 modular on intersecting approximate values (henceforth, quasi-sub-modular – see Proposition 314 7): this generalizes the fact that, on the (real-valued) metric space R, the diameter is modular 315 over intersecting closed intervals (see Fig. 5). 316 First, one can check that for all t, u ∈ΛA, δA t ∨u = dA(t, u), and that: 317 δReal(a) = sup{s −r \| s, r ∈R such that s, r ∈a}, 318 δA×B(p) = δA sup πLt \| t ∈p δB sup πRt \| t ∈p , 319 δA→B(b) = a 7→δB sup vt \| t ∈a, v ∈b . 320 δA→B(b) = a 7→δB sup vt \| t ∈a, v ∈b . 320 This leads then to the following: 321 ▶Proposition 7 (δA is quasi-sub-modular). For all simple types A and all a, b ∈JAK such 322 that a ∧b ̸= ∅, δ(a ∧b) + δ(a ∨b) ≤δ(a) + δ(b). 323 ▶Proposition 7 (δA is quasi-sub-modular). For all simple types A and all a, b ∈JAK such 322 that a ∧b ̸= ∅, δ(a ∧b) + δ(a ∨b) ≤δ(a) + δ(b). 323 Proof. We proceed by induction on types. 324 Proof. We proceed by induction on types. 324 Let a, b ∈JRealK such that a ∧b ̸= ∅. 3.2 A Partial Metric on Each Type 290 306 It would be tempting to deﬁne dA→B(t, u)(a) simply as sup {dB(tv, uw) \| v, w ∈a}, but 307 then the axiom “dA→B(t, t) ≤dA→B(t, u)” of partial metric spaces would fail. 308 It would be tempting to deﬁne dA→B(t, u)(a) simply as sup {dB(tv, uw) \| v, w ∈a}, but 307 then the axiom “dA→B(t, t) ≤dA→B(t, u)” of partial metric spaces would fail. 308 It would be tempting to deﬁne dA→B(t, u)(a) simply as sup {dB(tv, uw) \| v, w ∈a}, but 307 then the axiom “dA B(t t) ≤dA B(t u)” of partial metric spaces would fail 308 It would be tempting to deﬁne dA→B(t, u)(a) simply as sup {dB(tv, uw) \| v, w ∈a}, but 307 The maps dA are clearly compatible with observational equivalence (i.e. if a ≈A a′ and 309 b ≈A b′, then dA(a, b) = dA(a′, b′)). 310 The maps dA are clearly compatible with observational equivalence (i.e. if a ≈A a′ and 309 b ≈A b′, then dA(a, b) = dA(a′, b′)). 310 The maps dA are clearly compatible with observational equivalence (i.e. if a ≈A a′ and 309 b ≈A b′, then dA(a, b) = dA(a′, b′)). 310 Our objective is now to prove that (ΛA/ ≈A, LAM, dA) is a generalized partial metric space. 311 A δ JAK LAM Our objective is now to prove that (ΛA/ ≈A, LAM, dA) is a generalized partial metric space. 311 To this end, we deﬁne for all simple types A a monotone diameter function δA : JAK →LAM 312 Our objective is now to prove that (ΛA/ ≈A, LAM, dA) is a generalized partial metric space. 1 To this end we deﬁne for all simple types A a monotone diameter function δ : JAK →LAM Our objective is now to prove that (ΛA/ ≈A, LAM, dA) is a generalized partial metric space 311 To this end, we deﬁne for all simple types A a monotone diameter function δA : JAK →LAM 312 23:9 G. Geoﬀroy and P. Pistone As a result, ) ( ) ( )) ( ( ) ( ) ( ) ( )) ( ) ( ) Let f, g ∈JA →BK and a ∈JAK. For all h ∈JA →BK, let ha = sup{vt \| v ∈h, t ∈a}. 336 One can check that (f ∧g)a ⊆(fa) ∧(ga) and (f ∨g)a = (fa) ∨(ga). As a result, 337 (δ(f ∧g)+δ(f ∨g))(a) ≤δ((fa)∧(ga))+δ((fa)∨(ga)) ≤δ(fa)+δ(ga) = (δ(f)+δ(g))(a). ◀ 338 Let f, g ∈JA →BK and a ∈JAK. For all h ∈JA →BK, let ha = sup{vt \| v ∈h, t ∈a} 336 One can check that (f ∧g)a ⊆(fa) ∧(ga) and (f ∨g)a = (fa) ∨(ga). As a result, 337 Let f, g ∈JA →BK and a ∈JAK. For all h ∈JA →BK, let ha = sup{vt \| v ∈h, t ∈a}. 336 One can check that (f ∧g)a ⊆(fa) ∧(ga) and (f ∨g)a = (fa) ∨(ga). As a result, 337 (δ(f ∧g)+δ(f ∨g))(a) ≤δ((fa)∧(ga))+δ((fa)∨(ga)) ≤δ(fa)+δ(ga) = (δ(f)+δ(g))(a). ◀ 338 One can check that (f ∧g)a ⊆(fa) ∧(ga) and (f ∨g)a = (fa) ∨(ga). As a result, 337 (δ(f ∧g)+δ(f ∨g))(a) ≤δ((fa)∧(ga))+δ((fa)∨(ga)) ≤δ(fa)+δ(ga) = (δ(f)+δ(g))(a). ◀ 338 ( ) ( ) ( ) ( ) ( ) ( ) (δ(f ∧g)+δ(f ∨g))(a) ≤δ((fa)∧(ga))+δ((fa)∨(ga)) ≤δ(fa)+δ(ga) = (δ(f)+δ(g))(a). ◀ 338 It is well-known [39] that any function δ : L →[0, ∞] on a lattice L that is monotone 339 and sub-modular induces a pseudo-metric d : L × L →[0, ∞] by letting d∗(a, b) = 2δ(a ∨ 340 b) −δ(a) −δ(b). In fact, one can decompose this construction: ﬁrst, one deﬁnes a partial 341 pseudometric d on L by d(a, b) = δ(a ∨b), and then d∗is just the distance given by equation 342 (⋆): d∗(a, b) = 2d(a, b) −d(a, a) −d(b, b). We can use this way of reasoning to establish that 343 the maps dA are indeed partial metrics: 344 ▶Corollary 8. For all simple types A, (ΛA/ ≈A, LAM, dA) is a generalized partial metric 345 space, that is to say: 346 ▶Corollary 8. For all simple types A, (ΛA/ ≈A, LAM, dA) is a generalized partial metric 345 space, that is to say: 346 1. for all t, u ∈ΛA, dA(t, t) ≤dA(t, u), 347 1. for all t, u ∈ΛA, dA(t, t) ≤dA(t, u), 347 2. G. Geoﬀroy and P. Pistone Let I = {r ∈R \| r ∈a} and J = {s ∈R \| s ∈b}: 325 then I (respectively, J, I ∩J, I ∪J) is either R or a non-empty compact interval of R, 326 and its length in the usual sense is equal to δReal(a) (respectively, δReal(b), δReal(a ∧b), 327 δReal(a ∨b)). Note that the only reason we know that I ∪J is an interval is because 328 a ∧b ̸= ∅implies I ∩J ̸= ∅. The length of an interval of R is equal to its Lebesgue measure, 329 therefore length(I ∩J) +length(I ∪J) = length(I) +length(J), so δReal(a∧b) +δReal(a∨b) = 330 δReal(a) + δReal(b). 331 Let a, b ∈JAL ×ARK such that a∧b ̸= ∅. For all c ∈JAL ×ARK, let cL = sup{πLt \| t ∈c} 332 and cR = sup{πRt \| t ∈c}. One can check that (a ∧b)L = aL ∧bL, (a ∧b)R = aR ∧bR, 333 (a ∨b)L = aL ∨bL and (a ∨b)R = aR ∨bR, so δ(a ∧b) + δ(a ∨b) = (δ(aL ∧bL) + δ(aL ∨ 334 bL), δ(aR ∧bR) + δ(aR ∨bR)) ≤(δ(aL) + δ(bL), δ(aR) + δ(bR)) = δ(a) + δ(b). 335 Let a, b ∈JAL ×ARK such that a∧b ̸= ∅. For all c ∈JAL ×ARK, let cL = sup{πLt \| t ∈c} 332 and cR = sup{πRt \| t ∈c}. One can check that (a ∧b)L = aL ∧bL, (a ∧b)R = aR ∧bR, 333 (a ∨b)L = aL ∨bL and (a ∨b)R = aR ∨bR, so δ(a ∧b) + δ(a ∨b) = (δ(aL ∧bL) + δ(aL ∨ 334 bL), δ(aR ∧bR) + δ(aR ∨bR)) ≤(δ(aL) + δ(bL), δ(aR) + δ(bR)) = δ(a) + δ(b). 335 ) ( ) ( )) ( ( ) ( ) ( ) ( )) ( ) ( ) Let f, g ∈JA →BK and a ∈JAK. For all h ∈JA →BK, let ha = sup{vt \| v ∈h, t ∈a} 336 ) ( ) ( )) ( ( ) ( ) ( ) ( )) ( ) ( ) Let f, g ∈JA →BK and a ∈JAK. For all h ∈JA →BK, let ha = sup{vt \| v ∈h, t ∈a}. One can check that (f ∧g)a ⊆(fa) ∧(ga) and (f ∨g)a = (fa) ∨(ga). 2. for all t, u ∈ΛA, if dA(t, t) = dA(t, u) = dA(u, u), then t ≈A u, 348 1. for all t, u ∈ΛA, dA(t, t) ≤dA(t, u), 347 23:10 A Partial Metric Semantics of Higher-Order Programs 355 A( ) A( ) A( ), , A The triangular inequality is an immediate consequence of the quasi-sub-modularity of δA: 356 d(t, v)+d(u, u) = δ(t∨v)+δ(u) ≤δ((t∨u)∨(u∨v))+δ((t∨u)∧(u∨v)) ≤δ(t∨u)+δ(u∨v) = 357 d(t, u) + d(u, v). ◀ 358 The triangular inequality is an immediate consequence of the quasi-sub-modularity of δA: 356 d(t, v)+d(u, u) = δ(t∨v)+δ(u) ≤δ((t∨u)∨(u∨v))+δ((t∨u)∧(u∨v)) ≤δ(t∨u)+δ(u∨v) = 357 d(t, u) + d(u, v). ◀ 358 d(t, u) + d(u, v). 358 G. Geoﬀroy and P. Pistone for all t, u ∈ΛA, if dA(t, t) = dA(t, u) = dA(u, u), then t ≈A u, 348 CVIT 2016 4 Computing Program Distances using Partial Metrics 359 In the previous section we showed how to associate each simple type A with a partial metric 360 dA over the closed terms of type A. We now illustrate through a few basic examples how 361 the higher-order and metric features of this semantics can be used to formalize contextual 362 reasoning about program diﬀerences. 363 To make our examples more realistic, we will consider some natural extensions of 364 STλC(Fn). It is not diﬃcult to see that all constructions from Section 3 still work if 365 we add to STλC(Fn) some new base types. For example, we can add to our language a type 366 Nat for natural numbers, indicating for each n ∈N, the corresponding normal forms of Nat 367 as n. A natural choice is to let JNatK = {{t \| ∃n ∈a t ⇝n} \| a ﬁnite subset of N or a = N}, 368 LNatM = [0, ∞] and dNat(t, u) = \|n −m\|, where t →∗ β n and u →∗ β m. 369 β β Moreover, our constructions scale well also to extensions of STλC(Fn) obtained by adding 370 new program constructors, as soon as these do not compromise the existence and uniqueness 371 of normal forms (since the fact that closed programs of type Real have a normal form plays 372 an important role to deﬁne JRealK). For instance, if we suppose that all programs of type 373 Real →Real in STλC(Fn) are either diﬀerentiable or integrable (see Remark 3), we can 374 consider extension of STλC(Fn) with diﬀerential or integral operators, as in Real PCF [17, 18]. 375 We start with a classical example from approximate computing that we adapt from [44]. 376 ▶Example 9 (Loop perforation). We work in the extension of STλC(Fn) with a type Nat. 377 We discuss a transformation that replaces a program t which performs n iterations by a 378 program which only performs the iterations 0 k 2k 3k each repeated k times Suppose t : (A × A →A) →Nat →(A →A) →A, for n ≥1, is a term such that 380 thnf computes the n-times iteration of h as follows: th0f = h⟨f0, f0⟩and th(n + 1)f = 381 h⟨thnf, f(n + 1)⟩. 23:10 A Partial Metric Semantics of Higher-Order Programs 23:10 3. for all t, u ∈ΛA, dA(t, u) = dA(u, t), 349 3. for all t, u ∈ΛA, dA(t, u) = dA(u, t), 349 4. for all t, u, v ∈ΛA, dA(t, v) + dA(u, u) ≤dA(t, u) + dA(u, v). 350 4. for all t, u, v ∈ΛA, dA(t, v) + dA(u, u) ≤dA(t, u) + dA(u, v). 350 4. for all t, u, v ∈ΛA, dA(t, v) + dA(u, u) ≤dA(t, u) + dA(u, v). 350 Proof. As mentioned above, for all t, u ∈ΛA, dA(t, u) = δA(t ∨u), which immediately gives 351 point 3. Since δA is monotone and t ∨t ≤t ∨u, we also get point 1. 352 Proof. As mentioned above, for all t, u ∈ΛA, dA(t, u) = δA(t ∨u), which immediately gives 351 point 3. Since δA is monotone and t ∨t ≤t ∨u, we also get point 1. 352 Proof. As mentioned above, for all t, u ∈ΛA, dA(t, u) = δA(t ∨u), which immediately gives 351 point 3. Since δA is monotone and t ∨t ≤t ∨u, we also get point 1. 352 Proof. As mentioned above, for all t, u ∈ΛA, Proof. As mentioned above, for all t, u ∈ΛA, dA(t, u) = δA(t ∨u), which immediately gives 351 point 3. Since δA is monotone and t ∨t ≤t ∨u, we also get point 1. 352 One can check (by induction on types) that the restriction of δA to the ideal generated 353 by the t (for t ∈ΛA) is strictly monotone. Therefore, if dA(t, t) = dA(t, u) = dA(u, u), i.e. 354 δA(t) = δA(t ∨u) = δA(u), then t = t ∨u = u, so t ≈A u. 355 One can check (by induction on types) that the restriction of δA to the ideal generated 353 by the t (for t ∈ΛA) is strictly monotone. Therefore, if dA(t, t) = dA(t, u) = dA(u, u), i.e 354 δA(t) = δA(t ∨u) = δA(u), then t = t ∨u = u, so t ≈A u. 355 One can check (by induction on types) that the restriction of δA to the ideal generated 353 by the t (for t ∈ΛA) is strictly monotone. Therefore, if dA(t, t) = dA(t, u) = dA(u, u), i.e. 354 δA(t) = δA(t ∨u) = δA(u), then t = t ∨u = u, so t ≈A u. G. Geoﬀroy and P. Pistone 410 For example, if t is the function t = λx. sin(x), and a is an interval of 0, then using 408 standard analytic reasoning we can compute a bound dReal→Real(t, Tn(t, 0))(a) ≤δReal(a)n+1 (n+1)! , 409 which tends to 0 as the diameter of a tends to 0. 410 Observe that if, instead, we used the sup-distance dsup(t, u) = sup{dReal(tr, ur) \| r ∈ 411 ΛReal}, then we could not reason as above, since the sup-distance between λx. sin(x) and its 412 truncated Taylor polynomials is inﬁnite. 413 ▶Example 11 (Integral approximation). We now assume that all functions in Fn are integrable 414 and that we have (see [18]) at our disposal a program λfx.I[0,x](f) : (Real →Real) →Real → 415 Real such that I[0,r](t) computes (a precise enough approximation of) the deﬁnite integral 416 R \|r\| 0 tx dx. In many contexts we might prefer to replace the expensive computation of 417 I[0,r](t) by the (more economical but less precise) computation of a ﬁnite Riemann sum 418 Rn [0,r](t) = Pn i=1(txi) · \|r\|/n, where xi = i · \|r\|/n. 419 Suppose now that, in order to approximate the integral of some computationally expensive 420 program t on [0, r], we replace t by some more eﬃcient program u which, over [0, r], is very 421 close to t. Let εt(r) indicate the distance between the true integral of t over [0, r] and Rn [0,r](t), 422 and moreover let ηt,u(r) be the diameter of ∂(t)([0, r]) ∨∂(u)([0, r]). 423 Using the metric structure of Real we can then bound the error we incur in by replacing 424 the true integral of t with the Riemann sum of u. In fact, by standard calculation we can 425 compute the bound dReal(Rn [0,r](t), Rn [0,r](u)) ≤dReal→Real(t, u)([0, r]) · \|r\| = ηt,u(r) · \|r\|. Then, 426 using the triangular inequality of the standard metric on Real we deduce 427 dReal(I[0,r](t), Rn [0,r](u)) ≤dReal(I[0,r](t), Rn [0,r](t)) + dReal(R[0,r](t), Rn [0,r](u)) 428 ≤εt(r) + ηt,u(r) · \|r\| 429 430 429 430 Using the partial metric on Real →Real, we can also derive a bound expressing how much 431 the error above is sensitive to changes of r. First, using standard analytic techniques (under 432 suitable assumptions for t and its derivatives) one can ﬁnd a program v : Real →Real such 433 that vr computes an upper bound for εt(r). 4 Computing Program Distances using Partial Metrics 359 Let Perfk(t), the k-th perforation of t, be the program (Perfk(t))hnf = 382 t(λx.(h(k)x))⌊n⌋k(λx.f(x ∗k), where ⌊n⌋k indicates the least m ≤n such that m is divisible 383 by k, and x ∗k is the multiplication of x by k. 384 To compute the distance dA(vn, wn) between vn = thnf and its perforation wn = 385 Perfk(t)hnf we can reason as follows: 386 To compute the distance dA(vn, wn) between vn = thnf and its perforation wn = 385 Perfk(t)hnf we can reason as follows: 386 i. vn performs n-iterations while wn performs k⌊n⌋k ≤n iterations, and we can compute 387 dA(vn, v(k⌊n⌋k)) as the diameter of ∂(t)∂(h)([k⌊n⌋k, n]Nat)∂(f). 388 i. vn performs n-iterations while wn performs k⌊n⌋k ≤n iterations, and we can compute 387 dA(vn, v(k⌊n⌋k)) as the diameter of ∂(t)∂(h)([k⌊n⌋k, n]Nat)∂(f). 388 ii. If n is divisible by k, then for i ≤n, at the i-th iteration of vn the function f is applied 389 to i, while at the i-th iteration of wn, f is applied to ⌊i⌋k. Now, the error of replacing 390 fi by f⌊j⌋k, with i, j in some a ∈JNatK, is accounted for by the approximate program 391 c[y] = ∂(f)(y −k), where y −k = y ∨{u −k \| u ∈y}. We deduce then that dA(vn, wn) is 392 bounded by the diameter of ∂(t)∂(h)n(λy.c[y]). 393 23:11 G. Geoﬀroy and P. Pistone iii. From the fact that wn = w(k·⌊n⌋k) and the triangular inequality of the partial metric dA 394 we deduce dA(vn, wn) = dA(vn, w(k·⌊n⌋k)) ≤dA(vn, v(k·⌊n⌋k)) + dA(v(k·⌊n⌋k), w(k·⌊n⌋k)) − 395 dA(v(k·⌊n⌋k), v(k·⌊n⌋k)) 396 From facts i.-iii. we deduce an explicit bound for dA(vn, wn) in terms of ∂(t), ∂(f) and n: 397 dA(vn, wn) ≤δA(∂(t)∂(h)([k⌊n⌋k, n]Nat)∂(f)) + δA(∂(t)∂(h)n(λy.∂(f)(y −k))) −δA(∂(t)∂(h)n∂(f)). 398 We now show how the partial metric semantics can be used to reason about basic 399 approximation techniques from numerical analysis. 400 We now show how the partial metric semantics can be used to reason about basic 399 approximation techniques from numerical analysis. 400 ▶Example 10 (Taylor approximation). We assume that all programs of type Real →Real in 401 STλC(Fn) are diﬀerentiable and that for all n, program t : Real →Real and real number r, 402 we can deﬁne a term Tn(t, r) : Real →Real computing the n-th truncated Taylor polynomial 403 of t at r. The distance dReal→Real(t, Tn(t, 0)) is the map associating an interval a with the 404 diameter of the smallest interval containing the image of a under both t and Tn(t, 0). This 405 value will approximately converge to the self-distance of t when a is a small interval of 0, 406 and will tend to diverge when a contains points which are far enough from 0. 407 ▶Example 10 (Taylor approximation). We assume that all programs of type Real →Real in 401 STλC(Fn) are diﬀerentiable and that for all n, program t : Real →Real and real number r, 402 we can deﬁne a term Tn(t, r) : Real →Real computing the n-th truncated Taylor polynomial 403 of t at r. The distance dReal→Real(t, Tn(t, 0)) is the map associating an interval a with the 404 diameter of the smallest interval containing the image of a under both t and Tn(t, 0). This 405 value will approximately converge to the self-distance of t when a is a small interval of 0, 406 and will tend to diverge when a contains points which are far enough from 0. 407 For example, if t is the function t = λx. sin(x), and a is an interval of 0, then using 408 standard analytic reasoning we can compute a bound dReal→Real(t, Tn(t, 0))(a) ≤δReal(a)n+1 (n+1)! , 409 which tends to 0 as the diameter of a tends to 0. 23:12 A Partial Metric Semantics of Higher-Order Programs partial metric on Real →Real we deduce, for all interval a, the following bound: 435 dReal→Real(λx.I[0,x](t), λx.Rn [0,x](u))(a) 436 dReal→Real(λx.I[0,x](t), λx.Rn [0,x](u))(a) 436 ≤dReal→Real(λx.I[0,x](t), λx.Rn [0,x](t))(a) + dReal→Real(λx.R[0,x](t), λx.Rn [0,x](u))(a) 437 −dReal→Real(λx.R[0,x](t), λx.Rn 0,x](t))(a) 438 ≤dReal→Real(v, v)(a) + dReal→Real(t, u)(a) −dReal→Real(t, t)(a) · δReal(a) 439 440 ≤dReal→Real(v, v)(a) + dReal→Real(t, u)(a) −dReal→Real(t, t)(a) · δReal(a) 439 440 G. Geoﬀroy and P. Pistone Then, using the triangular inequality of the 434 CVIT 2016 Diameter Space Models Over a Cartesian Closed Category An exponential 464 (respectively, a lax-exponential) on C is the data of a map exp from Ob(C × C) to Ob(C) 465 and two families of monotone maps (evW,X,Y : C(W, exp(X, Y )) →C(W × X, Y )) and 466 (λW,X,Y : C(W × X, Y ) →C(W, exp(X, Y ))) such that: 467 evW,X,Y and λW,X,Y are natural with respect to W, 468 for all g ∈C(W × X, Y ), ev(λ(g)) = g (respectively, ev(λ(g)) ≤g), 469 for all f ∈C(W exp(X Y )) f = λ(ev(f)) (respectively f ≤λ(ev(f))) 470 ▶Deﬁnition 12. Let (C, ×, 1) be a cartesian poset-enriched category. An exponential 464 (respectively, a lax-exponential) on C is the data of a map exp from Ob(C × C) to Ob(C) 465 and two families of monotone maps (evW,X,Y : C(W, exp(X, Y )) →C(W × X, Y )) and 466 (λW,X,Y : C(W × X, Y ) →C(W, exp(X, Y ))) such that: 467 ▶Deﬁnition 12. Let (C, ×, 1) be a cartesian poset-enriched category. An exponential 464 (respectively, a lax-exponential) on C is the data of a map exp from Ob(C × C) to Ob(C) 465 and two families of monotone maps (evW,X,Y : C(W, exp(X, Y )) →C(W × X, Y )) and 466 (λW,X,Y : C(W × X, Y ) →C(W, exp(X, Y ))) such that: 467 evW,X,Y and λW,X,Y are natural with respect to W, 468 evW,X,Y and λW,X,Y are natural with respect to W, 468 , , , , for all g ∈C(W × X, Y ), ev(λ(g)) = g (respectively, ev(λ(g)) ≤g), 469 for all f ∈C(W, exp(X, Y )), f = λ(ev(f)) (respectively, f ≤λ(ev(f))). 470 for all f ∈C(W, exp(X, Y )), f = λ(ev(f)) (respectively, f ≤λ(ev(f))). 470 One can check that this deﬁnition makes exp a functor (respectively, a lax-functor) 471 from Ob(Cop × C) to Ob(C) (with exp(f, g) deﬁned as λ(g ◦ev(id) ◦(id ×f))). In addition, 472 this deﬁnition implies that ev and λ are natural, in the sense that ev(exp(α, β) ◦f ◦γ) = 473 β◦ev(f)◦(γ×α) and exp(α, β)◦λ(g)◦γ = λ(β◦g◦(γ×α)) (respectively, lax-natural [24], in the 474 sense that ev(exp(α, β)◦f ◦γ) ≤β◦ev(f)◦(γ×α) and exp(α, β)◦λ(g)◦γ ≤λ(β◦g◦(γ×α))). Diameter Space Models Over a Cartesian Closed Category The examples from the last section relied on the fact that our partial metric semantics scales 442 well to extensions of STλC(Fn) with new base types and new program constructors. In this 443 section we justify this fact in more general terms. In fact, we show that the constructions 444 from Section 3 can be reproduced starting from any model of the simply-typed λ-calculus. 445 First, we need a suitable notion of model of the simply-typed λ-calculus to start with. 446 Traditionally, one uses cartesian closed categories: cartesian categories where, for all objects 447 A, the functor A × −has a right adjoint (the exponential functor). However, since many 448 usual examples are in fact poset-enriched categories (e.g. Scott domains and continuous 449 functions, coherent spaces and stable functions), and since any (locally small) category can 450 be poset-enriched by using equality as the ordering, we will consider instead cartesian closed 451 poset-enriched categories. To give a counterpart to Proposition 4, we also need a notion of 452 “weak” model of the simply-typed λ-calculus: since poset-enriched categories are a particular 453 case of 2-categories (with a unique 2-arrow from f to g if and only if f ≤g), we follow Hilken 454 [24] and consider cartesian categories where, for all objects A, the functor A × −has a lax 455 right adjoint (the lax-exponential functor). 456 Products and exponentials, when they exist, are necessarily unique up to unique iso- 457 morphism: thus, traditionally, a cartesian closed category is deﬁned as a category in which 458 all ﬁnite products and exponentials exist, rather than a category equipped with products 459 and exponentials (i.e. it is a category with a given property, rather than a category with 460 additional structure). However, this is not the case for lax-exponentials, so for consistency 461 we will adopt the “structure” picture in both cases. Adapting Hilken’s deﬁnitions [24] to the 462 simpler case of poset-enriched categories, we obtain: 463 ▶Deﬁnition 12. Let (C, ×, 1) be a cartesian poset-enriched category. Diameter Space Models Over a Cartesian Closed Category 475 For the rest of this section, we ﬁx a cartesian poset-enriched category (C, ×, 1) (we denote 476 by ⟨−, −⟩the pairing transformation and by πL and πR the projections) and an exponential 477 (exp, ev, λ) on C. The morphisms of this category represent exact programs, so they play the 478 role of the terms from Section 3. 479 23:13 ∀a, b ∈JAK s.t. a ∧b ̸= ∅, δ(a ∧b) + δ(a ∨b) ≤δ(a) + δ(b), and such that for all t, u ∈ΛA, if δA(t) = δA(t ∨u), then t = t ∨u. 486 and such that for all t, u ∈ΛA, if δA(t) = δA(t ∨u), then t = t ∨u. 486 The role of the condition a ∧b ̸= ∅is illustrated by Fig. 5. 487 ▶Example 14. If C is the category whose objects are the simple types from Section 3 and 488 whose morphisms are the (open) terms modulo β-equivalence, then for all simple types A, 489 (A, JAK, LAM, δA) deﬁnes a C-diameter space. 490 ▶Example 14. If C is the category whose objects are the simple types from Section 3 and 488 whose morphisms are the (open) terms modulo β-equivalence, then for all simple types A, 489 (A, JAK, LAM, δA) deﬁnes a C-diameter space. G. Geoﬀroy and P. Pistone ▶Deﬁnition 13. A C-diameter space A is the data of 480 ▶Deﬁnition 13. A C-diameter space A is the data of 480 an object \|A\| of C. The poset C(1, \|A\|) will be denoted by ΛA; 481 an object \|A\| of C. The poset C(1, \|A\|) will be denoted by ΛA; 481 an object \|A\| of C. The poset C(1, \|A\|) will be denoted by ΛA; 481 a set JAK of downwards-closed subsets of ΛA that is closed under arbitrary intersections 482 In particular, JAK is a complete lattice whose meet is given by intersection, and for all 483 a set JAK of downwards-closed subsets of ΛA that is closed under arbitrary intersections. 482 In particular, JAK is a complete lattice whose meet is given by intersection, and for all 483 t ∈ΛA, there is a least element of JAK that contains t, which will be denoted by t; 484 In particular, JAK is a complete lattice whose meet is given by intersection, and for all 483 t ∈ΛA, there is a least element of JAK that contains t, which will be denoted by t; 484 J K t ∈ΛA, there is a least element of JAK that contains t, which will be denoted by t; 484 a commutative integral quantale (LAM, +, ≤); 485 a commutative integral quantale (LAM, +, ≤); 485 a commutative integral quantale (LAM, +, ≤); 485 a monotone function δA : JAK →LAM such that ∀a, b ∈JAK s.t. a ∧b ̸= ∅, δ(a ∧b) + δ(a ∨b) ≤δ(a) + δ(b), 490 Following Section 3, for all C-diameter spaces A and B, we deﬁne a C-diameter space A×B 491 such that \|A × B\| = \|A\| × \|B\| and a C-diameter space exp(A, B) such that \|exp(A, B)\| = 492 exp(\|A\| , \|B\|): 493 Following Section 3, for all C-diameter spaces A and B, we deﬁne a C-diameter space A×B 491 such that \|A × B\| = \|A\| × \|B\| and a C-diameter space exp(A, B) such that \|exp(A, B)\| = 492 exp(\|A\| , \|B\|): 493 JA × BK = {a × b \| a ∈JAK, b ∈JBK}, where a × b = {t ∈C(1, \|A\| × \|B\|) \| πL ◦t ∈ 494 a and πR ◦t ∈b}, 495 LA M LAM L M JA × BK = {a × b \| a ∈JAK, b ∈JBK}, where a × b = {t ∈C(1, \|A\| × \|B\|) \| πL ◦t ∈ 494 a and πR ◦t ∈b}, 495 JA × BK = {a × b \| a ∈JAK, b ∈JBK}, where a × b = {t ∈C(1, \|A\| × \|B\|) \| πL ◦t ∈ 494 a and πR ◦t ∈b}, 495 δA×B(c) = (δA({πL ◦t \| t ∈c}), δB({πR ◦t \| t ∈c})), 497 ( ) ( ({ \| }) ({ \| })) Jexp(A, B)K = {{t ∈C(1, exp(\|A\| , \|B\|)) \| ∀u ∈ΛA, ev(t)◦u ∈I(u)} \| I ∈Poset(ΛA, JBK)} 498 Lexp(A, B)M = Poset(JAK, LBM), 499 Lexp(A, B)M = Poset(JAK, LBM), 499 n L p( , )M (J K, L M), δexp(A,B)(c) = a 7→δB sup n ev(v) ◦t \| t ∈a, v ∈c o . 500 δexp(A,B)(c) = a 7→δB sup n ev(v) ◦t \| t ∈a, v ∈c o . 500 We need a counterpart to Proposition 4. As explained above, we obtain this by organizing 501 the C-diameter spaces as a cartesian poset-enriched category with a lax-exponential. First, 502 we need to deﬁne a notion of morphisms between two C-diameter spaces A and B (which 503 represent approximate programs). By analogy with Section 3, these will be monotone functions 504 from JAK to JBK; however, in order to actually obtain a cartesian category (which was not 505 an issue in Section 3), we will need to add an extra condition: 506 ▶Deﬁnition 15. ∀a, b ∈JAK s.t. a ∧b ̸= ∅, δ(a ∧b) + δ(a ∨b) ≤δ(a) + δ(b), We denote by Diam(C) the poset-enriched category deﬁned as follows: 507 the objects of Diam(C) are the C diameter spaces 508 ▶Deﬁnition 15. We denote by Diam(C) the poset-enriched category deﬁned as follows: 507 ▶Deﬁnition 15. We denote by Diam(C) the poset-enriched category deﬁned as follows: the objects of Diam(C) are the C-diameter spaces, y ( ) p g y ﬁ f the objects of Diam(C) are the C-diameter spaces, 508 for all C-diameter spaces A and B, Diam(C)(A, B) is the set of all monotone functions 509 ϕ : JAK →JBK such that there exists f ∈C(\|A\| , \|B\|) such that for all t ∈ΛA, f ◦t ∈ϕ t 510 (ordered by pointwise inclusion). 511 for all C-diameter spaces A and B, Diam(C)(A, B) is the set of all monotone functions 509 ϕ : JAK →JBK such that there exists f ∈C(\|A\| , \|B\|) such that for all t ∈ΛA, f ◦t ∈ϕ t 510 (ordered by pointwise inclusion). 511 One can check that the operation −×−deﬁned above on C-diameter spaces is a cartesian 512 product in Diam(C). In addition, one can check that there exists in Diam(C) a terminal 513 object 1Diam(C) such that 1Diam(C) = 1C. In other words, Diam(C) is cartesian. Here too, 514 we denote by ⟨−, −⟩the pairing transformation and by πL and πR the projections. 515 One can check that the operation −×−deﬁned above on C-diameter spaces is a cartesian 512 product in Diam(C). In addition, one can check that there exists in Diam(C) a terminal 513 object 1Diam(C) such that 1Diam(C) = 1C. In other words, Diam(C) is cartesian. ∀a, b ∈JAK s.t. a ∧b ̸= ∅, δ(a ∧b) + δ(a ∨b) ≤δ(a) + δ(b), Here too, 514 d t b ⟨ ⟩th i i t f ti d b d th j ti ⟨ ⟩ g Now, following Section 3, we can complete the deﬁnition of the lax-exponential: let 516 A, B, C be C-diameter spaces, 517 A, B, C be C-diameter spaces, 517 for all ϕ ∈Diam(C)(A, exp(B, C)), we deﬁne evA,B,C(ϕ) ∈Diam(C)(A × B, C) by 518 evA,B,C(ϕ)(p) = sup n ev(v) ◦u \| v ∈ϕ(πL(p)), u ∈πR(p) o , 519 for all ϕ ∈Diam(C)(A, exp(B, C)), we deﬁne evA,B,C(ϕ) ∈Diam(C)(A × B, C) by 518 evA,B,C(ϕ)(p) = sup n ev(v) ◦u \| v ∈ϕ(πL(p)), u ∈πR(p) o , 519 for all ϕ ∈Diam(C)(A, exp(B, C)), we deﬁne evA,B,C(ϕ) ∈Diam(C)(A × B, C) by 518 evA,B,C(ϕ)(p) = sup n ev(v) ◦u \| v ∈ϕ(πL(p)), u ∈πR(p) o , 519 CVIT 2016 Proof. Naturality with respect to A is immediate. 523 Proof. Naturality with respect to A is immediate. 523 Let p = a × b ∈JA × BK. For all v ∈λ(ψ)(a) and and u ∈b, by deﬁnition ev(u) ◦u ∈ 524 ψ(a × u) ⊆ψ(p). Therefore, ev(λ(ψ))(p) ⊆p. 525 Let a ∈JAK and v ∈ϕ(a). For all u ∈ΛB, by deﬁnition, ev(v) ◦u ∈λ(ϕ)(a × u), so 526 v ∈λ(ev(ϕ))(a). ◀ 527 As in Section 3, we can ﬁnd a kind of weak embedding from C to Diam(C). Namely, for 528 all C-diameter spaces A and B, we deﬁne a monotone map ∂: C(\|A\| , \|B\|) →Diam(C)(A, B) 529 by ∂(f)(a) = sup{f ◦t \| t ∈a}. The following compatibility result is immediate and oﬀers a 530 counterpart to Remark 6: 531 As in Section 3, we can ﬁnd a kind of weak embedding from C to Diam(C). Namely, for 528 all C-diameter spaces A and B, we deﬁne a monotone map ∂: C(\|A\| , \|B\|) →Diam(C)(A, B) 529 by ∂(f)(a) = sup{f ◦t \| t ∈a}. The following compatibility result is immediate and oﬀers a 530 counterpart to Remark 6: 531 ▶Proposition 17. For all C-diameter spaces A, B, C, all f ∈C(\|A\| , \|B\|) and all g ∈ 532 C(\|B\| , \|C\|), ∂(g ◦f) ≤∂(g) ◦∂(f). In addition, ∂(id\|A\|) = idA. 533 ▶Proposition 17. For all C-diameter spaces A, B, C, all f ∈C(\|A\| , \|B\|) and all g ∈ 532 C(\|B\| , \|C\|), ∂(g ◦f) ≤∂(g) ◦∂(f). In addition, ∂(id\|A\|) = idA. 533 One way to reformulate this result is that ∂induces an oplax-functor from the category 534 with the same objects as Diam(C) and the same morphisms as C, to Diam(C). 535 One way to reformulate this result is that ∂induces an oplax-functor from the category 534 with the same objects as Diam(C) and the same morphisms as C, to Diam(C). 535 O y ∂ p g y with the same objects as Diam(C) and the same morphisms as C, to Diam(C). 535 One can check that ∂preserves products, in the sense that ∂(⟨f, g⟩) = ⟨∂(f), ∂(g)⟩, 536 ∂(πL) = πL and ∂(πR) = πR. In addition ∂is weakly compatible with the exponential, which 537 corresponds to Remark 5: 538 ▶Proposition 18. Let A, B, C be C-diameter spaces, 539 for all f ∈C(\|A\| , exp(\|B\| , \|C\|)), ∂(ev(f)) ≤ev(∂(f)), 540 for all g ∈C(\|A\| × \|B\| , \|C\|), ∂(λ(g)) ≤λ(∂(g)). 23:14 A Partial Metric Semantics of Higher-Order Programs for all ψ ∈Diam(C)(A × B, C), we deﬁne λA,B,C(ψ) ∈Diam(C)(A, exp(B, C)) by 520 for all ψ ∈Diam(C)(A × B, C), we deﬁne λA,B,C(ψ) ∈Diam(C)(A, exp(B, C)) by 520 λA C(ψ)(a) = {v ∈Λ ( ) \| ∀u ∈Λ ev(v) ◦u ∈ψ(a × u)} 21 for all ψ ∈Diam(C)(A × B, C), we deﬁne λA,B,C(ψ) ∈Diam(C)(A, exp(B, C)) by 520 λA,B,C(ψ)(a) = {v ∈Λexp(B,C) \| ∀u ∈ΛB, ev(v) ◦u ∈ψ(a × u)}. 521 for all ψ ∈Diam(C)(A × B, C), we deﬁne λA,B,C(ψ) ∈Diam(C)(A, exp(B, C)) by 0 λA,B,C(ψ)(a) = {v ∈Λexp(B,C) \| ∀u ∈ΛB, ev(v) ◦u ∈ψ(a × u)}. 1 λA,B,C(ψ)(a) = {v ∈Λexp(B,C) \| ∀u ∈ΛB, ev(v) ◦u ∈ψ(a × u)}. 521 ▶Proposition 16. The triple (exp, ev, λ) is a lax-exponential on Diam(C). 522 Proof. Naturality with respect to A is immediate. 523 541 Finally, following Section 3, for all C-diameter spaces A and all t, u ∈ΛA, we write t ≈A u 542 if t = u. In addition, we deﬁne a function dA : ΛA × ΛA →LAM by dA(t, u) = δA(t ∨u). Then 543 the same arguments as in Corollary 8 show that: 544 ▶Proposition 19. For all C-diameter spaces A, (ΛA/ ≈A, LAM, dA) is a generalized partial 545 metric space. 546 One can check that what is described in Section 3 is indeed an instance of this construction. 547 Here are a couple more examples: 548 ▶Example 20. We can take C = Set (with the morphisms ordered by equality): Diam(Set) 549 contains an object RealSet that represents the real numbers with their standard metric 550 and the compact intervals (plus ∅and R) as approximate values, namely \|RealSet\| = R, 551 JRealSetK = {the compact intervals, ∅, R}, LRealSetM = [0, ∞] and δRealSet(I) = length(I). 552 In this case, \|exp(RealSet, RealSet)\| is the set of all functions from R to R, so dRealSet deﬁnes 3 a partial metric on all such functions. 4 ▶Example 21. We can take C = Eﬀ, the eﬀective topos [27]: Eﬀcontains an object REﬀ 555 of recursive reals, and we can deﬁne an object RealEﬀin Diam(Eﬀ) by \|RealEﬀ\| = REﬀ, 556 JRealEﬀK = {I ∩REﬀ\| I ∈JRealSetK}, LRealEﬀM = [0, ∞] and δRealEﬀ(I) = length(I). 557 In this case, \|exp(RealEﬀ, RealEﬀ)\| is the set of all recursive functions from RealEﬀto 558 R l d d ﬁ ti l t i ll h f ti ▶Example 21. We can take C = Eﬀ, the eﬀective topos [27]: Eﬀcontains an object REﬀ 555 of recursive reals, and we can deﬁne an object RealEﬀin Diam(Eﬀ) by \|RealEﬀ\| = REﬀ, 556 JRealEﬀK = {I ∩REﬀ\| I ∈JRealSetK}, LRealEﬀM = [0, ∞] and δRealEﬀ(I) = length(I). 557 In this case, \|exp(RealEﬀ, RealEﬀ)\| is the set of all recursive functions from RealEﬀto 558 RealEﬀ, so dRealEﬀdeﬁnes a partial metric on all such functions. 559 23:15 6 Conclusions 566 Related Work As stated in the introduction, diﬀerential logical relations [32] are a primary 567 source of inspiration for our approach. A related, but more syntactic approach to approximate 568 program transformations is that of Westbrook and Chauduri [44], who use a System F-based 569 type system with a type of real numbers and an explicit distinction between exact and 570 approximate programs. Most examples of contextual reasoning from [44] can be reformulated 571 in our framework (as the case of loop perforation discussed in Section 4). 572 The literature on program pseudo-metrics is vast. A major distinction can be made 573 between those approaches in which metrics account for extensional aspects of programs (like 574 ours), and approaches in which metrics are used to characterize more intensional aspects. To 575 the ﬁrst family belong all metric models developed for reasoning about diﬀerential privacy 576 [35, 3, 7], probabilistic computation [13, 14] and co-inductive models [16, 43, 11, 42]. To the 577 second class belong approaches like [19] which recovers the Scott model of PCF through a 578 ultrametric semantics, and most models based on partial metric spaces [9, 33], which rely on 579 a correspondence between continuous Scott domains and the T0 topology of partial metrics. 580 From a more mathematical viewpoint, [12] discusses a characterization of exponentiable 581 GPMS, showing that no such category can both be cartesian closed and contain the standard 582 metric on R. This result seems to add further evidence of the necessity of considering 583 metrics over varying quantales in order to model higher-order languages. Finally, the elegant 584 categorical approach to GPMS based on quantaloid-enriched categories from [26] seems to 585 provide the relevant structure to develop explicit typing rules for our approximate programs. 586 The literature on program pseudo-metrics is vast. A major distinction can be made 573 between those approaches in which metrics account for extensional aspects of programs (like 574 ours), and approaches in which metrics are used to characterize more intensional aspects. To 575 the ﬁrst family belong all metric models developed for reasoning about diﬀerential privacy 576 [35, 3, 7], probabilistic computation [13, 14] and co-inductive models [16, 43, 11, 42]. G. Geoﬀroy and P. Pistone ▶Example 22. We can take C = Scott, the poset-enriched category of Scott domains and 560 continuous functions. It contains an object representing the reals: RScott = (R ∪{⊥}, ⊑), 561 with r ⊑s iﬀr = s or r = ⊥. Again, we can deﬁne in Diam(Scott) an object RealScott that 562 represents the real numbers with their standard metric, and this deﬁnes a partial metric 563 on \|exp(RealScott, RealScott)\|, the set of all Scott continuous functions from RScott to RScott, 564 which are essentially the partial functions from R to R. 565 ▶Example 22. We can take C = Scott, the poset-enriched category of Scott domains and 560 continuous functions. It contains an object representing the reals: RScott = (R ∪{⊥}, ⊑), 561 with r ⊑s iﬀr = s or r = ⊥. Again, we can deﬁne in Diam(Scott) an object RealScott that 562 represents the real numbers with their standard metric, and this deﬁnes a partial metric 563 on \|exp(RealScott, RealScott)\|, the set of all Scott continuous functions from RScott to RScott, 564 which are essentially the partial functions from R to R. 565 23:16 A Partial Metric Semantics of Higher-Order Programs 1 S. Abramsky, D.M. Gabbay, and T.S.E. Maibaum. Handbook of Logic in Computer Science: 605 Volume 2. Background: Computational Structures. Handbook of Logic in Computer Science. 606 Clarendon Press, 1992. URL: https://books.google.fr/books?id=zqkXKMNXVi0C. 607 1 S. Abramsky, D.M. Gabbay, and T.S.E. Maibaum. Handbook of Logic in Computer Science 605 Volume 2. Background: Computational Structures. 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ACM SIGPLAN Not., 638 49:145–155, 2014. 6 Conclusions 566 To the 577 second class belong approaches like [19] which recovers the Scott model of PCF through a 578 ultrametric semantics, and most models based on partial metric spaces [9, 33], which rely on 579 a correspondence between continuous Scott domains and the T0 topology of partial metrics. 580 From a more mathematical viewpoint, [12] discusses a characterization of exponentiable 581 GPMS, showing that no such category can both be cartesian closed and contain the standard 582 metric on R. This result seems to add further evidence of the necessity of considering 583 metrics over varying quantales in order to model higher-order languages. Finally, the elegant 584 categorical approach to GPMS based on quantaloid-enriched categories from [26] seems to 585 provide the relevant structure to develop explicit typing rules for our approximate programs. 586 Future Work The approach we presented lends itself to further extensions and general- 587 izations. First, we would like to investigate the interpretation of more type constructions 588 than those of STλC(Fn) (e.g. coproducts, recursive types, eﬀects). Moreover, we would like 589 to explore the possibility of exploiting the structure of the category Diam(C) to construct 590 new and more reﬁned notions of approximations. For example (we work in Diam(Set) for 591 simplicity), starting from the “standard” set of approximate values I on RX×X (with elements 592 of I being families of compact intervals Ux,x′ ⊆R indexed by elements of X and X′), one 593 can deﬁne a new family ∆∗I of approximate values for RX by “pulling back” the exact map 594 ∆: RX →RX×X deﬁned by ∆f(x, x′) = f(x′) −f(x), i.e. letting ∆∗I = {∆−1(a) \| a ∈I}. 595 The new approximate values then correspond to sets of functions f ∈RX with a controlled 596 variation, that is, such that f(x′) −f(x) is bounded by some family of intervals Ux,x′ ∈I. 597 Another interesting research direction concerns probabilistic extensions of STλC(Fn). 598 Probabilistic metrics [15, 30, 13, 14] have been the object of much research in recent years, due 599 to the relevance of metric reasoning in some areas of computer science in which probabilistic 600 computation plays a key role (e.g. in cryptography [22] and machine learning [31]). 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https://openalex.org/W1708067740	https://europepmc.org/articles/pmc3691389?pdf=render	English	null	Symptomatic Bradycardia Caused By Premature Atrial Contractions Originating From Right Atrial Appendage	Indian pacing and electrophysiology journal/Indian Pacing and Electrophysiology Journal	2,013	cc-by	1,133	114 114 114 114 Introduction Palpitation is the main complaint in patients with supraventricular arrhythmias. Hemodynamic compromise secondary to high ventricular rates may also occur. In this report, we present a patient with symptomatic bradycardia which developed secondary to blocked premature atrial contractions (PAC) originating from right atrial appendage and was successfully treated with radiofrequency ablation (RFA). Abstract Premature atrial contraction is a common form of supraventricular arrhythmias. In rare cases, severe symptoms other than palpitation may occur. In this report, we present a patient with symptomatic bradycardia which developed secondary to blocked premature atrial contractions and was successfully treated with radiofrequency ablation. Key words: bradycardia, premature atrial contraction, radiofrequency ablation Symptomatic Bradycardia Caused By Premature Atrial Contractions Originating From Right Atrial Appendage Alper AT, MD; Gungor B, MD; Turkkan C, MD; Tekkesin AI, MD. Alper AT, MD; Gungor B, MD; Turkkan C, MD; Tekkesin AI, MD. Department of Cardiology, Siyami Ersek Thoracic and Cardiovascular Surgery Center, Training and Research Hospital, Istanbul, Turkey Address for Correspondence: Baris Gungor, MD, Mehmet Akif Mah. Hicret Sok. Merit Life Bulvar, A/3 Umraniye/Istanbul Turkey. Email: drbarisgungor/at/gmail.com Case Report A 35-year-old woman presented to our clinic with dyspnea and fatigue since last two months. Her blood pressure was 90/50 mmHg and her pulse was regular at a rate of 40 beats/min. The physical examination was otherwise normal. Surface ECG was compatible with sinus bradycardia with R-R and P-P wave intervals of 1400 ms and PR interval of 120 ms. But careful observation of the terminal portion of the T waves revealed non-conducted P waves with subsequent prolonged pauses (Figure 1). The morphology of the non-conducted P waves was negative in lead V1,V2, and positive in the inferior leads. A 24-hour Holter monitoring showed more than twenty thousand PACs most of which were not conducted to the ventricle. Her echocardiographic examination and laboratory tests including thyroid function were in normal range. Indian Pacing and Electrophysiology Journal (ISSN 0972-6292), 13 (3): 114-117 (2013) Alper AT et el, “Symptomatic-bradycardia secondary to premature atrial contractions” 116 Alper AT et el, “Symptomatic-bradycardia secondary to premature atrial contractions” 116 There were frequent PACs that any maneuver to induce sustained arrhythmia was not needed. Endocardial mapping demonstrated atrioventricular block at suprahisian level and the earliest endocardial activation site was in the right atrial appendage (RAA) where the local intracardiac electrocardiogram was 60 ms prior to the onset of the P waves (Figure 2). Application of the radiofrequency current at this site resulted in termination of the PACs and within 3 seconds surface ECG returned to normal sinus rhythm (Figure 3). The rest of the hospitalization was uneventful and the patient did not have any cardiac symptoms during follow-up of 6 months. Figure 3. ECG after the ablation procedure showing normal sinus rhythm without any premature atrial contractions. Figure 3. ECG after the ablation procedure showing normal sinus rhythm without any premature atrial contractions. Discussion Premature atrial contractions are one of the most common forms of arrhythmias. Atrial bigeminy is rarely associated with severe symptoms and palpitation is the only complaint in most of the cases. In our case, the main complaint of the patient was exercise intolerance and fatigue due to bradycardia caused by blocked PACs. In some cases, non-conducted P waves may superimpose on the T waves of the preceding sinus beats and may be difficult to differentiate from the T and U waves. Thus, the bradycardia may be misdiagnosed as sinoatrial block. Furthermore, blocked atrial bigeminy may be misdiagnosed as second-degree AV block [1]. In these cases unnecessary pacemaker implantation can be avoided by careful ECG examination. In second-degree AV block P-P interval remains constant, and the P wave morphology is unchanged. Previous case reports have shown that antiarrhythmic drugs [2] and catheter ablation [3] could be alternatives for treatment of atrial bigeminy in symptomatic patients [1-3]. Because of the severe symptoms and high number of PACs, we considered RF ablation as a first line therapy. In our case the origin of the atrial bigeminy was RAA. In literature, there are no studies about the origins of the atrial bigeminy. But, studies on atrial tachycardia (AT) reported RAA as the site of focal AT in 3.8% of the cases [4]. This study has reported that P wave morphology of AT's originating from RAA is characteristic which is negative in leads V1,V2 and a transition to positivity occurs in the other precordial leads [4]. The P waves in our patient also had similar morphologic features. Indian Pacing and Electrophysiology Journal (ISSN 0972-6292), 13 (3): 114-117 (2013) Alper AT et el, “Symptomatic-bradycardia secondary to premature atrial contractions” 115 Alper AT et el, “Symptomatic-bradycardia secondary to premature atrial contractions” 115 Figure 1. ECG showing bradycardia caused by blocked atrial bigeminy. Arrows indicate non-conducted P waves. Figure 1. ECG showing bradycardia caused by blocked atrial bigeminy. Arrows indicate non-conducted P waves. After obtaining informed consent, the patient was transferred to the electrophysiology laboratory. The procedure was performed with the patient in the fasting, non-sedated state. One 6-French Josephson catheter, one 7-French ablation catheter and one 7-French mapping catheter were advanced through femoral veins and placed at right ventricular apex and right atrium (Figure 2). re 2. (A) posteroanterior and (B) right anterior oblique flouroscopic views of the catheters during the on procedure (C) intracardiac recordings at the site of successful radiofrequency ablation showing that st atrial activation precedes the onset of non-conducted P wave by 60 ms in the distal bipolar recordings. adiofrequency ablation catheter; MAP, mapping catheter; RV, right ventricle catheter. Figure 2. (A) posteroanterior and (B) right anterior oblique flouroscopic views of the catheters during the ablation procedure (C) intracardiac recordings at the site of successful radiofrequency ablation showing that earliest atrial activation precedes the onset of non-conducted P wave by 60 ms in the distal bipolar recordings. RF, radiofrequency ablation catheter; MAP, mapping catheter; RV, right ventricle catheter. Indian Pacing and Electrophysiology Journal (ISSN 0972-6292), 13 (3): 114-117 (2013) Conclusion Atrial bigeminy with blocked PACs can result in significant bradycardia with symptoms. Radiofrequency catheter ablation may be considered as the first line of treatment in these patients. References 1. Gaudio C, Di Michele S, Ferri FM, Mirabelli F, Franchitto S, Alessandri N. A case of non- conducted atrial bigeminy simulating a second- degree atrioventricular block. A holter ECG diagnosis. Eur Rev Med Pharmacol Sci. 2004;8(4):169-71. 2. Veress G. Infra-His blocked premature atrial contractions simulating 2:1 sinoatrial block in a patient with an atrio-His bypass tract. Chest. 1993;104:1306–1308. 3. Vora A, Lokhandwala Y. ECG of the month. Radiofrequency ablation for bradycardia! Indian Heart J. 2009;61:108. 4. Freixa X, Berruezo A, Mont L, Magnani S, Benito B, Tolosana JM, Perafan P, Tamborero D, Brugada J. Characterization of focal right atrial appendage tachycardia. Europace 2008; 10:105–109. Indian Pacing and Electrophysiology Journal (ISSN 0972-6292), 13 (3): 114-117 (2013)
https://openalex.org/W3092525543	https://www.nature.com/articles/s41598-020-73628-6.pdf	English	null	Identifying patterns in the multitrophic community and food-web structure of a low-turbidity temperate estuarine bay	Scientific reports	2,020	cc-by	18,257	estuarine bay Hee Yoon Kang1, Changseong Kim1, Dongyoung Kim1, Young‑Jae Lee1, Hyun Je Park2, Goutam K. Kundu1, Young Kyun Kim1, Riaz Bibi1, Jaebin Jang1, Kwang‑Hun Lee1, Hyun‑Woo Kim3, Sung‑Gyu Yun4, Heeyong Kim5 & Chang‑Keun Kang 1* Food web dynamics outline the ecosystem processes that regulate community structure. Challenges in the approaches used to capture topological descriptions of food webs arise due to the difficulties in collecting extensive empirical data with temporal and spatial variations in community structure and predator–prey interactions. Here, we use a Kohonen self-organizing map algorithm (as a measure of community pattern) and stable isotope-mixing models (as a measure of trophic interaction) to identify food web patterns across a low-turbidity water channel of a temperate estuarine-coastal continuum. We find a spatial difference in the patterns of community compositions between the estuarine and deep-bay channels and a seasonal difference in the plankton pattern but less in the macrobenthos and nekton communities. Dietary mixing models of co-occurring dominant taxa reveal site-specific but unchanging food web topologies and the prominent role of phytoplankton in the trophic base of pelagic and prevalent-detrital benthic pathways. Our approach provides realistic frameworks for linking key nodes from producers to predators in trophic networks. Estuaries form an intermediate transition zone between rivers and seas1. These coastal ecotones are characterized by highly dynamic physical, biogeochemical, and biological processes, and the functioning of these transitional ecosystems involves the complex interactions of those processes2,3. Fluctuations in the processes create distinct community patterns along the salinity gradient and thus the scale and magnitude of community patterns are subject to seasonal changes in freshwater discharge4–6. This wide spectrum of variability with space and time constructs the structural and dynamic properties of estuarine food webs7,8. Consequently, the understanding of food web dynamics in estuaries is fundamental for predicting the responses of communities (and populations) under both natural and anthropogenic forcing.hi The identification of patterns in structure (e.g., links between species) and processes (e.g., energy or matter flux, interaction strengths) within food webs is crucial for understanding function and further constructing quantitative food webs for more detailed predictions of ecosystem-level responses9,10. Indeed, diverse network models have been applied to quantitatively analyse matter flows through trophic pathways within and among estuarine ecosystems, providing useful tools for assessing response patterns to environmental stresses11–13. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ and identifying patterns of co-occurrence of different trophic groups of organisms that create different types of interactions (i.e., competition, mutualism, and consumption) in a real community15. In addition, quantitative data on the diets of species are essential in terms of deciding trophic nodes in the food web and for partitioning the total consumption of each node into the relative contributions by other nodes, characterizing the sets of feeding links within a food web16,17. Seasonal and longitudinal patterns in community structure in an estuarine-coastal continuum allow for the classification of benthic and pelagic community compositions along steep environmental gradients and the establishment of a solid basis of temporal and spatial scales for the further modelling of food webs. Spatial and temporal variation in typology within estuaries gives rise to concomitant changes in predator–prey interactions, making the generalization of estuarine food webs inappropriate16,18,19. Furthermore, while benthic suspension feeders rely on diets from the overlying water column, demersal fish often feed more on benthic prey than on pelagic prey20,21. Diets of predators also display ontogenetic as well as seasonal changes reflecting size selectivity and prey availability19,22. As a result, analyses of community composition should consider almost all taxonomic and/or functional groups colonizing both water and sediment at the appropriate spatial and temporal scales because of their potential interactions. A variety of ordination techniques have been used to simply compile community patterns from highly com- plex community data. Considering the prevalent non-linearity of biological processes in estuaries23,24, we con- veyed the abundances of phyto- and zooplankton, benthos, and nekton to the Kohonen self-organized map (SOM) algorithm25,26 to characterize community patterns along a salinity gradient that creates unique physical, chemical, and biological features. The SOM is an unsupervised neural network that has been widely applied to patterning communities15,27,28. The SOM finally performs clustering analysis of the input data through competi- tive learning and visualizes species assemblages on a bi-dimensional plane. g g Once typological characteristics with time and space were determined, we used carbon and nitrogen stable isotope ratios of flora and fauna to infer the trophic relationships and the flow pattern of energy through the food webs29. This approach has been employed as an alternative tool for obtaining direct empirical information on consumer diets (e.g., gut contents). www.nature.com/scientificreports/ δ13C and δ15N have been commonly used to provide time- and space- integrated insights into trophic relationships between organisms, allowing for a comprehensive review of a priori assumptions of the trophic roles of individual organisms29. δ13C values can be used to trace original sources of dietary carbon because primary producers have distinct values from each other, and these values are conservative during trophic transfers, with little or no trophic enrichment (≤ 1‰)30. In contrast, the δ15N values of consumers manifest significant trophic-step fractionation in 15N (2–4‰, average 3.4‰ heavier than those of their prey) and are thus used as an estimate of the trophic position of consumers and the food chain length31. Estuarine ecosys- tems function in association with the large detrital pool derived from various types of vegetation32. Therefore, this approach is often more effective than other traditional empirical techniques in tracing the trophic pathways of carbon/energy and the trophic connectivity between habitats within estuaries33. Here, we combined the SOM algorithm and stable isotope techniques to explore the food web structure in the water channel along the estuarine-coastal marine continuum of a temperate coastal embayment, Gwangyang Bay, Korea, which is subject to low-turbidity riverine discharge and a short water residence time (Fig. 1). We synthesized multitrophic community patterns encompassing plankton, benthos, and nekton and determined subsets of co-occurring dominant taxa in different types of community associations with space and season. We then evaluated the relative contributions of dominant flora and river-borne organic matter in the landscape of the embayment to the dominant primary consumers (suspension and deposit feeders) that allowed for the subsequent calculations of the trophic links with benthic vs. pelagic pathways29. Using these mixing models, we integrated trophic interactions between main taxa that constitute key nodes of food-web networks. We found that a combination of both approaches enabled us to identify realistic food-web patterns at seasonal as well as spatial scales across the coastal ecotone. estuarine bay To construct better food web models, it may be necessary to have empirical information on the underlying archi- tecture (the map of predator–prey interactions) and interaction strength8,14. An estimation of the abundance and biomass of organisms often serves as a key component in computing the production of trophic groups8 1School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea. 2Department of Marine Bioscience, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea. 3Department of Marine Biology, Pukyong National University, Busan 48513, Republic of Korea. 4Department of Science Education, Daegu University, Gyeongsan 38453, Republic of Korea. 5South Sea Fisheries Research Institute, National Institute of Fisheries Science, Yeosu 59780, Republic of Korea. *email: ckkang@gist.ac.kr Scientific Reports \| (2020) 10:16637 \| https://doi.org/10.1038/s41598-020-73628-6 www.nature.com/scientificreports/ Results P tt i 145 km2 and experiences a semidiurnal tidal cycle, with tidal ranges of 3.40 and 1.10 m during spring and neap tides, respectively. The Seomjin River discharges an annual mean of ca. 120 m3 s−1 (range: 30–95 m3 s−1 in winter base flows to 300–400 m3 s−1 in summer monsoon flows) of freshwater into the bay34. The northern estuarine channel is characterized by shallow depths (depth range: 2.4–8.0 m), short residence times (flushing time: 7.0 d)35, and low-turbidity water (extremely low concentrations, less than 20 mg l−1, of suspended particulate matter) following a lack of input of river-borne particles36,37. Three stations for the estuarine and six stations for the deep-bay channel were chosen for the field survey along a longitudinal water-channel trajectory of the estuarine-coastal marine continuum. See the Supplementary information for additional details. subclusters (3a, 3b, and 3c; MRPP, A = 0.09 to 0.24, P = 0.001 for all cases). The distributions of clusters reflected a clear longitudinal pattern with no perceptible seasonal changes in the macrobenthic community (Fig. 2f). The estuarine-channel stations were divided into clusters 1 and 2. Of the deep-bay stations (3–9) belonging to cluster 3, subclusters 3b and 3c corresponded mostly to stations 5 and 8, respectively. When the SOM was trained on 16 datasets (3 sites × 6 times) of nekton assemblages, their community pat- terns were visualized on a two-dimensional map (4 × 4 neurons, Fig. 2g). A visual inspection of the SOM map primarily revealed a clear spatial pattern in the nekton community by an array of estuarine channel stations in the upper right corner and deep bay stations in the other regions. The cluster analysis classified those two groups (MRPP, A = 0.17 to 0.44, P < 0.02 for clusters I vs. II, III or IV, Fig. 2h) and further divided the deep-bay samples into the lower right (cluster II), the lower left (III), and the upper left (IV) corners. The sample-cluster association then clarified the spatial pattern of the nekton community (Fig. 2i). Cluster I included samples collected at the estuarine station throughout the year, whereas clusters II, III and IV corresponded to the assemblages sampled at the deep-bay stations in August-November, February, and May, respectively. Seasonal discrimination in com- munity composition was non-significant between clusters II vs. IV (MRPP, A = 0.38, P = 0.067) and clusters III vs. Results P tt i Patterning communities. Based on 54 individual datasets (9 sites × 6 times) of plankton assemblages (Supplementary Table 1), we trained the SOM and condensed them on the map (6 × 6 rectangular grid, Fig. 2a). The map configuration supported a clear segregation of seasonal samples from distributions in different areas on the map as well as a spatial separation of the estuarine stations in the upper left part of the map from the deep- bay stations. A hierarchical cluster analysis classified the SOM units into five groups (A, B, C, D, and E; Fig. 2b) that showed significant differences in terms of species composition (multi-response permutation procedure [MRPP], A = 0.10 to 0.42, P < 0.03 for all cases). The association between samples (individual datasets collected at a given site and time) and clusters (to which samples belong) depicted the seasonal and spatial patterns of clus- ters (Fig. 2c). In the estuarine channel (stations 1–2), the plankton assemblages displayed a seasonal succession alternating between clusters A and B, driving the differentiation from the deep-bay clusters (C, D, and E). The plankton assemblages in the deep bay (stations 3–9) showed a clear seasonal succession. In February and May 2015, most samples belonged to cluster E. The dominant cluster was replaced in turn by clusters B (November), C (February 2016), D (August), and B (November). Interestingly, plankton community at station 3, which is located at the mouth of the estuarine channel, alternated between estuarine and deep-bay clusters, forming the estuarine–coastal transition or mixing zone.t g After training the SOM, the map (6 × 6 neurons) allowed us to visualize the information in the 54 datasets of macrobenthos assemblages (Fig. 2d). The spatial distribution of the samples on the map revealed a longitudinal pattern from the estuarine-channel stations in the lower part of the map to the deep-bay stations in the upper part of the map, further clustering the SOM units into three major groups (clusters 1, 2, and 3; MRPP, A = 0.09 to 0.24, P < 0.01 for all cases, Fig. 2e). Results P tt i Cluster 3 contained the samples from the deep bay and was subdivided into three Scientific Reports \| (2020) 10:16637 \| https://doi.org/10.1038/s41598-020-73628-6 www.nature.com/scientificreports/ N 127o 80 127o 60 E 34o 80 N 35o 00 Seomjin River Gwangyang Bay Yeosu Sound 10 20 10 20 10 20 10 10 1 km Seomjin River 127o 80 E 5 km N 35o 00 + + + + + + + + 1 2 3 4 5 6 7 8 9 Phragmites bed 127o 128o 129o 35o 34o KOREA South Sea of Korea N 126o E + a b c POSCO Figure 1. Map of study location and river-estuary-coastal sea continuum. (a) Map showing the location of Gwangyang Bay, Korea. (b) Sampling stations in the deep bay. The brown area represents the intertidal bed, and the dark green area represents the Zostera bed. (c) Sampling stations in the estuarine channel. The yellow-green area represents the Phragmites bed. The bay has an area of ca. 145 km2 and experiences a semidiurnal tidal cycle, with tidal ranges of 3.40 and 1.10 m during spring and neap tides, respectively. The Seomjin River discharges an annual mean of ca. 120 m3 s−1 (range: 30–95 m3 s−1 in winter base flows to 300–400 m3 s−1 in summer monsoon flows) of freshwater into the bay34. The northern estuarine channel is characterized by shallow depths (depth range: 2.4–8.0 m), short residence times (flushing time: 7.0 d)35, and low-turbidity water (extremely low concentrations, less than 20 mg l−1, of suspended particulate matter) following a lack of input of river-borne particles36,37. Three stations for the estuarine and six stations for the deep-bay channel were chosen for the field survey along a longitudinal water-channel trajectory of the estuarine-coastal marine continuum. See the Supplementary information for additional details. N 127o 80 127o 60 E 34o 80 N 35o 00 Seomjin River Gwangyang Bay Yeosu Sound 10 20 10 20 10 20 10 10 5 km + + + + + 4 5 6 7 8 9 + b POSCO Figure 1. Map of study location and river-estuary-coastal sea continuum. (a) Map showing the location of Gwangyang Bay, Korea. (b) Sampling stations in the deep bay. The brown area represents the intertidal bed, and the dark green area represents the Zostera bed. (c) Sampling stations in the estuarine channel. The yellow-green area represents the Phragmites bed. The bay has an area of ca. www.nature.com/scientificreports/ www.nature.com/scientificreports/ The association of dominant taxa40 in accordance with the configurations of cluster arrays illustrates the co- occurrence patterns of major food-web components within the multitrophic communities (Fig. 3, Supplementary Table 3). Clusters constituting the plankton community had an approximately equal number of dominant taxa (10–11 and 7–10 for phyto- and zooplankton, respectively). Although there were a few common dominant taxa in all clusters, there was a clear distinction in the dominant plankton taxa between the estuarine channel and the deep bay clusters. In the deep-bay clusters (B–E), some dominant taxa showed a varying frequency of occurrence with season. Clusters 1 and 2 of the macrobenthos community, which appeared in the estuarine channel, had only a restricted number (3 taxa for both) of dominant taxa compared to those (5–9 taxa) of deep-bay cluster 3. Three deep-bay subclusters shared the majority of dominant benthos taxa that created the clear separation from those in the estuarine clusters. Cluster I of the nekton community, present in the estuarine channel, recorded a lack of indicator taxa as well as a restricted number (6 taxa) of dominant taxa compared to those (13 taxa for each) in the deep-bay clusters II–IV. The median abundances of nektonic taxa in cluster I were much lower than those in the deep bay. The deep-bay clusters II–IV shared the majority of dominant taxa, some of which had an occurrence frequency that varied with season. Environmental characterization. Comparisons of the environmental attributes of the water column were made among the values of the measured variables for the five clusters of plankton communities that repre- sented both seasonal and spatial patterns (Fig. 4). A Kruskal–Wallis test identified significant differences (H-test, P < 0.001) in most variables. The following Mann–Whitney pairwise comparison test (at P = 0.05) revealed that the temperature peaked in the summer cluster D and was lowest in the winter cluster C; salinity was lowest in cluster A and consistently high in the other clusters (medians, > 29.5); dissolved inorganic nutrient concentra- tions were inversely related to salinity, being extremely high in clusters A and B and low in the other clusters; and chlorophyll a concentration displayed bimodal peaks in clusters A (estuarine channel) and E (spring). Sus- pended particulate matter concentrations were lowest in cluster A and highest in cluster E (with a median of 12.6 mg l−1). www.nature.com/scientificreports/ Most of the variables were characterized by broad variation ranges in clusters A (estuarine channel) and B (covering the whole bay area in fall) but by narrow ranges and comparatively low levels in the deep-bay clusters. Stable isotope measurements. Analysis of variance (ANOVA) revealed a great seasonal uniformity in the δ13C and δ15N values of wetland producers (Phragmites australis, microphytobenthos, and Zostera marina) and riverine suspended particulate organic matter (RPOM, F3,40 = 0.724 and 2.590, P = 0.544 and 0.066, respec- tively), which had ranges from − 27.5 ± 0.6‰ (annual mean ± 1 SD) to − 8.6 ± 1.0‰ and 5.4 ± 0.4‰ to 8.5 ± 0.8‰, respectively (F3,40 = 554.8 and 37.3, P < 0.001 for both; Fig. 5, Supplementary Table 4). y , g y ANOVA and the following post hoc Tukey test (P < 0.05) indicated that the mean δ13C values of phytoplank- ton were clearly distinguished between clusters A–B (the estuarine channel, − 22.9 ± 2.6‰) and clusters B–E (the deep bay, − 19.9 ± 1.8‰) (F5,44 = 6.153, P < 0.001), while their δ15N values displayed undetectable seasonal and spatial variation (F5,44 = 0.403, P = 0.844), averaging 7.8 ± 2.1‰ (Fig. 5, Supplementary Table 5). ANOVA also showed that the δ13C values of zooplankton varied from a pooled mean − 23.8 ± 2.8‰ for clusters A–B (the estuarine channel) to one of − 19.5 ± 1.2‰ for clusters B–E (the deep bay) (F5,38 = 12.125, P < 0.001). The close proximity (a post hoc Tukey multiple comparison, P > 0.05) of the δ13C values between clusters in each location exhibited no seasonality. ANOVA revealed a non-significant difference in the δ15N values of zooplankton among clusters (F5,44 = 1.371, P = 0.257), with an overall mean of 9.5 ± 1.3‰. These area-pooled mean δ13C and δ15N values of zooplankton were used as isotopic baselines of the pelagic pathway in the subsequent isotope-mixing model for higher-level consumers.h The mean δ13C values of the dominant primary consumers (suspension feeders and deposit feeders) in each cluster of the macrobenthos community ranged from − 24.4 ± 0.5‰ to − 15.9 ± 0.5‰ (Supplementary Table 6). Their mean δ15N values varied little, ranging from 10.2 ± 0.4‰ to 11.8‰. www.nature.com/scientificreports/ Suspension feeders in the estuarine channel (Clusters 1–2) had slightly more 13C- and 15N-depeleted values (− 24.4 ± 0.5‰ to − 22.1 ± 0.7‰ and 10.2 ± 0.4‰ to 10.4 ± 0.9‰, respectively) than those (− 21.5 ± 1.0‰ to − 21.2 ± 0.4‰ and 10.9 ± 0.9‰ to 11.5 ± 1.2‰, respectively) of deposit feeders (Mann–Whitney U test, U = 6.5, P < 0.001 for δ13C; Student’s t test, t18 = − 3.737, P = 0.002 for δ15N, Fig. 5a). Both δ13C and δ15N values of deposit feeders in the deep-bay clusters (3a–c) fell within very narrow ranges of − 16.7‰ to − 15.9 ± 0.5‰ and 9.6 ± 0.8‰ to 12.1‰, respectively (Fig. 5b). A clear spatial shift in the δ13C and δ15N values was found for deposit feeders between the estuarine channel (Clusters 1–2, mean − 21.3 ± 0.6‰ and 11.4 ± 0.6‰, respectively) and the deep bay (Clusters 3a–c, − 16.7 ± 0.8‰ and 10.5 ± 0.8‰) (Student’s t test, t42 = 16.637, P < 0.001 for δ13C; t42 = − 2.990, P = 0.005 for δ15N). These area- pooled mean δ13C and δ15N values of deposit feeders were applied as isotopic baselines of the benthic pathway in the isotope-mixing model for higher-level consumers.hi p g g The mean δ13C values of dominant predatory taxa of macrobenthos and nekton clusters exhibited a significant spatial difference between the estuarine bay and the deep bay (Student’s t-test, t27 = − 4.436, P < 0.001, Table 1). In the deep bay, their δ13C values were very consistent between taxa, with a narrow range from − 16.2 ± 0.6‰ to − 14.1 ± 0.5‰ (with only the exception of − 17.7 ± 2.6‰ observed for T. kammalensis), overlapping with the range of the deep-bay deposit feeders (Fig. 5b). The δ13C values of dominant nektonic taxa in the estuarine chan- nel varied from − 18.5 ± 0.5‰ to − 16.0 ± 0.1‰, being, on average, 1.7‰ more negative than those of the deep-bay taxa but closer to the values of primary consumers of the deep bay than those of the estuarine channel (Fig. 5a). Isotope mixing model. Results P tt i IV (A = 0.30, P = 1.000); the comparison showed a difference between the groups II and III (A = 0.22, P = 0.022), of which the result was less clear than the spatial pattern.h The visualization of the spatial distribution patterns of planktonic, benthic, and nektonic taxa on the SOM units manifested the associations of species with clusters (Supplementary Fig. 1). As measured by the IndVal index38,39, 16 phytoplankton, 20 zooplankton, 15 macrobenthos, 13 nektonic invertebrates, and 8 fish of common taxa considered in the indicator species analysis were found to be significantly (P < 0.05) associated with one or more clusters of taxonomic groups to which the species belonged, enabling us to summarize the assemblages characterizing each cluster (Supplementary Table 2). The relative abundance (A) of a species in a site group over all site groups and the relative frequency of occurrence (B) of that species inside the target cluster group recorded higher values than 0.52 (Diatoma sp.) and 0.50 (Tortanus dextrilobatus), respectively, in the present investigation. The square root of the IndVal index ranged from 0.71 for zooplankton (Evadne nordmanni) to 1.00 for benthic bivalves (Corbicula japonica) and fish (Thryssa kammalensis). Only a few indicator taxa were associated with the estuarine-channel clusters (A and 1–2) and were differentiated from the taxa of the deep-bay counterparts in the corresponding taxonomic groups. In contrast, the deep-bay clusters shared many indicator species and had a few unique characteristic taxa. Scientific Reports \| (2020) 10:16637 \| https://doi.org/10.1038/s41598-020-73628-6 www.nature.com/scientificreports/ Taxon Estuarine channel Deep bay δ13C (‰) δ15N (‰) f TP δ13C (‰) δ15N (‰) f TP Macrobenthos Glycera chirori − 15.9 ± 0.7 13.6 ± 0.7 (14) 0.82 3.0 Sigambra tentaculata − 16.1 13.2 (1) 0.75 2.9 Scoletoma longifolia − 16.1 ± 0.8 12.9 ± 0.5 (5) 0.75 2.8 Crustaceans Charybdis japonica − 17.2 ± 0.2 13.9 ± 0.7 (4) 0.58 3.2 Crangon hakodatei − 17.4 ± 0.7 12.9 ± 0.6 (3) 0.38 2.9 Oratosquilla oratoria − 15.5 ± 0.7 13.2 ± 0.4 (6) 0.96 2.8 Charybdis bimaculata − 15.7 ± 0.4 12.0 ± 0.2 (5) 0.89 2.5 Parapenaeopsis tenella − 16.0 ± 0.5 12.0 ± 0.4 (7) 0.79 2.5 Metapenaeus joyneri − 16.0 ± 0.3 11.9 ± 0.4 (6) 0.79 2.5 Trachysalambria curvirostris − 17.3 13.2 (1) 0.51 3.0 − 14.4 ± 0.6 12.6 ± 0.8 (5) 1.00 2.6 Portunus trituberculatus − 15.0 ± 0.4 12.8 ± 0.4 (4) 1.00 2.7 Palaemon gravieri − 14.4 ± 0.1 13.5 ± 0.4 (2) 1.00 2.9 Alpheus japonicus − 14.2 ± 0.4 12.9 ± 0.4 (3) 1.00 2.7 Alpheus digitalis − 14.1 ± 0.5 12.6 ± 0.7 (4) 1.00 2.6 Cephalopods Loligo japonica − 15.8 ± 0.3 14.3 ± 0.7 (3) 0.86 3.2 Octopus variabilis − 15.5 ± 0.4 13.6 ± 0.6 (4) 0.96 3.0 Euprymna morsei − 14.7 ± 0.8 13.0 ± 0.4 (4) 1.00 2.8 Fish Pleuronectes yokohamae − 16.0 ± 0.1 15.4 ± 0.3 (3) 0.79 3.5 Leiognathus nuchalis − 18.5 ± 0.5 14.7 ± 0.4 (3) 0.29 3.2 − 16.2 ± 0.6 15.0 ± 0.9 (6) 0.71 3.5 Konosirus punctatus − 16.1 13.3 (1) 0.78 2.9 Cynoglossus joyneri − 15.0 ± 0.5 13.6 ± 0.5 (8) 1.00 2.9 Amblychaeturichthys hexanema − 15.1 ± 0.4 14.2 ± 0.4 (6) 1.00 3.1 Pennahia argentata − 15.2 ± 0.6 14.9 ± 1.0 (6) 1.00 3.3 Thryssa kammalensis − 17.7 ± 2.6 14.6 ± 1.1 (6) 0.18 3.5 Okamejei kenojei − 14.4 ± 0.5 13.4 ± 0.9 (5) 1.00 2.9 Ctenotrypauchen microcephalus − 15.7 ± 0.5 14.1 ± 0.2 (4) 0.89 3.1 Johnius grypotus − 14.7 ± 0.1 14.4 ± 0.8 (4) 1.00 3.2 Table 1. δ13C and δ15N values, dependence on benthic affinity prey (f), and trophic position (TP) of dominant predatory consumers. www.nature.com/scientificreports/ The IsoSource mixing model calculation revealed that estuarine phytoplankton played a dominant role as the main nutritional contributor (median 81% [range: 74–86%] to suspension feeders, 68% [27–100%] to deposit feeders, and 46% [2–91%] to filter-feeding zooplankton) to the dominant primary https://doi.org/10.1038/s41598-020-73628-6 Scientific Reports \| (2020) 10:16637 \| www.nature.com/scientificreports/ Taxon Estuarine channel Deep bay δ13C (‰) δ15N (‰) f TP δ13C (‰) δ15N (‰) f TP Macrobenthos Glycera chirori − 15.9 ± 0.7 13.6 ± 0.7 (14) 0.82 3.0 Sigambra tentaculata − 16.1 13.2 (1) 0.75 2.9 Scoletoma longifolia − 16.1 ± 0.8 12.9 ± 0.5 (5) 0.75 2.8 Crustaceans Charybdis japonica − 17.2 ± 0.2 13.9 ± 0.7 (4) 0.58 3.2 Crangon hakodatei − 17.4 ± 0.7 12.9 ± 0.6 (3) 0.38 2.9 Oratosquilla oratoria − 15.5 ± 0.7 13.2 ± 0.4 (6) 0.96 2.8 Charybdis bimaculata − 15.7 ± 0.4 12.0 ± 0.2 (5) 0.89 2.5 Parapenaeopsis tenella − 16.0 ± 0.5 12.0 ± 0.4 (7) 0.79 2.5 Metapenaeus joyneri − 16.0 ± 0.3 11.9 ± 0.4 (6) 0.79 2.5 Trachysalambria curvirostris − 17.3 13.2 (1) 0.51 3.0 − 14.4 ± 0.6 12.6 ± 0.8 (5) 1.00 2.6 Portunus trituberculatus − 15.0 ± 0.4 12.8 ± 0.4 (4) 1.00 2.7 Palaemon gravieri − 14.4 ± 0.1 13.5 ± 0.4 (2) 1.00 2.9 Alpheus japonicus − 14.2 ± 0.4 12.9 ± 0.4 (3) 1.00 2.7 Alpheus digitalis − 14.1 ± 0.5 12.6 ± 0.7 (4) 1.00 2.6 Cephalopods Loligo japonica − 15.8 ± 0.3 14.3 ± 0.7 (3) 0.86 3.2 Octopus variabilis − 15.5 ± 0.4 13.6 ± 0.6 (4) 0.96 3.0 Euprymna morsei − 14.7 ± 0.8 13.0 ± 0.4 (4) 1.00 2.8 Fish Pleuronectes yokohamae − 16.0 ± 0.1 15.4 ± 0.3 (3) 0.79 3.5 Leiognathus nuchalis − 18.5 ± 0.5 14.7 ± 0.4 (3) 0.29 3.2 − 16.2 ± 0.6 15.0 ± 0.9 (6) 0.71 3.5 Konosirus punctatus − 16.1 13.3 (1) 0.78 2.9 Cynoglossus joyneri − 15.0 ± 0.5 13.6 ± 0.5 (8) 1.00 2.9 Amblychaeturichthys hexanema − 15.1 ± 0.4 14.2 ± 0.4 (6) 1.00 3.1 Pennahia argentata − 15.2 ± 0.6 14.9 ± 1.0 (6) 1.00 3.3 Thryssa kammalensis − 17.7 ± 2.6 14.6 ± 1.1 (6) 0.18 3.5 Okamejei kenojei − 14.4 ± 0.5 13.4 ± 0.9 (5) 1.00 2.9 Ctenotrypauchen microcephalus − 15.7 ± 0.5 14.1 ± 0.2 (4) 0.89 3.1 Johnius grypotus − 14.7 ± 0.1 14.4 ± 0.8 (4) 1.00 3.2 Table 1. www.nature.com/scientificreports/ δ13C and δ15N values, dependence on benthic affinity prey (f), and trophic position (TP) of dominan predatory consumers. Differences in the δ13C values of predatory macrobenthic and nektonic taxa between the estuarine channel and the deep bay were significant (Student’s t-test, t27 = − 4.436, P < 0.001) but the same pattern was not observed for the δ15N values (t27 = 1.182, P = 0.248). The δ13C values of nektonic taxa collected from the estuarine channel were located outside the range of the end-member values (i.e., benthic and pelagic baselines). Therefore, considering their active movement characteristics, the dependence (f) of nektonic migrants in the estuarine channel on deep-bay benthic affinity prey was calculated using the IsoSource mixing model, using the isotope values of both benthic and pelagic baselines in the estuarine channel and the deep bay as end-members, and the model provided the median values (see Supplementary Table 8). When f 1, the f value was assigned to 1. The TP was estimated in the same manner as was done for the deep-bay predatory consumers, based on the δ15N value calculated from the contributions of four (benthic and pelagic) baseline end-members. www.nature.com/scientificreports/ As a result, 26 25 macrobenthic invertebrates, 17 nektonic invertebrates, and 15 fish taxa w patterns. www.nature.com/scientificreports/ Patterning communities on the Kohonen self-organizing map (SOM). (a), (d), (g) Ordination of samples for plankton, benthos, and nekton, respectively, on the SOM. Acronyms of individual samples are denoted by a set of the sampling year (201515 or 201616), month (February [F], May [M], August [A], November [N]), the name of station (1–9). (b), (e), (h) Clustering of the trained SOM units. Numbers go from top to bottom and from left to right on the map. Clusters are indicated by different colours and corresponding numbers. (c), (f), (i) Distribution of clusters in time and space. A total of 225 taxa (species or genus) of phytoplankton, 66 of zooplankton, 77 of benthic invertebrates, and 105 of nekton (including fish, decapods and cephalopods) were identified. Among these, breakpoints of rank-frequency curves on each sampling occasion were first set to reduce the bias from rare species and left common taxa accounting for at least 5% of total abundances. Considering differences in mobility and sampling units between planktonic, benthic, and nektonic assemblages, community analyses were conducted separately. As a result, 26 phytoplankton, 28 zooplankton and 25 macrobenthic invertebrates, 17 nektonic invertebrates, and 15 fish taxa were selected to analyse community patterns. (predatory) consumers exclusively prey (71–100%), with only the exception of 18% of T. kammalensis, on benthic affinity prey in the deep bay (Table 1). The δ13C values of the nektonic taxa collected from the estuarine channel were located outside the range of the end-member values (i.e., the benthic and pelagic baselines). Therefore, considering the motile characteristics of nekton influenced by tidal movement, the dependence (f) of the estu- arine-channel nekton on the deep-bay benthic affinity prey was calculated using the IsoSource mixing model based on both benthic and pelagic baseline values in the estuarine channel and the deep bay as end-members. The deep-bay benthic affinity prey was estimated to play an important role (median values of 51–79%) as dietary contributors to the estuarine-channel motile consumers, with the exception of Crangon hakodatei (38%) and Leiognathus nuchalis (29%), which had dependence values of 49% and 62% on the deep-bay pelagic and the estuarine-channel benthic affinity prey, respectively. (predatory) consumers exclusively prey (71–100%), with only the exception of 18% of T. kammalensis, on benthic affinity prey in the deep bay (Table 1). www.nature.com/scientificreports/ 2127 1514 1920 2526 3132 33 3 9 4 510 11 6 1 2 8 713 1217 1816 2223 2428 2930 3534 36 1 2 3 4 5 6 7 8 9 10 A C D B E Euclidian distance b 15M2 16F4 16F3 16F6 16F7 16F8 16F9 15F2 16A1 16A2 16F2 16F5 15F5 15F3 15M1 16N1 16A3 16A4 16A5 16A6 16A7 15F1 16N2 16N3 16N4 16N6 16N7 16N9 15M3 16N8 15M4 16A8 15N1 15N2 16A9 16N5 15M7 15M8 15M9 15M5 15M6 15N3 15N4 15N5 15N6 15N7 15N8 15N9 16F1 15F8 15F9 15F4 15F6 15F7 A C D B E a B A B B A A A A B A A B A B B C B B E D B C D B D E B C D B E E B C D B E E B C D B B E B C D B B E B C B B May Nov Feb Aug Nov Feb 2015 2016 1 2 3 4 5 6 7 8 9 Station c b a c Station Euclidian distance f 1 1 1 1 1 1 2 1 2 3a 2 2 3a 3a 3a 3a 3a 3a 3a 3a 3a 3c 3a 3a 3b 3b 3b 3b 3b 3b 3b 3b 3a 3a 3a 3a 3a 3c 3a 3a 3a 3a 3b 3c 3c 3c 3c 3a 3a 3a 3a 3a 3b 3a 1 2 3 4 5 6 7 8 9 Station May Nov Feb Aug Nov Feb 2015 2016 f d Euclidian distance Euclidian distance 15 2110 1622 23 4 17 1 2 3 9 7 818 2813 1419 2027 25 26 33 31 32 5 12 11 624 2934 3035 36 0 0.2 0.4 0.6 0.8 1.0 1.2 3 2 1 a b c e 15F3 16F2 15N4 15F7 15F9 15M9 15N7 16F3 16F7 16F9 16A2 16N2 15M4 15N9 16N9 15F2 15N2 16F5 16A5 16N5 15M3 15N3 15N6 16F6 16A3 16A6 16A7 16N3 16N6 16N7 16N8 15F5 15M5 15M6 15N5 15F4 16A4 16N4 16N1 15N8 15F6 15F8 16A9 15N1 16A1 15M7 16F4 16F8 16A8 15M8 16F1 15F1 15M1 15M2 2 1 3c 3a 3b d e Euclidian distance Feb 2 5 Station 7 May Nov Feb 2015 2016 Aug Nov I I I I I I I I I IV IV III III III II II II II i i Euclidian distance 13 14 9 10 11 I II III IV 15 12 16 1 2 5 6 3 4 7 8 1 2 3 Euclidean distance 4 5 6 0 7 h 15F5 15M5 15M7 16F7 16F5 15F7 I II III IV 15N7 15F2 15N2 16F2 16J2 16J5 16N2 15M2 15N5 16J7 16N5 16N7 g h g Figure 2. www.nature.com/scientificreports/ Differences in the δ13C values of predatory macrobenthic and nektonic taxa between the estuarine channel and the deep bay were significant (Student’s t-test, t27 = − 4.436, P < 0.001) but the same pattern was not observed for the δ15N values (t27 = 1.182, P = 0.248). The δ13C values of nektonic taxa collected from the estuarine channel were located outside the range of the end-member values (i.e., benthic and pelagic baselines). Therefore, considering their active movement characteristics, the dependence (f) of nektonic migrants in the estuarine channel on deep-bay benthic affinity prey was calculated using the IsoSource mixing model, using the isotope values of both benthic and pelagic baselines in the estuarine channel and the deep bay as end-members, and the model provided the median values (see Supplementary Table 8). When f 1, the f value was assigned to 1. The TP was estimated in the same manner as was done for the deep-bay predatory consumers, based on the δ15N value calculated from the contributions of four (benthic and pelagic) baseline end-members. consumers in the estuarine channel (Fig. 6, Supplementary Table 7). Phragmites is the next most influential con- tributor (16% [10–20%]) to food for the suspension feeders, but Phragmites and deep-bay phytoplankton share overlapping distributions of potential contributions (10% [0–30%] and 9% [0–54%], respectively, to deposit feeders; 18% [0–46%] and 14% [0–72%], respectively, to zooplankton) to the primary consumers. Riverine SPOM, microphytobenthos and Zostera, were minor nutritional contributors to estuarine channel consumers. In the deep bay, deep-bay phytoplankton generated the most important contributions of 69% (22–97%) and 47% (4–87%) to the nutrition of deposit feeders and zooplankton, respectively. Microphytobenthos made a consid- erable contribution of 14% (0–49%), but the others (riverine SPOM, Phragmites, estuarine phytoplankton, and Zostera) were only minor contributors (medians > 2–6%) to the nutrition of deposit feeders. The nutritional contributions of riverine SPOM, Phragmites, estuarine phytoplankton, and Zostera to the deep-bay zooplankton were equally substantial, with values of 8% (0–38%), 13% (0–44%), 6% (0–36%), 11% (0–54%), and 7% (0–34%), respectively. www.nature.com/scientificreports/ p y Using the δ13C and δ15N values of the deep-bay zooplankton and deposit feeders as the isotopic basel of benthic and pelagic trophic pathways, our two-source mixing model calculations revealed that higher-l Scientific Reports \| (2020) 10:16637 \| https://doi.org/10.1038/s41598-020-73628-6 www.nature.com/scientificreports/ 2127 1514 1920 2526 3132 33 3 9 4 510 11 6 1 2 8 713 1217 1816 2223 2428 2930 3534 36 1 2 3 4 5 6 7 8 9 10 15M2 16F4 16F3 16F6 16F7 16F8 16F9 15F2 16A1 16A2 16F2 16F5 15F5 15F3 15M1 16N1 16A3 16A4 16A5 16A6 16A7 15F1 16N2 16N3 16N4 16N6 16N7 16N9 15M3 16N8 15M4 16A8 15N1 15N2 16A9 16N5 15M7 15M8 15M9 15M5 15M6 15N3 15N4 15N5 15N6 15N7 15N8 15N9 16F1 15F8 15F9 15F4 15F6 15F7 A C D B E A C D B E Euclidian distance a B A B B A A A A B A A B A B B C B B E D B C D B D E B C D B E E B C D B E E B C D B B E B C D B B E B C B B May Nov Feb Aug Nov Feb 2015 2016 1 2 3 4 5 6 7 8 9 Station c Euclidian distance 15F3 16F2 15N4 15F7 15F9 15M9 15N7 16F3 16F7 16F9 16A2 16N2 15M4 15N9 16N9 15F2 15N2 16F5 16A5 16N5 15M3 15N3 15N6 16F6 16A3 16A6 16A7 16N3 16N6 16N7 16N8 15F5 15M5 15M6 15N5 15F4 16A4 16N4 16N1 15N8 15F6 15F8 16A9 15N1 16A1 15M7 16F4 16F8 16A8 15M8 16F1 15F1 15M1 15M2 15 2110 1622 23 4 17 1 2 3 9 7 818 2813 1419 2027 25 26 33 31 32 5 12 11 624 2934 3035 36 0 0.2 0.4 0.6 0.8 1.0 1.2 3 2 1 2 1 a b c 3c 3a 3b 1 1 1 1 1 1 2 1 2 3a 2 2 3a 3a 3a 3a 3a 3a 3a 3a 3a 3c 3a 3a 3b 3b 3b 3b 3b 3b 3b 3b 3a 3a 3a 3a 3a 3c 3a 3a 3a 3a 3b 3c 3c 3c 3c 3a 3a 3a 3a 3a 3b 3a 1 2 3 4 5 6 7 8 9 Station May Nov Feb Aug Nov Feb 2015 2016 13 14 9 10 11 I II III IV 15 12 16 1 2 5 6 3 4 7 8 1 2 3 Euclidean distance 4 5 6 0 7 Feb 2 5 Station 7 May Nov Feb 2015 2016 Aug Nov 15F5 15M5 15M7 16F7 16F5 15F7 I II III IV 15N7 15F2 15N2 16F2 16J2 16J5 16N2 15M2 15N5 16J7 16N5 16N7 I I I I I I I I I IV IV III III III II II II II b d f e g i h Figure 2. www.nature.com/scientificreports/ Patterning communities on the Kohonen self-organizing map (SOM). (a), (d), (g) Ordination of samples for plankton, benthos, and nekton, respectively, on the SOM. Acronyms of individual samples are denoted by a set of the sampling year (201515 or 201616), month (February [F], May [M], August [A], November [N]), the name of station (1–9). (b), (e), (h) Clustering of the trained SOM units. Numbers go from top to bottom and from left to right on the map. Clusters are indicated by different colours and corresponding numbers. (c), (f), (i) Distribution of clusters in time and space. A total of 225 taxa (species or genus) of phytoplankton, 66 of zooplankton, 77 of benthic invertebrates, and 105 of nekton (including fish, decapods and cephalopods) were identified. Among these, breakpoints of rank-frequency curves on each sampling occasion were first set to reduce the bias from rare species and left common taxa accounting for at least 5% of total abundances. Considering differences in mobility and sampling units between planktonic, benthic, and nektonic assemblages, community analyses were conducted separately. As a result, 26 phytoplankton, 28 zooplankton and 25 macrobenthic invertebrates, 17 nektonic invertebrates, and 15 fish taxa were selected to analyse community patterns. www.nature.com/scientificreports/ 2127 1514 1920 2526 3132 33 3 9 4 510 11 6 1 2 8 713 1217 1816 2223 2428 2930 3534 36 1 2 3 4 5 6 7 8 9 10 15M2 16F4 16F3 16F6 16F7 16F8 16F9 15F2 16A1 16A2 16F2 16F5 15F5 15F3 15M1 16N1 16A3 16A4 16A5 16A6 16A7 15F1 16N2 16N3 16N4 16N6 16N7 16N9 15M3 16N8 15M4 16A8 15N1 15N2 16A9 16N5 15M7 15M8 15M9 15M5 15M6 15N3 15N4 15N5 15N6 15N7 15N8 15N9 16F1 15F8 15F9 15F4 15F6 15F7 A C D B E A C D B E Euclidian distance a Station c Euclidian distance 15F3 16F2 15N4 15F7 15F9 15M9 15N7 16F3 16F7 16F9 16A2 16N2 15M4 15N9 16N9 15F2 15N2 16F5 16A5 16N5 15M3 15N3 15N6 16F6 16A3 16A6 16A7 16N3 16N6 16N7 16N8 15F5 15M5 15M6 15N5 15F4 16A4 16N4 16N1 15N8 15F6 15F8 16A9 15N1 16A1 15M7 16F4 16F8 16A8 15M8 16F1 15F1 15M1 15M2 15 2110 1622 23 4 17 1 2 3 9 7 818 2813 1419 2027 25 26 33 31 32 5 12 11 624 2934 3035 36 0 0.2 0.4 0.6 0.8 1.0 1.2 3 2 1 2 1 a b c 3c 3a 3b Station 13 14 9 10 11 I II III IV 15 12 16 1 2 5 6 3 4 7 8 1 2 3 Euclidean distance 4 5 6 0 7 St ti 15F5 15M5 15M7 16F7 16F5 15F7 I II III IV 15N7 15F2 15N2 16F2 16J2 16J5 16N2 15M2 15N5 16J7 16N5 16N7 b d f e g i h Figure 2. Patterning communities on the Kohonen self-organizing map (S of samples for plankton, benthos, and nekton, respectively, on the SOM. Ac are denoted by a set of the sampling year (201515 or 201616), month (Februa November [N]), the name of station (1–9). (b), (e), (h) Clustering of the tra top to bottom and from left to right on the map. Clusters are indicated by df numbers. (c), (f), (i) Distribution of clusters in time and space. A total of 22 phytoplankton, 66 of zooplankton, 77 of benthic invertebrates, and 105 of n cephalopods) were identified. Among these, breakpoints of rank-frequency were first set to reduce the bias from rare species and left common taxa acco abundances. Considering differences in mobility and sampling units betwee assemblages, community analyses were conducted separately. www.nature.com/scientificreports/ The resultant associations of clusters generated multitrophic communities with arrays of two or four contrasting types for the estuarine channel and the deep bay, respectively, classifying the seasonal typology of biocenoses in respective localities. The number of dominant taxa in different taxonomic groups is indicated for cluster associations. The dominant taxa, expressed in numbers in the figure, are listed in Supplementary Table 3. Figure 3. The associations of clusters and the number of dominant taxa constituting multitrophic communities. Based on the co-occurrence patterns at seasonal and spatial scales, we integrated sympatric clusters constituting plankton, benthos, and nekton communities during a given time period. The spatial characterization of the clusters was achieved between the estuarine channel and the deep bay but less clearly within the deep bay, except for the plankton assemblage in February 2015. The resultant associations of clusters generated multitrophic communities with arrays of two or four contrasting types for the estuarine channel and the deep bay, respectively, classifying the seasonal typology of biocenoses in respective localities. The number of dominant taxa in different taxonomic groups is indicated for cluster associations. The dominant taxa, expressed in numbers in the figure, are listed in Supplementary Table 3. A B C D E Temperature (oC) 0 7 14 21 28 35 A B C D E Salinity (psu) 0 7 14 21 28 35 A B C D E Chl-a (μg l-1) 0 1 2 3 4 5 6 A B C D E SPM (mg l-1) 0 10 20 30 40 50 A B C D E SPOM (mg l-1) 0 2 4 6 8 10 A B C D E PO4 (μM) 0.0 0.3 0.6 0.9 1.2 1.5 1.8 A B C D E NH4 (μM) 0 2 4 6 8 10 12 A B C D E NO2 (μM) 0 1 2 3 4 5 A B C D E NO3 (μM) 0 20 40 60 80 100 120 A B C D E SiO2 (μM) 0 30 60 90 120 150 c b b a ab a b b b a ab b c a b c b b ab a a a b bc c a a b bc c b a c bc c a b bc bc c ab a b b b Clusters Figure 4. Environmental attributes of the water column in five clusters of plankton assemblages. www.nature.com/scientificreports/ Box-and- whisker plots of temperature, salinity, phosphate (PO4), ammonium (NH4), nitrate (NO2), nitrate (NO3), silicate (SiO2), chlorophyll a (Chl-a), suspended particulate matter (SPM), and particulate organic matter (SPOM) in the water column of five clusters (groups of stations). The median value of each cluster is displayed with the horizontal bar inside the box. Since cluster groupings of the plankton community represented seasonal as well as spatial patterns, we tested significant differences in environmental variables of the water column among those clusters using a Kruskal–Wallis test followed by a Mann–Whitney pairwise comparison test. The same superscript indicates a non-significant difference between medians (P > 0.05). A B C D E Temperature (oC) 0 7 14 21 28 35 A B C D E Salinity (psu) 0 7 14 21 28 35 A B C D E PO4 (μM) 0.0 0.3 0.6 0.9 1.2 1.5 1.8 A B C D E NH4 (μM) 0 2 4 6 8 10 12 A B C D E NO2 (μM) 0 1 2 3 4 5 c b b a ab ab b c a b b a c bc c a b bc bc c ab a b b b A B C D E Chl-a (μg l-1) 0 1 2 3 4 5 6 A B C D E SPM (mg l-1) 0 10 20 30 40 50 A B C D E SPOM (mg l-1) 0 2 4 6 8 10 B C D E a b b b a c b b ab a a b bc c Clusters A B C D E Chl-a (μg l-1) 0 1 2 3 4 5 6 A B C D E SPM (mg l-1) 0 10 20 30 40 50 A B C D E SPOM (mg l-1) 0 2 4 6 8 10 A B C D E NO3 (μM) 0 20 40 60 80 100 120 A B C D E SiO2 (μM) 0 30 60 90 120 150 a b b b a c b b ab a a a b bc c a a b bc c Clusters Figure 4. Environmental attributes of the water column in five clusters of plankton assemblages. www.nature.com/scientificreports/ B d h l d i l l i d i l i i Phytoplankton / Zooplankton Bivalves / Crustaceans / Polychaetes Crustaceans / Cephalpods / Fish Estuarine channel Deep bay May-August November-February November February May August A A B B C D E 1 2 1 2 I I 3a b c 3a b c 3a b c 3a b c I II I II III IV Phytoplankton / Zooplankton Bivalves / Crustaceans / Polychaetes Crustaceans / Cephalpods / Fish 11 / 7 15 / 14 1 / 0 / 3 1 / 0 / 3 3 / 0 / 3 3 / 0 / 3 11 / 10 0 / 2 / 8 8 / 2 / 7 10 / 9 0 / 2 / 8 6 / 2 / 5 11 / 10 0 / 2 / 8 8 / 2 / 7 11 / 9 0 / 2 / 8 6 / 1 / 6 Figure 3. The associations of clusters and the number of dominant taxa constituting multitrophic communities. Based on the co-occurrence patterns at seasonal and spatial scales, we integrated sympatric clusters constituting plankton, benthos, and nekton communities during a given time period. The spatial characterization of the clusters was achieved between the estuarine channel and the deep bay but less clearly within the deep bay, except for the plankton assemblage in February 2015. The resultant associations of clusters generated multitrophic communities with arrays of two or four contrasting types for the estuarine channel and the deep bay, respectively, classifying the seasonal typology of biocenoses in respective localities. The number of dominant taxa in different taxonomic groups is indicated for cluster associations. The dominant taxa, expressed in numbers in the figure, are listed in Supplementary Table 3. 35 35 1.8 12 5 b b a ab a Figure 3. The associations of clusters and the number of dominant taxa constituting multitrophic communities. Based on the co-occurrence patterns at seasonal and spatial scales, we integrated sympatric clusters constituting plankton, benthos, and nekton communities during a given time period. The spatial characterization of the clusters was achieved between the estuarine channel and the deep bay but less clearly within the deep bay, except for the plankton assemblage in February 2015. www.nature.com/scientificreports/ The δ13C values of the nektonic taxa collected from the estuarine channel were located outside the range of the end-member values (i.e., the benthic and pelagic baselines). Therefore, considering the motile characteristics of nekton influenced by tidal movement, the dependence (f) of the estu- arine-channel nekton on the deep-bay benthic affinity prey was calculated using the IsoSource mixing model based on both benthic and pelagic baseline values in the estuarine channel and the deep bay as end-members. The deep-bay benthic affinity prey was estimated to play an important role (median values of 51–79%) as dietary contributors to the estuarine-channel motile consumers, with the exception of Crangon hakodatei (38%) and Leiognathus nuchalis (29%), which had dependence values of 49% and 62% on the deep-bay pelagic and the estuarine-channel benthic affinity prey, respectively. https://doi.org/10.1038/s41598-020-73628-6 Scientific Reports \| (2020) 10:16637 \| www.nature.com/scientificreports/ Phytoplankton / Zooplankton Bivalves / Crustaceans / Polychaetes Crustaceans / Cephalpods / Fish Estuarine channel Deep bay May-August November-February November February May August A A B B C D E 1 2 1 2 I I 3a b c 3a b c 3a b c 3a b c I II I II III IV Phytoplankton / Zooplankton Bivalves / Crustaceans / Polychaetes Crustaceans / Cephalpods / Fish 11 / 7 15 / 14 1 / 0 / 3 1 / 0 / 3 3 / 0 / 3 3 / 0 / 3 11 / 10 0 / 2 / 8 8 / 2 / 7 10 / 9 0 / 2 / 8 6 / 2 / 5 11 / 10 0 / 2 / 8 8 / 2 / 7 11 / 9 0 / 2 / 8 6 / 1 / 6 Figure 3. The associations of clusters and the number of dominant taxa constituting multitrophic communities. www.nature.com/scientificreports/ Biplots of δ13C and δ15N of primary sources of organic matter and dominant taxa. (a) The estuarine h Figure 5. Biplots of δ13C and δ15N of primary sources of organic matter and dominant taxa. (a) The estuarine channel. (b) The deep bay. Strokes indicate the corresponding feeding mode: sky blue = deposit feeder, red = suspension feeder, dark blue = predator. List of dominant primary consumers: Bivalve Corbicula japonica (COJ); Crustaceans Melita sp. (MEL), Xenophthalmus pinnotheroides (XEP); Polychaetes Neanthes japonica (NEP), Prionospio japonicas (PRJ), Capitella capitata (CAC), Magelona japonica (MAJ), Sternaspis scutata (STS), Paraprionospio pinnata (PAP), Chaetozone sp. (CHA), and Praxillella affinis (PRA). Predatory taxa are listed in Table 1, and species codes are given by acronyms of first two letters of genus name and first letter of species name. The acronyms of organic matter sources are: Phragmites australis (PHA), locally produced (estuarine vs. deep-bay) phytoplankton, microphytobenthos (MPB), Zostera marina (Zostera), and riverine suspended particulate organic matter (RPOM). Figure 5. Biplots of δ13C and δ15N of primary sources of organic matter and dominant taxa. (a) The estuarine channel. (b) The deep bay. Strokes indicate the corresponding feeding mode: sky blue = deposit feeder, red = suspension feeder, dark blue = predator. List of dominant primary consumers: Bivalve Corbicula japonica (COJ); Crustaceans Melita sp. (MEL), Xenophthalmus pinnotheroides (XEP); Polychaetes Neanthes japonica (NEP), Prionospio japonicas (PRJ), Capitella capitata (CAC), Magelona japonica (MAJ), Sternaspis scutata (STS), Paraprionospio pinnata (PAP), Chaetozone sp. (CHA), and Praxillella affinis (PRA). Predatory taxa are listed in Table 1, and species codes are given by acronyms of first two letters of genus name and first letter of species name. The acronyms of organic matter sources are: Phragmites australis (PHA), locally produced (estuarine vs. deep-bay) phytoplankton, microphytobenthos (MPB), Zostera marina (Zostera), and riverine suspended particulate organic matter (RPOM). Source contribution (%) 0 20 40 60 80 100 Estuarine-channel suspension feeders Estuarine-channel deposit feeders Estuarine-channel zooplankton Deep-bay deposit feeders Deep-bay zooplankton Estuarine phytoplankton Microphytobenthos Deep-bay phytoplankton Phragmites australis RPOM Zostera marina Figure 6. Feasible contributions (%) of primary producers to the nutrition of primary consumers. Primary producers include estuarine-channel suspension feeders, estuarine-channel deposit feeders, estuarine-channel zooplankton, deep-bay deposit feeders, and deep-bay zooplankton. The dominant primary producers considered include Phragmites australis, locally produced (estuarine vs. deep-bay) phytoplankton, microphytobenthos, Zostera marina, and riverine suspended particulate organic matter (RPOM). Feasible contributions represent median values. www.nature.com/scientificreports/ Box-and- whisker plots of temperature, salinity, phosphate (PO4), ammonium (NH4), nitrate (NO2), nitrate (NO3), silicate (SiO2), chlorophyll a (Chl-a), suspended particulate matter (SPM), and particulate organic matter (SPOM) in the water column of five clusters (groups of stations). The median value of each cluster is displayed with the horizontal bar inside the box. Since cluster groupings of the plankton community represented seasonal as well as spatial patterns, we tested significant differences in environmental variables of the water column among those clusters using a Kruskal–Wallis test followed by a Mann–Whitney pairwise comparison test. The same superscript indicates a non-significant difference between medians (P > 0.05). Figure 4. Environmental attributes of the water column in five clusters of plankton assemblages. Box-and- whisker plots of temperature, salinity, phosphate (PO4), ammonium (NH4), nitrate (NO2), nitrate (NO3), silicate (SiO2), chlorophyll a (Chl-a), suspended particulate matter (SPM), and particulate organic matter (SPOM) in the water column of five clusters (groups of stations). The median value of each cluster is displayed with the horizontal bar inside the box. Since cluster groupings of the plankton community represented seasonal as well as spatial patterns, we tested significant differences in environmental variables of the water column among those clusters using a Kruskal–Wallis test followed by a Mann–Whitney pairwise comparison test. The same superscript indicates a non-significant difference between medians (P > 0.05). Dominant predatory consumers displayed an average dietary trophic position estimate between 2.5 and 3.5 in the estuarine and deep-bay channels (Table 1). Trophic position values of macrobenthos and crustaceans fell within the range from 2.5 to 3.2, making them omnivorous and secondary consumers. The average trophic position estimates of fish ranged from 2.9 to 3.5. being on average ~ 0.4 higher than those of macrobenthos and crustaceans. The observed spatial variation in fish trophic position exhibited a high degree of overlap in their trophic position between estuarine and deep-bay channels. www.nature.com/scientificreports/ 1–99 percentile ranges for the distribution of feasible contributions of each source are given in Supplementary Table 7. Detailed procedures for estimation are given in the Methods. Figure 6. Feasible contributions (%) of primary producers to the nutrition of primary consumers. Primary producers include estuarine-channel suspension feeders, estuarine-channel deposit feeders, estuarine-channel zooplankton, deep-bay deposit feeders, and deep-bay zooplankton. The dominant primary producers considered include Phragmites australis, locally produced (estuarine vs. deep-bay) phytoplankton, microphytobenthos, Zostera marina, and riverine suspended particulate organic matter (RPOM). Feasible contributions represent median values. 1–99 percentile ranges for the distribution of feasible contributions of each source are given in Supplementary Table 7. Detailed procedures for estimation are given in the Methods. www.nature.com/scientificreports/ Scientific Reports \| (2020) 10:16637 \| https://doi.org/10.1038/s41598-020-73628-6 www.nature.com/scientificreports/ δ13C (o/oo) -28 -26 -24 -22 -20 -18 -16 -14 -12 -10 -8 -6 δ15N (o/oo) 4 6 8 10 12 14 16 δ13C (o/oo) -28 -26 -24 -22 -20 -18 -16 -14 -12 -10 -8 -6 δ15N (o/oo) 4 6 8 10 12 14 16 PHA RPOM Estuarine phytoplankton Deep-bay phytoplankton MPB Zostera PHA RPOM MPB Zostera PLY LEN KOP CHJ CRH TRC PRJ CAC NEJ COJ COJ Zooplankton Zooplankton MEL XEP CAC MAJ STC PAP CHA PRA SCL SIT GLC THK OKK CYJ CTM AMH JOG PEA LEN PAG MEJ PAT CHB LOJ ORO ALJ POTTRC ALD OCV EUM Zooplankton Macrobenthos Nektonic crucstceans Cephalopods Fish PRJ a b Deep-bay phytoplankton Estuarine phytoplankton Figure 5. Biplots of δ13C and δ15N of primary sources of organic matter and dominant taxa. (a) The estuarine channel. (b) The deep bay. Strokes indicate the corresponding feeding mode: sky blue = deposit feeder, red = suspension feeder, dark blue = predator. List of dominant primary consumers: Bivalve Corbicula japonica (COJ); Crustaceans Melita sp. (MEL), Xenophthalmus pinnotheroides (XEP); Polychaetes Neanthes japonica (NEP), Prionospio japonicas (PRJ), Capitella capitata (CAC), Magelona japonica (MAJ), Sternaspis scutata (STS), Paraprionospio pinnata (PAP), Chaetozone sp. (CHA), and Praxillella affinis (PRA). Predatory taxa are listed in Table 1, and species codes are given by acronyms of first two letters of genus name and first letter of species name. The acronyms of organic matter sources are: Phragmites australis (PHA), locally produced (estuarine vs. deep-bay) phytoplankton, microphytobenthos (MPB), Zostera marina (Zostera), and riverine suspended particulate organic matter (RPOM). δ13C (o/oo) -28 -26 -24 -22 -20 -18 -16 -14 -12 -10 -8 -6 δ15N (o/oo) 4 6 8 10 12 14 16 PHA RPOM MPB Zostera PLY LEN KOP CHJ CRH TRC PRJ CAC NEJ COJ COJ Zooplankton Zooplankton Macrobenthos PRJ a Deep-bay phytoplankton Estuarine phytoplankton 6 δ13C (o/oo) -28 -26 -24 -22 -20 -18 -16 -14 -12 -10 -8 -6 δ15N (o/oo) 4 6 8 10 12 14 16 PHA RPOM Estuarine phytoplankton Deep-bay phytoplankton MPB Zostera Zooplankton MEL XEP CAC MAJ STC PAP CHA PRA SCL SIT GLC THK OKK CYJ CTM AMH JOG PEA LEN PAG MEJ PAT CHB LOJ ORO ALJ POTTRC ALD OCV EUM N kt i t C h l d Fi h b b Zooplankton Nektonic crucstceans Cephalopods igure 5. www.nature.com/scientificreports/ also with low salinity and high inorganic nutrient and chlorophyll a concentrations. Along with the seasonal reduction of river discharge in November and February, the plankton community in the estuarine channel shifted towards deep-bay cluster B. The sample-cluster association further clarified the importance of seasonality in the transition of the deep-bay plankton community. The spatial uniformity of the plankton community in each season and the prevalence of marine taxa in indicator species provide clear indications of marine ecosystem attributes45. Consistently high salinity and low inorganic nutrient concentrations and turbidity in the deep bay reflect that river discharge is rapidly diluted with and diffused into inflowing marine water that is highly dynamic and quickly renewed owing to the short water residence time of 2.5–3.4 d driven by semidiurnal tides46. Previous studies also proposed seasonal succession in the phytoplankton community (i.e., relative abundances of centric and pennate diatoms and flagellates) in response to temperature fluctuation, inorganic nutrient loading status and N:P stoichiometry47 as well as zooplankton assemblages linked to temperature, salinity and Noctiluca vs. diatom densities43,48.f Our SOM training intuitively differentiated the macrobenthic community into clusters 1 and 2 (estuarine channel) and cluster 3 (deep bay), reflecting a salinity gradient49. The dominance of the brackish-water indica- tor species C. japonica and Prionospio japonicus in the estuarine-channel clusters supports the longitudinal transition of the macrobenthic community. Although patchy distributions of some indicator taxa in the central area (stations 5 and 8) of the deep bay created subclusters, many indicator and dominant taxa were widespread in deep-bay cluster 3 throughout the year, further highlighting a lack of seasonality in the succession of the macrobenthic community. Historically, the deep bay has experienced extensive anthropogenic activity, such as large-scale reclamation and dredging in the 1980s and 1990s, which led to changes in sediment granulometry (from sand to fine-grained clay and silt). Alterations in habitat features due to major geological events have modified the spatial distribution patterns and dominant taxa of macrobenthic assemblages50. Given that most macrobenthic taxa are sedentary with little mobility, seasonally persisting macrobenthic community patterns appear to reflect the stabilized sedimentary condition of the bay habitats50.h ppl y y The nekton community patterns indicated the occurrence of four assemblage types that represent the spatial variation (estuarine channel vs. deep bay) and seasonal variation (within the deep bay). www.nature.com/scientificreports/ Cluster I, representative of the estuarine channel, was characterized by a very restricted number of dominant taxa without an assign- ment of an indicator species due to a random distribution (low frequency) and low abundances of species. Clusters II–IV mainly depicted the seasonal transition of the deep-bay nekton assemblages. However, our IndVal analysis highlights extensive overlap in indicator species between clusters (at least two clusters share 18 of 21 taxa), and further multi-response permutation procedure (MRPP) testing concludes less clear seasonal than habitat environment-based patterns as previously observed51. The observed seasonal succession of the deep- bay assemblages, although not great, seems to be attributed to the addition of a few seasonal (or temporary) migrants. As a result, the nekton assemblages were characterized by rare species in the estuarine channel and by the predominance of year-round resident species, which occupied over 80% of the total abundances51,52, with a few transient migrants in the deep bay.h g p y The association of co-occurring clusters of plankton, benthos, and nekton assemblages illustrates multitrophic communities that can be formed by any type of interspecific interactions in a given site and time (Fig. 3). As discussed earlier, the distributions of indicator taxa vary systematically across the estuarine channel and deep bay, characterizing the spatial and seasonal organization of biocenoses in accordance with environmental condi- tions. Therefore, the association between the identified indicator taxa and clusters underpins the intrinsic linkage between the SOM clusters. While trophic networks have long been recognized as a key component in ecological systems, the estimation of food web functioning has been challenging because of the difficulties in measuring interactions between species that constitute the nodes of such networks7,53. In this context, while taxa with high IndVal values represent an indicator of particular environmental conditions, their role in trophic transfers would be equivocal because they are found at smaller abundances than dominant taxa40. Dominant taxa with high abundances and frequencies in the associations of clusters can be assigned to major trophic components that are most influential in community dynamics and constitute key nodes of multitrophic levels in complex ecological networks15,42. g Stable isotopes reveal trophism and trophic connections of the co-occurring dominant taxa in the multi- trophic community. Consistent with the spatial characterization of the associations of co-occurring clusters, the occurrence of dominant taxa differed considerably between the estuarine channel and the deep bay. Discussion h l l While a large variety of theoretical stimulations and empirical tests have been adopted to construct community trophic networks, identifying key species and feeding links is crucial to the architecture of a food web diagram that further characterizes ecological dynamics9,41,42. We combined community patterns with stable isotope deter- mination to characterize typologies in a community-wide food web structure in a temperate estuarine embay- ment. Our community pattern analyses, based on the SOM procedure, revealed distinct typological patterns between the estuarine channel and the deep bay as well as differing seasonal patterns among plankton, benthos, and nekton assemblages. Subsequent isotope mixing-model estimation of trophic linkages between co-occurring dominant taxa (as key nodes of networks) highlighted a spatial variation in food web architecture that was in accordance with community typologies.hih y yp g The plankton community was first divided into estuarine-channel and deep-bay clusters. The estuarine-chan- nel clusters were clearly associated not only with the occurrence of freshwater, brackish, and benthic indicator taxa (e.g., Diatoma sp., Nitzchia sp., Cylindrotheca closterium, Sinocalanus tenellus, and T. dextrilobatus)43,44 but Scientific Reports \| (2020) 10:16637 \| https://doi.org/10.1038/s41598-020-73628-6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ In the estuarine channel, the dominant plankton taxa varied between the summer wet and winter dry periods, but the macrobenthos and nekton taxa were seasonally identical with a very restricted number. Our isotope mixing- model calculations demonstrated that pelagic primary production forms the important base of the estuarine trophic network. This result is supported by the high chlorophyll a levels retained by pelagic and benthic diatoms throughout the year. High abundances of both herbivorous and predatory zooplankton complicate the plank- tonic food webs and links with other predators. Dominant zooplankton had consistent δ13C values with those of phytoplankton throughout the year. This result suggests that switching plankton assemblages guarantees the persistence of patterns of planktonic food chains (e.g., trophic base, resource utilization, and trophic niche)43. Th l l f δ13 l b d d k h h l The split alignment of δ13C values between primary consumers and dominant nekton in the estuarine channel indicated the lack of trophic connection between them. Indeed, our mixing-model calculations denoted that most of the nutrition of these motile consumers was derived from the deep-bay benthic prey (Table 1), most likely reflecting their rapid channel-bay movement due to the high tidal prism and short residence time35,46. An increase in the consumption of estuarine prey was found only for the fish L. nuchalis, which feeds mainly on benthic prey at the adult stage in the estuary54, and the shrimp C. hakodatei. The overall combination of the mixing-model calculations allowed us to draw a generalized food web structure that remains unchanged with season in the estuarine community (Fig. 7a). The low number and abundances of dominant nekton suggest that Scientific Reports \| (2020) 10:16637 \| https://doi.org/10.1038/s41598-020-73628-6 www.nature.com/scientificreports/ Trophic position 1 Suspension feeder 2 Phragmites River POM Estuary Bay Phytoplankton Micro- phytobenthos Zostera Deposit feeder Zooplankton 3 4 Crustacean Demersal fish Pelagic fish Deep bay Trophic position 1 2 Phragmites River POM Estuary Bay Phytoplankton Micro- phytobenthos Zostera Deposit feeder Zooplankton 3 4 Crustacean Pelagic fish Piscivorous & demersal fish a b δ13C δ13C Median > 41% 21 to 40% 6 to 20% δ15N δ15N Figure 7. Representative food web of water channel community of Gwangyang Bay. (a) Estuarine channel. (b) Deep bay. Sizes of linkage arrows indicate the relative contribution of prey to consumer nutrition. Sky blue arrows indicate pelagic pathways and orange benthic pathways. www.nature.com/scientificreports/ biomass transfer to higher-level consumers is modest, simplifying the higher-level feeding links in the channel community.i Our overall mixing-model calculations for the highly diversified deep-bay community are summarized by the seasonally consistent architecture of the food web in the deep-bay ecosystem (Fig. 7b). Chlorophyll a concentra- tions peaked in spring and remained at relatively low levels despite high photosynthetic activity37 during the rest of the year, supporting high grazing activity. Despite compositional changes in the dominant phytoplankton taxa, the consistent and close proximity in the δ13C values between bay phytoplankton and primary consum- ers throughout the year suggests a functional redundancy of phytoplankton assemblages, as was discussed for the estuarine channel. Indeed, our isotope mixing-model calculations emphasized the trophic importance of deep-bay phytoplankton to both benthic deposit feeders and zooplankton. The discrimination of the δ13C values between benthic and pelagic feeders may be explained by the feeding on 13C-enriched sources (e.g., sedimenta- tion of phytoplankton blooms55,56 and zooplankton faecal pellets57 or some fractions of microbial and microalgal biomass16,58) by deposit feeders, taxon-specific differences in trophic fractionation of C isotopes59,60, and the degradation or microbial recycling status of sedimentary organic matter61,62.h g y g y g The general pattern of the δ13C alignment and δ15N increase between the benthic and nektonic consumers reveals their strong trophic linkages, emphasizing the prevalence of benthic pathways. Furthermore, the relatively stabilized numbers and abundances of dominant benthic taxa reject seasonal dietary shifts of nektonic predators in response to changes in prey resource availability. The estimated trophic positions of motile predators indicate prevalent omnivory. Such a functional role of diverse benthic taxa as trophic mediators in the mid-trophic levels would attest to food-web stability in response to compositional changes in the lower trophic-level assemblages. The only exception of benthic feeding was observed for the planktivorous fish T. kammalensis63. An increased reliance of L. nuchalis, the most abundant fish, on benthic prey likely reflects an ontogenetic dietary shift from planktivorous feeding in the estuarine channel to feeding on polychaetes and crab larvae in the deep bay54.f To conclude, our results successfully differentiated two contrasting community structures between the estua- rine channel and the deep bay. Our results also indicated that the autochthonous production of phytoplankton serves as a principal basal resource supporting the unvegetated, low-turbidity estuarine and deep-bay food webs, contrasting with diversified food-web bases found in marsh-covered or other highly turbid coastal systems16,64,65. www.nature.com/scientificreports/ Conceptual food-web models were generated for two longitudinally different communities, based on co-occurring dominant taxa in the associations of clusters constituting multitrophic communities presented in Fig. 3. Despite seasonal changes in community composition (largely plankton assemblages) and population size structure of certain species, most trophic connections and food-web pathways remain unchanged in two different localities. Dominant taxa in each trophic group are summarized in Supplementary Table 9. Trophic position 1 Suspension feeder 2 Phragmites River POM Estuary Bay Phytoplankton Micro- phytobenthos Zostera Deposit feeder Zooplankton 3 4 Crustacean Demersal fish Pelagic fish Deep bay a δ13C Median > 41% δ15N Trophic position 1 2 Phragmites River POM Estuary Bay Phytoplankton Micro- phytobenthos Zostera Deposit feeder Zooplankton 3 4 Crustacean Pelagic fish Piscivorous & demersal fish b δ13C 21 to 40% 6 to 20% δ15N b a Median > 41% Figure 7. Representative food web of water channel community of Gwangyang Bay. (a) Estuarine channel. Figure 7. Representative food web of water channel community of Gwangyang Bay. (a) Estuarine channel. (b) Deep bay. Sizes of linkage arrows indicate the relative contribution of prey to consumer nutrition. Sky blue arrows indicate pelagic pathways and orange benthic pathways. Conceptual food-web models were generated for two longitudinally different communities, based on co-occurring dominant taxa in the associations of clusters constituting multitrophic communities presented in Fig. 3. Despite seasonal changes in community composition (largely plankton assemblages) and population size structure of certain species, most trophic connections and food-web pathways remain unchanged in two different localities. Dominant taxa in each trophic group are summarized in Supplementary Table 9. Figure 7. Representative food web of water channel community of Gwangyang Bay. (a) Estuarine channel. (b) Deep bay. Sizes of linkage arrows indicate the relative contribution of prey to consumer nutrition. Sky blue arrows indicate pelagic pathways and orange benthic pathways. Conceptual food-web models were generated for two longitudinally different communities, based on co-occurring dominant taxa in the associations of clusters constituting multitrophic communities presented in Fig. 3. Despite seasonal changes in community composition (largely plankton assemblages) and population size structure of certain species, most trophic connections and food-web pathways remain unchanged in two different localities. Dominant taxa in each trophic group are summarized in Supplementary Table 9. Methods Field samplings and data acquisition. Nine sampling stations were chosen along a main water-channel trajectory of the river-estuary-coastal sea continuum in the bay (Fig. 1, Supplementary information). Sampling and data collection were conducted seasonally (February, May, and November 2015, and February, August, and November 2016) at nine stations for two years. Sampling months were chosen as representatives of seasonal physical characteristics according to clear seasonal variations in precipitation, freshwater discharge, water tem- perature, and salinity typical of summer rainy and winter dry conditions, and spring and autumn transitional conditions in the temperate northeast Asian monsoon climate zone37. Data included physical and chemical parameters and abundances of phytoplankton, mesozooplankton, and benthic invertebrates. Nektonic (fish and invertebrate) samples were collected at only three locations (stations 2, 5, and 7) because of their migration characteristics and long field collection area (tens of metres). Additional collections of specimens for dominant taxa were conducted to analyse the stable isotope ratios. To acquire stable isotope data of potential sources of organic matter, suspended particulate organic matter (SPOM), the dominant marsh plant Phragmites australis, microphytobenthos, and the seagrass Zostera marina were also collected at respective locations. On each sampling occasion, the water temperature and salinity were measured in situ using a YSI Model 85 probe (YSI Inc., Yellow Springs, OH, USA). The dissolved oxygen concentrations were high (over 6.5 mg l−1) in both the surface and the bottom layers throughout the year, with no hypoxia; thus, they were not reported in the present study. Water was collected using a 10 l van Dorn water sampler at the subsurface (1 m below the water surface) and at the bottom (variable with depth: 2.4–8.0 m in the northern estuarine channel and 10–30 m in the deep-bay channel) of each station. Water samples for suspended particulate matter (SPM), chlorophyll a (Chl a), and dissolved inorganic nutrient measurements were immediately prefiltered with a 180-μm Nitex mesh to eliminate large particles and zooplankton; samples were collected in acid-washed plastic bottles and stored in the dark on dry ice. For phytoplankton species abundances, 1 l of seawater collected was contained in glass dark bottles, fixed with Lugol’s solution (3% of final concentration), and concentrated for later analysis. Methods Additional details of the analytical procedures and adopted methods for the quantification of SPM (SPOM), Chl a, and dis- solved inorganic nutrient concentrations and the identification of phytoplankton species after transportation of water samples to the laboratory are given in the Supplementary information. Mesozooplankton samples were collected using a conical plankton net (mesh sizes 200 μm, mouth diameter of 45 cm) equipped with a Model 2030R Mechanical Flowmeter (General Oceanics Inc., Miami, FL). The net was obliquely towed several times from the bottom to the surface at a speed of 1 m s−1. After collection, the specimens were poured into 300 ml plastic bottles and immediately fixed with a 10% solution of borax-buffered formalin.t if Macrobenthic invertebrates were collected by sieving sediment through a 1-mm mesh net after collection using a 0.12 m2 van Veen grab. Three to five replicates at each station were sampled to analyse the benthic com- munity of the bay. Because of the dominance of fine sediments, sediments collected were broken up in water inside the bucket by adding seawater and stirring gently before sieving. The sieved material was placed in 2 l plastic containers and immediately preserved with a 10% solution of borax-buffered formalin. After a few days, the samples were removed from the fixing solution, rinsed and preserved in an ethanol solution (70% ethanol and 5% glycerin) in the laboratory.i g y y Nekton (both fish and invertebrates) were collected using a beam trawl, the opening (mouth) of which was 8.5 m long and 1.1 m (maximum 3 m) high. The mesh size of the trawling net was 18 mm on the wings and belly of the net and 10 mm in the cod end. A much smaller mesh net than that used in commercial fishing gear (25–240 mm mesh net) was used to capture small specimens. Two replicates were conducted at each site for approximately 1 h per trawl set at a speed of 4.0 km h−1. All materials captured were placed in labelled plastic containers and fixed in 10% formalin as described above. The detailed laboratory procedures for the identifica- tion and numeration of animal samples are presented in the Supplementary information. Sample processing for isotopic measurement. Additional samplings for stable isotope analyses were conducted by adopting the same manner and time as used for the collection of environmental parameters and community analysis. www.nature.com/scientificreports/ Furthermore, food-web topologies persist across seasons in accordance with the great compositional consist- ency of benthic and nektonic communities in respective areas, posing a challenge to the formation of a general consensus of seasonality in the web structure16. It would be expected that ontogenetic changes in the food web components may change interaction strengths at the species level, adding complexity9. Various approaches for ecological network analysis have attempted to quantitatively assess the interactions among species (or guilds), their demographic effects and the dynamics of communities7,66. Our empirical analysis provides the basis of the food web structure to select nodes at the species and/or guild levels, construct networks, and quantify ecosystem functioning58; additionally, we were able to identify unchanging food-web architecture of merit for ecological modelling in our model system67. Finally, isotope measurements can partition primary source contribution to dominant detritivory in benthic pathways and provide potential to depict complete trophic transfer in end-to- end food webs (from microbes to large metazoans) by determining microbial compartments16,58. Scientific Reports \| (2020) 10:16637 \| https://doi.org/10.1038/s41598-020-73628-6 www.nature.com/scientificreports/ Methods After sorting and identification, only muscle tissues of live and intact molluscs, crabs and shrimp of macroinvertebrates were collected to minimize con- tamination with other materials. Whole body tissues of polychaetes and only white muscle tissues of the dorsal region of fish were prepared after the removal of their viscera by dissection. They were not acid treated. Copep- ods, amphipods, and other small invertebrates, including crustaceans, gastropods, and bivalves, were pooled to provide enough material for isotope analysis. They were decalcified with 0.12N HCl solution until the bubbling stopped to remove the probable effects of carbonates. Another series of their tissues was not acid-treated for nitrogen isotopic analysis. The δ13C values of zooplankton and fish tissues displayed substantial shifts before and after lipid extraction in the present study, reflecting the species’ differences in their contents of isotopically lighter lipids70. We used the δ13C data for the defatted samples of zooplankton and fish tissues, and the detailed defatting procedures before analysis are given in the Supplementary information. Stable isotope analysis. All the pre-treated floral and faunal samples were kept frozen, lyophilized and then pulverized to a fine powder with a ball mill before analysis (Retsch MM200 Mixer Mill, Hyland Scientific, WA). Filters containing SPOM were wrapped with tin foil, and the powdered samples were packed into tin combustion cups (8 × 5 mm). For all sealed samples, the δ13C and δ15N values were measured using a continu- ous flow isotope ratio mass spectrometer (CF-IRMS; Isoprime, GV Instrument, Manchester, UK) connected to an elemental analyser (Eurovector 3000 Series, Milan, Italy). The carbon and nitrogen elemental concentrations were analysed simultaneously with isotopic composition. Isotopic values were expressed in the conventional delta as deviations from standards (i.e., Pee Dee Belemnite for carbon and atmospheric air for nitrogen) follow- ing the formula: δX(‰) = Rsample/Rstandard −1 × 103 , where X is the 13C or 15N and R is the 13C/12C or 15N/14N ratio. Accuracy was established through repeated analyses of internal laboratory standards calibrated against International Atomic Energy Agency standards CH-6 (sucrose) and IAEA-N1 (ammonium sulphate). The instrument precision was 0.15‰ for δ13C and 0.2‰ for δ15N based on replicate measurements of these reference materials. Self‑organizing map (SOM) modelling. A self-organizing map (SOM) with a Kohonen’s competitive and unsupervised artificial neural network algorithm, which approximates the probability density function of the input data26, was employed to characterize the distribution patterns of communities. Methods The SOM performs a nonlinear projection of multivariate datasets onto a two-dimensional space. This algorithm constitutes two layers. In the present study, the input layer includes 54 nodes (one by taxa) connected to the sampling datasets of 9 sites × 6 times for plankton and benthos and 18 nodes of 3 sites × 6 times for nekton. The initialized virtual sites drawn from these input datasets are updated by an unsupervised learning procedure based on an iterative method. The subsequent learning phase determines the ‘best matching unit’ based on Euclidean distance, and the further ordering and tuning phases are repeated to adjust the neighbouring units. In the training phase, the weights of the whole neighbourhood are moved in the same direction. Finally, each input sample is linked to the corresponding hexagon (each unit) of the map. Neighbouring map units on the two-dimensional grid are similar and thus expected to be clustered together. p g Consequently, the output layer is composed of 36 neurons for plankton and benthos, organized in a rectan- gular grid of 6 × 6, and 16 neurons for nekton, in a grid of 4 × 4. The number of output neurons was chosen by two approaches. The grid size was first chosen according to Vesanto’s heuristic rule71 of 5 √ N , where N is the size of the dataset, and finally, the actual size of the SOM map was decided based on the minimum values of the quantization error (QE) and topographic error (TE) by running the entire procedure several times72. The QE and TE values were 0.110 and 0.037 for plankton, 0.108 and 0.074 for macrobenthos, and 0.148 and < 0.001 for nekton assemblages, respectively.t g p y Training of the SOM and the clustering procedures were performed using MATLAB software (Version 6.1, MathWorks, Natick, MA). Prior to training, the species abundance data were scaled to a range of 0–1 to reduce the variation and skewness of abundance and normalized to the interval ZEW to impose the same weight on different taxa that appear in different ways (e.g., order, number, units). A unified-matrix displays the Euclidean distance between weight vectors of neighbouring units of the SOM and allows us to detect the cluster boundaries on the map (data not shown here). After training, Ward’s minimum variance method with the Euclidean distance measure was applied to a hierarchical cluster analysis of the SOM units73. Methods For SPOM samples, approximately 20 l of water was collected at each station and a river discharge area using a van Dorn water sampler, prefiltered in situ with a 180-μm Nitex mesh, and filtered again onto the GF/F filter (ϕ25 mm). In the laboratory, the filter samples were acidified by fuming for 24 h in a desic- cator saturated with HCl to remove the inorganic carbon68. Particulate organic carbon (POC) and nitrogen (PN) concentrations and δ13C and δ15N values were determined on filtered and dried (at 60 °C) particulate samples. To determine isotope ratios of micro-size phytoplankton such as diatoms, a conical plankton net (mesh size of 20 μm, mouth diameter of 45 cm) was vertically towed at each station. Some phytoplankton samples concen- trated into individual test tubes were acidified by adding several drops of 0.12N HCl for δ13C measurements before drying at 60 °C, and those for δ15N were not acidified.l y gi To detect a possible cross-boundary resource flux by the isotopic signatures of consumers, Phragmites, micro- phytobenthos, and Zostera samples were collected in the upper estuarine wetland, on the northern intertidal muddy sand flat, and on the fringe of the sand flat, respectively (Fig. 1). The Phragmites and Zostera leaves were collected by hand, and MPB was collected by scraping the visible mat of benthic diatoms from the sediment surface during low tide. Pure microphytobenthos was extracted by collecting benthic diatoms attached to silica powder69. These plant samples were washed with Milli-Q water and lyophilized at − 70 °C. To determine the stable isotope ratios of sedimentary organic matter, slices of the surface sediments (top 2 cm) were put into 20 ml glass tubes after collection using a van Veen grab and kept on dry ice in the dark. After thawing in the laboratory, sediment samples were treated with 10% HCl solution until the bubbling to remove carbonates stopped, oven dried for 72 h at 60 °C, and homogenized by pulverizing. Scientific Reports \| (2020) 10:16637 \| https://doi.org/10.1038/s41598-020-73628-6 www.nature.com/scientificreports/ Zooplankton and macrobenthic invertebrates collected for isotope analysis were kept alive for at least 6 h in filtered seawater on board to allow them to evacuate their gut materials. All animal samples were submerged in cold (< 5 °C) water containers and transported to the laboratory. www.nature.com/scientificreports/ In this mixing model calculation, the composition of the nutritional source of consumers was estimated at the individual level. Second, we calculated the relative contribution of benthic affinity prey (f) to the nutrition of higher-level consumers (i.e., predators) to identify their trophic links with benthic vs. pelagic pathways, as follows: f = δ13Cconsumer −δ13Czooplankton −TEF / δ13Cbenthicprey −δ13Czooplankton where δ13Czooplankton and δ13Cbenthicprey represent the δ13C values of zooplankton and deposit feeders as isotopic baselines of pelagic and benthic pathways, respectively. The previously published TEF values for diet–animal tissue isotopic fractionations were employed: δ13C of 1.3 ± 0.3‰ and δ15N of 2.2 ± 0.3‰ for primary consumers and δ15N of 3.3 ± 0.26‰ for carnivorous species78. This estimation incorporates variance in the diet composition of individual consumers and illustrates the variability of trophic structure of different community typologies. The trophic position (TP) of individual consumers was also estimated using the following formulas31,79: y pf The trophic position (TP) of individual consumers was also estimated using the follo TPconsumer = δ15Nconsumer −δ15Nbaseline /TEF + baseline; δ15Nbaseline = (δ15Nbenthicprey × f ) + [δ15Nzooplankton × 1 −f ] δ15Nbaseline = (δ15Nbenthicprey × f ) + [δ15Nzooplankton × 1 −f ] where TPconsumer and δ15Nconsumer represent the TP and δ15N value of the tested consumer, respectively; δ15Nbaseline and baseline represent the δ15N value and TP (= 2 in this study) of the baseline organism, respectively; and TEF is the trophic enrichment factor (= 3.3 ± 0.26‰). Statistical analyses of environmental and isotopic data. Before statistical analyses, all data were checked for normality and homoscedasticity of variance using the Shapiro–Wilk procedure and Levene’s test, respectively. We tested for significant differences in environmental variables among clusters of the plankton community because of recognizable seasonal as well as spatial patterns. Since individual environmental vari- ables did not meet the assumption for parametric tests, a Kruskal–Wallis test was employed to compare the environmental characteristics among clusters, followed by a Mann–Whitney pairwise comparison test. Analysis of variance followed by a post hoc Tukey multiple comparison was employed to test for significant differences in the δ13C and δ15N values among primary sources of organic matter and among cluster groups of the plankton community. When necessary to meet homoscedasticity, the δ13C values of plankton were transformed to 1/ square root ( \|x\| ). www.nature.com/scientificreports/ significance of the relationship was tested by using a Monte Carlo permutation test39. The resulting association index returns the square root of the IndVal index (IndVal.g)38. The maximum indicator value (= 1) can be seen when all occurrences of a species are found in either individual groups of sites or a combination of groups of sites and when the species occurs in all sites of those groups. Indicator species analyses were run using the package “indicpecies” (version 1.7.6) in R75. In the present study, the IndVal values were separately obtained according to the five taxonomic groups.i i g p Considering that dominant taxa would serve as an important trophic mediator in food webs, we identified the dominant taxa of individual taxonomic groups (Supplementary Table 3) to characterize the most important trophic relations in each cluster using the following formula40: D′ ij = Fij × Dij × 100 = Pij/Pj × 100 ×     Pj k=1 (Nik/Nk)  /Pj  × 100 where Pij = number of samples including species i in cluster j; Pj = total number of samples in cluster j;Nik = density of species i in the kth sample of cluster j; and Nk = total density of the kth sample. All taxa with a frequency ( Fij ) higher than 50% and an abundance higher than the median value of Dij in individual clusters were considered dominant. Then, the trophic interactions between co-occurring dominant taxa in each cluster and/or a combina- tion of clusters were evaluated by employing a stable isotope mixing model. Isotope mixing model. Two separate steps were applied to mixing model calculations to delineate pictures of the structure of trophic networks of individual community typologies. First, the relative contributions of puta- tive sources of primary organic matter (riverine SPOM, estuarine and coastal-bay phytoplankton, P. australis, microphytobenthos, and Z. marina) to the nutrition of primary consumers (suspension feeders and deposit feeders) were evaluated using an IsoSource mixing model76. The δ13C and δ15N values of estuarine and coastal- bay phytoplankton were obtained from site-specific phytoplankton data from stations 1–2 and 5–977. We ran the IsoSource mixing model using the three required input datasets: the mean isotopic values and standard devia- tions of end-members of organic matter sources, the isotopic values of primary consumer species, and the aver- age trophic enrichment factors (TEFs) for aquatic consumers. Methods The significance among the clusters was tested by a multi-response permutation procedure, which is a nonparametric method used to test differences between groups, using R 3.4.1 software (vegan library74). In addition, the trend of occurrence of each species in the clusters was displayed by the degree of colour on the SOM, making the importance of a given species to particular clusters identifiable. Indicator and dominant species. The visual map after the training of the SOM provides a trend of spe- cies occurrence instead of statistical indication and classifies map units into clusters that consist of similar assemblages. 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Jeong Bae Kim and Jong-Bin Kim (National Institute of Fisheries Science, Korea) for allowing access to an automated nutrient analyser and fishing boat to cast a beam trawl. This research was supported by ″Long-term change of structure and function in marine ecosystems of Korea″ funded by the Ministry of Oceans and Fisheries Korea. We thank Drs. Jeong Bae Kim and Jong-Bin Kim (National Institute of Fisheries Science, Korea) for allowing access to an automated nutrient analyser and fishing boat to cast a beam trawl. References C., Cho, Y. K., Kim, T. W. & Valle-Levinson, A. Spatio-temporal variation of flushing time in the Sumjin River estuary Terr. Atmos. Ocean. Sci. 23, 119–130 (2012). 6. Kwon, K. Y., Moon, C. H., Kang, C. K. & Kim, Y. N. Distribution of particulate organic matters along the salinity gradient in the Seomjin River estuary. J. Korean Fish. Soc. 35, 86–96 (2002). Seomjin River estuary. J. Korean Fish. Soc. 35, 86–96 (2002). 37. Bibi, R. et al. 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Coast. 43, 496–511 (2020).t g g g 78. McCutchan, J. H. Jr., Lewis, W. M. Jr., Kendall, C. & McGrath, C. C. Variation in trophic shift for stable isotope ratios of car nitrogen, and sulphur. Oikos 102, 378–390 (2003). g p 9. Post, D. M. Using stable isotopes to estimate trophic position: models, methods, and assumptions. Ecology 83, 703–718 (2002). g 79. Post, D. M. Using stable isotopes to estimate trophic position: models, methods, and assumptions. Author contributions H.Y.K., H.W.K., S.G.Y., and C.K.K. designed research. Y.K.K., D.K., and Y.J.L. performed the hydrodynamic observation. H.Y.K., R.B., and K.H.L. carried out biogeochemical measurement. D.K., H.W.K., and J.J. were responsible for the field survey and data acquisition of plankton. S.G.Y., H.J.P., and C.K. performed sample and Scientific Reports \| (2020) 10:16637 \| https://doi.org/10.1038/s41598-020-73628-6 www.nature.com/scientificreports/ data collection of benthos. G.K.K., H.K., and Y.J.L. carried out the nekton survey and sample collection. D.K., C.K., J.J., and H.J.P. performed stable isotope-ratio mass-spectrometer measurement. H.Y.K., C.K., and C.K.K. interpreted data and performed simulation of self-organizing map and stable isotope-mixing models. H.Y.K. and C.K.K wrote the manuscript. H.W.K., H.K., H.J.P., and S.G.Y. revised the manuscript. data collection of benthos. G.K.K., H.K., and Y.J.L. carried out the nekton survey and sample collection. D.K., C.K., J.J., and H.J.P. performed stable isotope-ratio mass-spectrometer measurement. H.Y.K., C.K., and C.K.K. interpreted data and performed simulation of self-organizing map and stable isotope-mixing models. H.Y.K. and C.K.K wrote the manuscript. H.W.K., H.K., H.J.P., and S.G.Y. revised the manuscript. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-73628-6. Supplementary information is available for this paper at https://doi.org/10.10 Correspondence and requests for materials should be addressed to C.-K.K. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 Scientific Reports \| (2020) 10:16637 \| https://doi.org/10.1038/s41598-020-73628-6
https://openalex.org/W2478112303	http://oceanrep.geomar.de/35845/1/fmars-03-00132.pdf	English	null	Identifying the Sources and Sinks of CDOM/FDOM across the Mauritanian Shelf and Their Potential Role in the Decomposition of Superoxide (O2-)	Frontiers in marine science	2,016	cc-by	17,533	Identifying the Sources and Sinks of CDOM/FDOM across the Mauritanian Shelf and Their Potential Role in the Decomposition of Superoxide (O−) 2 Maija I. Heller 1, 2, Kathrin Wuttig 1, 3 and Peter L. Croot 1, 4 1 Marine Biogeochemistry, GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel, Germany, 2 Department of Ocean Sciences, University of California, Santa Cruz, Santa Cruz, CA, USA, 3 Antarctic Climate and Ecosystems Cooperative Research Centre, University of Tasmania, Hobart, TAS, Australia, 4 Earth and Ocean Sciences, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Superoxide (O−) is a short lived reactive oxygen species (ROS) formed in seawater 2 by photochemical or biological sources, it is important in the redox cycling of trace elements and organic matter in the ocean. The photoproduction of O−is now thought 2 to involve reactions between O2 and reactive reducing (radical) intermediates formed from dissolved organic matter (DOM) via intramolecular reactions between excited singlet state donors and ground-state acceptors (Zhang et al., 2012). In seawater the main pathways identiﬁed for the decomposition of O−into H 2 2O2 and O2, involve reactions with Cu, Mn, and DOM. In productive regions of the ocean, the reaction between DOM and O−can be a signiﬁcant sink for O−. Thus, DOM is a key component of both the 2 2 formation and decomposition of O−and formation of H2O2. In the present work we 2 examined the relationships between O−decay rates and parameters associated with 2 chromophoric dissolved organic matter (CDOM) and ﬂuorescent dissolved organic matter (FDOM) by using the thermal O−source SOTS-1. Filtered samples (0.2 µm) were run 2 both in the presence, and absence, of the metal chelator diethylenetriaminepentaacetic acid (DTPA) to determine the contribution from DOM. Samples were collected along a transect across the continental shelf of the Mauritanian continental shelf during a period of upwelling. In this region we found that reactions with DOM, are a signiﬁcant sink for O−in the Mauritanian Upwelling, constituting on average 58 ± 13% of the O−loss rates. 2 2 Superoxide reactivity with organic matter showed no clear correlation with bulk CDOM or FDOM properties (as assessed by PARAFAC analysis) suggesting that future work should concentrate at the functional group level to clearly elucidate which molecular species are involved as bulk properties represent a wide spread of chemical moieties with different O−reactivities. Analysis of FDOM parameters indicates that many of the markers used 2 previously for terrestrial sources of DOM and FDOM are called into question as marine sources exist. Identifying the Sources and Sinks of CDOM/FDOM across the Mauritanian Shelf and Their Potential Role in the Decomposition of Superoxide (O−) 2 In particular recent work (Rico et al., 2013) indicates that algal species may also produce syringic, vanillic, and cinnamic acids, which had previously been ascribed solely to terrestrial vegetation. Reviewed by: Reviewed by: Yi Zhang, University of Maryland, College Park, USA Christopher James Miller, University of New South Wales, Australia Correspondence: Maija I. Heller maijaheller@gmail.com Specialty section: This article was submitted to Marine Biogeochemistry, a section of the journal Frontiers in Marine Science Specialty section: This article was submitted to Marine Biogeochemistry, a section of the journal Frontiers in Marine Science Received: 31 May 2016 Accepted: 18 July 2016 Published: 03 August 2016 Received: 31 May 2016 Accepted: 18 July 2016 Published: 03 August 2016 Keywords: reactive oxygen species, parafac, colored dissolved organic matter (CDOM), Atlantic Ocean, excitation emission matrix ﬂuorescence, ﬂuorescence dissolved organic matter (FDOM), superoxide dismutase, hydrogen peroxide ORIGINAL RESEARCH published: 03 August 2016 doi: 10.3389/fmars.2016.00132 ORIGINAL RESEARCH Edited by: Edited by: Leanne C. Powers, Skidaway Institute of Oceanography, USA Reactive Oxygen Species in Seawater—Superoxide (O− 2 ) and Peroxide (H2O2) Superoxide (O− 2 ) is an important transient reactive oxygen species (ROS) in the ocean formed as a reactive intermediate in photosynthesis and respiration and with this the conversion of oxygen (O2) into water and vice versa. O− 2 is a highly reactive and the short-lived radical anion can be produced both via photochemical (Micinski et al., 1993) and biological processes in the ocean (Diaz et al., 2013; Roe et al., 2016). O− 2 and H2O2 are directly involved in degradation of organic pollutants and photobleaching of CDOM (Scully et al., 2003; Chen et al., 2009), cause oxidative stress in aquatic organisms and alter the redox cycling of trace metals like Fe, Cu, and Mn (Moﬀett and Zika, 1987; Wuttig et al., 2013b). In earlier work, where we examined the decomposition rate of O− 2 throughout the water column in the Eastern Tropical North Atlantic (ETNA) Ocean, we found that in the surface ocean, which is in this area strongly impacted by Saharan aerosols and coastal sediment resuspension, the main decay pathways for superoxide (Figure 1) were reactions with Mn(II) and DOM (Wuttig et al., 2013a,b). Pioneering work by Coble (1996) showed that the Excitation Emission Matrix (EEM) measurements of CDOM ﬂuorescence (often referred to as FDOM) can generally be divided into two categories—humic-type or protein/amino acid-type ﬂuorescence. Furthermore, Coble (1996) deﬁned 5 major ﬂuorescence regions as per the excitation/emission spectra as follows: Humics—peak A (λex/λem ∼260/380–460 nm), Peak C (λex/λem ∼350/420–480 nm), peak M (λex/λem ∼312/380– 420 nm); Proteins—peak B (λex/λem ∼275/310 nm) and peak T (λex/λem ∼275/340 nm). More recently EEM has been combined with parallel factor (PARAFAC) data analysis (Stedmon and Bro, 2008) to independently determine multiple components of the CDOM pool–many, but not all, of which are related INTRODUCTION of DOM that absorbs light and this can be characterized by its absorbance and ﬂuorescence properties (Coble, 2007). CDOM is a ubiquitous component of the open ocean dissolved matter pool, and is important because of its inﬂuence on the optical properties of the water column, its role in photochemistry and photobiology, and its utility as a tracer of deep ocean biogeochemical processes and circulation. The general distribution of CDOM in the global ocean is controlled by a balance between production and photolysis, with vertical circulation playing an important role in the transport of CDOM to and from intermediate water masses. Fluctuations in the abundance of CDOM in the global surface ocean have been observed, indicating a potentially important role for CDOM in ocean-climate connections because of its impact on photochemistry and photobiology (Nelson and Siegel, 2013). Reactive Oxygen Species in Seawater—Superoxide (O− 2 ) and Peroxide (H2O2) Citation: Heller MI, Wuttig K and Croot PL (2016) Identifying the Sources and Sinks of CDOM/FDOM across the Mauritanian Shelf and Their Potential Role in the Decomposition of Superoxide (O− 2 ). Front. Mar. Sci. 3:132. Keywords: reactive oxygen species, parafac, colored dissolved organic matter (CDOM), Atlantic Ocean, excitation emission matrix ﬂuorescence, ﬂuorescence dissolved organic matter (FDOM), superoxide dismutase, hydrogen peroxide August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org FDOM/O− 2 Decay - Mauritanian Shelf Heller et al. dy Region—Mauritanian Upwelling Study Region—Mauritanian Upwelling In the present work we focus on the Mauritanian upwelling system which stretches from the Iberian Peninsula to about 10◦N along the Northwest African coast. The Mauritanian upwelling is one of the main Eastern Boundary Upwelling Systems (EBUS), where nutrient rich waters are upwelled by the trade winds to fuel one of the most biologically productive regions in the global ocean (Messié and Chavez, 2015). Due to changes in wind forcing the coastal upwelling oﬀMauritania exhibits a pronounced seasonal cycle and the Mauritanian upwelling is the most productive branch of the Canary Current upwelling system (Tanhua and Liu, 2015). Upwelling between 20◦N and 25◦N is persistent throughout the year (Schafstall et al., 2010). In contrast, upwelling north and south of this area is strongly seasonal due to wind forcing associated with the migration of the ITCZ (Tomczak and Godfrey, 1994). Vertical mixing induced by bottom turbulence is also an important transport process for supplying nutrients to the euphotic zone (Schafstall et al., 2010). Primary production is high year-round (80–200 mmol m−2 d−1 of C) and elevated beyond the shelf break (Huntsman and Barber, 1977). Aims and Objectives of the Present Study Aims and Objectives of the Present Study Our main aim in this study was to assess the role of DOM in the decomposition of O− 2 along a transect across the continental shelf in the Mauritanian upwelling region by comparing O− 2 decay rates to bulk CDOM and FDOM properties. A further objective was to identify CDOM or FDOM parameters that may indicate what type of organic material is responsible for the production or decomposition of O− 2 in the ocean. The ﬁnal objective was to examine the inﬂuence of primary production, photobleaching, and microbial activity on CDOM and FDOM properties in an upwelling region devoid of riverine input. It is well-known that photo-oxidation of proteins such as Tryptophan produces O− 2 and subsequently H2O2 (Mccormick and Thomason, 1978), reactions with other photo-produced reactive oxygen species (ROS) (e.g., 1O2 and OH) may also be important pathways for the destruction of proteins, and hence the loss of protein-like ﬂuorescence, in the ocean (Boreen et al., 2008). Similarly the loss of FDOM in surface waters is often Role of CDOM in the Production and Decomposition of ROS in Seawater Our understanding of how ROS species are generated in seawater by CDOM absorption of sunlight in the euphotic zone of the ocean has advanced substantially in recent years. In particular the paradigm that existed until recently, that excited triplet states of CDOM reacted with 3O2 to form O− 2 and carbocations (O’sullivan et al., 2005) has been replaced with a new mechanism in which a low-eﬃciency intramolecular electron transfer from an excited singlet donor (e.g., substituted phenol) to a ground- state acceptor (e.g., quinone), that produces a radical species that reacts with O2 to form O− 2 and subsequently H2O2 (Zhang et al., 2012; Sharpless and Blough, 2014). The seasonality in the upwelling strength also impacts phytoplankton dynamics of the Senegal-Mauritanian upwelling region (Farikou et al., 2015), as a seasonal cycle is observed beginning with the onset of the upwelling (December–February), mainly nanoeukaryote type phytoplankton are found in the coastal area; while in April–May, the period corresponding to the maximum chlorophyll a concentration, the nanoeukaryotes were replaced by diatoms. Recent work by Powers and Miller (2014) estimated using satellite climatologies, using average apparent quantum yield (AQY) spectrum determined from laboratory irradiations, found that daily H2O2 photoproduction rates (averaged over an annual cycle) were highest in equatorial regions and lowest at the poles (range: 0.07–93.2; average: 40.4; median; 39.5 nM per day). The same group in a further paper, Powers et al. (2015), also reevaluated the photoreactivity of refractory DOC by investigating the photochemical production H2O2 and O− 2 , using controlled irradiations at sea and in the laboratory. They found that in the open ocean, a large fraction of photoproduced O− 2 does not lead to H2O2, which means, that the relationship between these two ROS involve complex pathways. In particular, the apparent stoichiometry of formation was found to be closer to 4:1 (Powers et al., 2015) instead of the 2:1 which would be expected solely from dismutation. This may in part be explained by the photo generation of oxidized CDOM species, which can act as an electron acceptor, and react with O− 2 to form O2 (Garg et al., 2011; Zhang et al., 2012). This in turn may be a signiﬁcant sink for refractory DOC as it circulates through the surface ocean (Mopper et al., 1991; Stubbins et al., 2012). Frontiers in Marine Science \| www.frontiersin.org Chromophoric Dissolved Organic Matter (CDOM) DOM is a complex mix of organic molecules and is poorly described in terms of it composition. CDOM is the proportion FIGURE 1 \| Schematic of the different decay pathways for O− 2 decay in the ocean. O− 2 is biologically and photo produced. Modiﬁed from Wuttig et al. (2013a). rent decay pathways for O− 2 decay in the ocean. O− 2 is biologically and photo produced. Modiﬁed from Wuttig et al. (2013a). FIGURE 1 \| Schematic of the different decay pathways for O− 2 decay in the ocean. O− 2 is biologically and photo produced. Modiﬁed from Wuttig et al. (2013a). FIGURE 1 \| Schematic of the different decay pathways for O− 2 decay in the ocean. O− 2 is biologically and photo prod August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org Frontiers in Marine Science \| www.frontiersin.org 2 FDOM/O− 2 Decay - Mauritanian Shelf Heller et al. to the peaks found in the original Coble analysis. Humic-like ﬂuorescence (FDOMH: typically λex/λem ∼320/420 nm) has been observed in a wide range of marine environments correlates in general well with nutrients (NO− 3 , PO3− 4 ) and apparent oxygen utilization (AOU) in diﬀerent water masses (Hayase and Shinozuka, 1995; Kuma et al., 1998; Yamashita et al., 2007; Yamashita and Tanoue, 2008). These correlations suggest that some of the components that make up FDOM are formed by the remineralization of settling organic particles and are destroyed or modiﬁed by irradiation. However, marine humic substances are composed of a large fraction of the uncharacterized DOM pool in the ocean (Zaﬁriou et al., 1984) and the relative contribution of these complex substances to seawater ﬂuorescence is still unclear. ascribed to photo-bleaching which may be due to reactions with O− 2 (Omori et al., 2011). Thus, quantitative information on the production, sinks, and concentrations of O− 2 and H2O2 in the open ocean is fundamental to fully understand their role in global biogeochemical cycles (Powers and Miller, 2014). Role of CDOM in the Production and Decomposition of ROS in Seawater The Oxygen Minimum Zone (OMZ) in the ETNA appears to be undergoing a signiﬁcant water column deoxygenation of 0.5 µmol kg−1 yr−1 (Stramma et al., 2009). MATERIALS AND METHODS In order to prevent trace metal contamination all laboratory work was performed at sea in a trace metal clean chemistry laboratory under ISO Class 5 conditions using a specially designed containerized clean room (Clean Modules UK). All August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org 3 Heller et al. FDOM/O− 2 Decay - Mauritanian Shelf chemicals that were used in this study were of ultrapure grade unless noted. Ultrapure (UP) water (resistivity > 18.2 M cm−1) was obtained in the laboratory and in the ship going clean container via a Millipore Synergy 185 system that was fed by an Elix-3 (Millipore) reverse osmosis system connected to the mains supply. Pipettes (Finnpipette) were calibrated monthly and trace metal clean pipette tips (Rainin RT-250, RT-1000, and Finntip 10) were used as supplied. An inoLab pH 720 (WTW) was used to determine pH values on the NBS scale (pHNBS). All plasticware and bottles (low density high polyethylene (LDPE) and Polytetraﬂuoroethylene (PTFE)) were extensively cleaned according to the GEOTRACES trace metal clean protocols (Cutter et al., 2010). Satellite Data Satellite chlorophyll-a data was obtained from OBPG MODIS- Aqua Monthly Global 9-km Products via GIOVANNI (http:// giovanni.gsfc.nasa.gov/giovanni/) using the Ocean Color Time- Series Online Visualization and Analysis platform. Analyses and visualizations used in this paper were produced with the Giovanni online data system, developed and maintained by the NASA Goddard Earth Sciences (GES) Data and Information Services Center (DISC). All satellite images were ﬁnally displayed as postscript images using the Generic Mapping Tools (GMT) software (Wessel and Smith, 1998). Post-processing of the complete EEM data set (Go-Flo and Niskin bottles) was performed according to accepted protocols (Lawaetz and Stedmon, 2009; Murphy et al., 2010) in the following sequence: (i) correction of instrument bias using the correction ﬁles provided by the manufacturer, (ii) subtraction of the EEM of UP water, and ﬁnally, (iii) the ﬂuorescence intensity was normalized to the area under the UP water Raman peak (excitation 350 nm) run with each sample batch. Note as the sample absorbance was low, no correction was made for the internal absorption of the samples. Seawater Sampling Seawater samples were collected using Go-Flo sampling bottles deployed on a Kevlar line at 5 stations (Figure 2) occupied during the RV Maria S. Merian expedition MSM17/4 from March 13 to GURE 2 \| (Top) Bathymetry of the Mauritanian upwelling region, the 5 stations occupied are shown as purple circles, note the presence of the Banc Arguin along the Mauritanian coast (bottom left) Chlorophyll composite map derived from MODIS-AQUA images for March 2011. The black box dicates the location of the main study region (bottom right) Composite image (bathymetry and satellite chlorophyll) of the study region with the location of the 5 ain stations sampled in this work (purple circles). FIGURE 2 \| (Top) Bathymetry of the Mauritanian upwelling region, the 5 stations occupied are shown as purple circles, note the presence of the Banc d’Arguin along the Mauritanian coast (bottom left) Chlorophyll composite map derived from MODIS-AQUA images for March 2011. The black box indicates the location of the main study region (bottom right) Composite image (bathymetry and satellite chlorophyll) of the study region with the location of the 5 main stations sampled in this work (purple circles). August 2016 \| Volume 3 \| Article 132 4 Frontiers in Marine Science \| www.frontiersin.org FDOM/O− 2 Decay - Mauritanian Shelf Heller et al. the present work we measured CDOM absorbance over the wavelength (λ) range 280–800 nm. April 5 2011 (boreal spring, upwelling season). The E-W transect along 18◦N across the shelf/slope at the Mauritanian upwelling region covered a distance of 50 km where water depth increases from ∼50 to ∼1100 m (please See Table S1 for the details on all of the stations occupied and samples taken). The spectral slope parameter (Helms et al., 2008), S, was calculated over a range of 275–295 nm (S275–295) and 350–400 nm (S350–400) using a non-linear least squares ﬁtting procedure in Matlab. S275–295 is commonly used as a proxy for molecular weight with increasing values indicating decreasing molecular weight and aromaticity (Helms et al., 2008). Similarly the E2:E3 ratio, also used to track changes in the relative size of CDOM, was calculated as the ratio of CDOM absorption at 250 to 365 nm (De Haan and De Boer, 1987). CDOM Fluorescence—Excitation Emission Matrix CDOM Fluorescence—Excitation Emission Matrix Samples for CDOM ﬂuorescence measurements were syringe ﬁltered through 0.2 µm ﬁlters (Sarstedt) as described above for the absorbance measurements. Humic-type ﬂuorescence measurements were performed with a Cary Eclipse Fluorometer using a 1 cm quartz cell. Measurements of FDOMH (Tani et al., 2003) were performed by analysis of samples using excitation at 320 nm and emission at 420 nm (10 nm slit widths). Each sample was also analyzed as Excitation Emission Matrix (EEM) on the Cary Eclipse Fluorometer using the same 1 cm quartz cell as for the FDOMH measurements. For the EEM analysis, excitation wavelengths were scanned (12000 nm/min) from 250 to 500 nm (5 nm slit width and 5 nm increments) and emission wavelengths (5 nm slit width and 5 nm increments) from 280 to 600 nm, the photon multiplier tube (PMT) voltage was set at 700 V (maximum) and the response time 0.08 s. Day to day variation in the instrument was monitored by daily measurements of the Raman scattering of UP water (excitation 350 nm; Stedmon et al., 2003; Heller et al., 2013) and the use of a standard of quinine sulfate (1 ppm in 0.05 N H2SO4) which was also diluted to form a calibration series for quinine ﬂuorescence (QSU) and run daily (Mopper and Schultz, 1993). CDOM Absorbance and Fluorescence CDOM Absorbance CDOM absorbance measurements were performed using a liquid waveguide capillary cell (LWCC) (LWCC-2100 World Precision Instruments, Sarasota, FL, USA) and an Ocean Optics USB4000 UV-VIS spectrophotometer in conjunction with an Ocean Optics DT-MINI-2-GS light source. Samples were ﬁltered through an 0.2 µm syringe ﬁlter (Sarstedt) using a 10 mL Telfon syringe (Savillex), the ﬁrst ∼10 mL were discarded and the absorbance of the afterwards ﬁltered solution then measured by direct injection into the LWCC. Absorbance measurements were made relative to UP and corrected for the refractive index of seawater based on the procedure outlined in Nelson et al. (2007). The resulting dimensionless optical density spectra were converted to absorption coeﬃcient (m−1): aCDOM(λ) = 2.303 Aλ/l, where 2.303 converts decadal logarithmic absorbance to base e, and l is the eﬀective optical pathlength of the waveguide (here 100.3 ± 0.5 cm as determined by the manufacturer). In Hydrography and Nutrients Hydrographical data was obtained using a Seabird Conductivity- Temperature-Depth (CTD) rosette system. This system consisted of a SBE911plus CTD system in combination with a carousel water sampler SBE32 with 24 12-L bottles. The CTD system was equipped with a CT sensor pair, two O2 sensors (SBE43 SN871 and SBE43 SN950 -calibrated by comparison to Winkler O2 titrations), a turbidity sensor and a chlorophyll sensor (chlorophyll a ﬂuorescence—calibrated according to the manufacturers protocols). CDOM and FDOM samples were obtained from Go-Flo bottles and Niskins on the CTD system. Samples for dissolved macro nutrients were subsampled from the Go-Flo bottles and either analyzed onboard for low level (nM) concentrations of nitrite (NO− 2 ) and ammonium (NH+ 4 ) using photometric (Grasshoﬀet al., 1999) or ﬂuorometric method (Holmes et al., 1999), respectively. Samples for silica (H4SiO4), phosphate (PO3− 4 ) and nitrate (NO− 2 ) were frozen, transported back to Germany for analysis in the Nutrient laboratory of the MPI-Bremen (Grasshoﬀet al., 1999). Frontiers in Marine Science \| www.frontiersin.org Determination of O− 2 Decay Rates Experimental Design In the present study we employed the thermal O− 2 source SOTS- 1 [di(4-carboxybenzyl) hyponitrite—molecular weight 330.3 gmol−1; Ingold et al., 1997] as described by us previously (Heller and Croot, 2010a). SOTS-1 has some advantages over KO2 and other currently used methods which generate O− 2 at µM concentrations as it produces O− 2 slowly and continuously over a longer duration to be able to mimic the in vivo situation and additionally there is little or no H2O2 formed upon introduction to the sample. It also avoids the problem of adding the chelator DTPA, in order to complex metals, prior to irradiating ketone solutions to produce O− 2 (Mcdowell et al., 1983), as photodegradation products of DTPA will be formed and cause problems with calibration and speciation analysis (Heller and Croot, 2011). Analysis of O− 2 Decay Using SOTS-1 as O− 2 Source In this work the rate of the superoxide reaction with unamended seawater is reported as kSW while that of the DTPA amended is listed as kDTPA. It is assumed that the rate of reaction with organic matter is equal to the reaction in the presence of DTPA, i.e., korg = kDTPA. Other assumptions include: (i) That the response of the system is overall ﬁrst order with respect to O−. 2 The decomposition rate of SOTS-1 is well-described in seawater and was shown to follow a ﬁrst order decay with a 40 mol% yield of O−− 2 (Ingold et al., 1997). (ii) That all the metal species are made inert by complexation with DTPA, leaving only reactions with organic species and the uncatalysed self dismutation reaction as the pathways for O− 2 decay. y 2 g 500 µg aliquots of SOTS-1 were used as received (Cayman Chemicals) and stored at −80◦C until use. Immediately prior to the start of any experiment a primary stock of SOTS- 1 was prepared by the dissolution of the 500 µg SOTS-1 aliquots in DMSO (Fluka, puriss p.a.=99.9%) before further dilution in seawater. Final starting concentrations for SOTS- 1 ([SOTS-1]0) at the beginning of each experiment in this study were between 0.86 and 1.78 µM (Table S2). Experiments were performed in Teﬂon bottles (Nalgene) which were either left unamended or had DTPA, Cu (0.79, 1.58 nM), Mn (1.00, 2.00 nM), or Fe (0.90, 1.79 nM) added. Determination of H2O2 Samples for H2O2 were analyzed directly using a ﬂow injection chemiluminescence (FIA-CL) reagent injection method (Yuan and Shiller, 1999) as described previously (Croot et al., 2004). Samples were analyzed using 5 replicates: typical precision was 2–3% through the concentration range 0.5–100 nM, the detection limit (3 s) is typically 0.2 nM. Determination of O− 2 Decay Rates Experimental Design All samples were equilibrated for at least 12 h before the experiment was initiated by the addition of a speciﬁc amount of SOTS-1 from the primary standard to a known volume of seawater. All reagents and samples were kept at a constant temperature (21.5 ± 0.2◦C) throughout the course of the experiment in the temperature controlled class 100 clean laboratory. Only the unamended and DTPA results are reported in the present work, the results from the trace metal additions will be reported elsewhere. (iii) That the 2nd order self-dismutation reaction is signiﬁcantly small that it can be ignored. In the present case this a reasonable assumption as this reaction is well-described in seawater as a function of pH (Zaﬁriou, 1990; Heller and Croot, 2010c) and the nM to pM levels of O− 2 generated using µM concentrations of SOTS-1 indicates that results in a pseudo ﬁrst order reaction on the order of 1 × 10−4 s−1 or less. The analysis of the time dependent concentration of O− 2 generated in seawater samples due to the additions of SOTS-1 was performed as previously described (Heller and Croot, 2010a). Brieﬂy raw photon counts are ﬁrstly corrected for the signal blank due to MCLA auto-oxidation and then converted to a concentration of O− 2 by applying the calculated sensitivity factor previously determined by calibration of a seawater sample with additions of KO2. The analysis of the time dependent concentration of O− 2 generated in seawater samples due to the additions of SOTS-1 was performed as previously described (Heller and Croot, 2010a). Brieﬂy raw photon counts are ﬁrstly corrected for the signal blank due to MCLA auto-oxidation and then converted to a concentration of O− 2 by applying the calculated sensitivity factor previously determined by calibration of a seawater sample with additions of KO2. The rate equation derived for the formation of O− 2 from SOTS-1 previously (Heller and Croot, 2010a) is shown below: PARAFAC Analysis of 3D Excitation Emission Matrix (EEM) The normalized EEMs were analyzed by PARAFAC in MATLAB under application of the DOMFluor toolbox (Stedmon and Bro, 2008) using models constrained to non-negative values. Outlier identiﬁcation was performed using the outlier test function provided with the DOMFluor toolbox. No samples with extreme leverage were found, indicating no extreme, and potentially outlying, EEMs in the dataset. Determination of the most suitable number of components was achieved by the split-half analysis and random initialization where by both halves were successfully August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org 5 FDOM/O− 2 Decay - Mauritanian Shelf Heller et al. -3-one, HCl]] (MCLA) (Fluka) (Nakano et al., 1986). In the present work we used the same method as we had used previously (Heller and Croot, 2010a,c). A brief description follows; MCLA was used as received, a primary 1 mM MCLA standard was prepared by dissolving 10 mg MCLA in 34.5 mL MQ water, where upon 1 mL aliquots of this solution were then pipetted into 2 mL polyethylene vials and frozen at −80◦C until required for use. The working MCLA standard, 1 µM, was prepared from a thawed vial of the primary stock by dilution into a 1 L solution of 0.05 M Sodium acetate (Sigma Ultra) buﬀered (4.1 g) in MQ water adjusted to pHNBS 6. Several time points between 0 and 23 h were taken from the experimental solutions as described above, and were drawn directly into the ﬂow cell of the chemiluminescence detector as described before (Heller and Croot, 2010c). The sensitivity of the MCLA method is very strongly temperature dependent and for this reason all samples and reagents were kept at constant temperature (21.5 ± 0.2◦C) throughout the course of the experiments. validated. No systematic residual was found in the modeled EEMs. validated. No systematic residual was found in the modeled EEMs. CDOM Absorbance Vertical proﬁles of CDOM absorbance, a325, at the 5 GO-FLO stations are shown in Figure 3. Highest values of a325 were found near the shelf break (Stn 14–15) and decreased oﬀshore. [O− 2 ]i = 0.4k[SOTS]0 ( e−kt −e−kobst kobs −k ) (2) (2) Values of E2:E3 (Figure 4) were ∼6 at stations on the shelf and in surface waters oﬀthe shelf. At stations 8–9 there was a monotonic increase in E2:E3 with depth which may have been caused by strong mixing at depth across the shelf as evidenced by the transmission proﬁles (not shown). Thus, it is likely that material from the sediments with a higher E2:E3 (∼9) was being resuspended and mixed through the water column in the vicinity of the shelf break (Stns 8–9 and 6–7). There is limited data for E2:E3 values of marine porewaters but what data there is suggests that values increase with sediment depth (Dang et al., 2014), though it is likely that this is dependent on the rate of carbon and O2 supply. As an increase in E2:E3 is thought to reﬂect a shift toward lower molecular weight compounds this may also represent diﬀusion of such material from the shelf sediments. When kobs >> k, as would be expected under most circumstances in seawater, Equation 2 reduces to: 0.4k[SOTS]0e−kt = kobs[O− 2 ]i (3) (3) Taking the natural logarithm of both sides then gives: −kt = ln[O− 2 ]i + ln kobs 0.4k[SOTS]0 (4) (4) Thus, in this case a plot of ln[O− 2 ]i vs. time will have a slope of k, the thermal decomposition rate of SOTS-1. Note that the value of kobs can also be determined here from the value of the intercept as the value for [SOTS]0 is known. Rearranging Equation 3 gives the following: Thus, in this case a plot of ln[O− 2 ]i vs. time will have a slope of k, the thermal decomposition rate of SOTS-1. Note that the value of kobs can also be determined here from the value of the intercept as the value for [SOTS]0 is known. Rearranging Equation 3 gives the following: Determination of O− 2 Concentrations using MCLA Chemiluminescence ∂[O− 2 ] ∂t = 0.4k[SOTS]0e−kt −kobs[O− 2 ] (1) (1) The most widely used approach to measuring O− 2 in seawater (Rose et al., 2008; Heller and Croot, 2010a) is via the use of the chemiluminescence probe Cypridina luciferin analog [[2- methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin where t is the time since the introduction of the SOTS-1, k is the rate of thermal decomposition of SOTS-1, [SOTS]0 is the initial Frontiers in Marine Science \| www.frontiersin.org August 2016 \| Volume 3 \| Article 132 6 Heller et al. FDOM/O− 2 Decay - Mauritanian Shelf concentration of SOTS-1 and kobs is the observed 1st order loss rate of O− 2 . If the initial concentration of O− 2 is zero (a reasonable assumption when the seawater is ﬁltered and kept in the dark), then Equation 1 has the exact solution (Harcourt and Esson, 1866): the continental shelf. Shelf waters were still oxygenated however with a strong vertical gradient present between surface and depth. PARAFAC Analysis and FDOMH PARAFAC analysis of our complete 3D EEM FDOM data set identiﬁed 3 independent components (Table 1; Table S3 and Figures 5, 6). The calculated excitation and emission spectra for each of the 3 components is shown in Figure S1. Component C1 was similar to a marine fulvic (M-peak), λex/λem = 240/412 and 320/412. Component C2 had elements that were similar to both classical terrestrial humics (A and C-peaks), λex/λem = 240/412 and 320/412. Lastly component C3 was similar to the Tryptophan-like T-peak, λex/λem = 280/330. In the present case it was not clear if the data was of suﬃcient resolution for PARAFAC to separate out more components, other approaches which assume the presence of speciﬁc chromophores may be able to resolve this better but would be reliant on assumptions made regarding the presence of speciﬁc chromophores in solution. Direct measurements of humic ﬂuorescence (not shown) or FDOMH (λex/λem = 320/420) correlated [Spearman’s rho, calculated using corr function in Matlab (Mathworks)] most strongly with C1 (ρ = 0.59, n = 252, p < 0.001) but also had weaker correlations with C2 (ρ = 0.21, n = 251, p < 0.001) and C3 (r = 0.41, n = 251, p < 0.001) (Tables 1 and S3, Figures 5, 6). This diﬀered from our earlier work (Heller et al., 2013) where only one of the components identiﬁed by PARAFAC was strongly correlated to FDOMH. kobs = 0.4k[SOTS]0e−kt [O− 2 ]i (5) (5) Operationally the determination of kobs is optimal once the maximum value of [O− 2 ]i is reached: Operationally the determination of kobs is optimal once the maximum value of [O− 2 ]i is reached: t = ln k kobs k −kobs (6) (6) Typically it takes 10 min to reach the maximum [O− 2 ]i at 25◦C if kobs = 0.01 s−1. Thus, in the present case we used data collected after 30 min and up to 23 h since the initiation of the experiment to determine kobs. Frontiers in Marine Science \| www.frontiersin.org Hydrography and Nutrients FIGURE 4 \| Vertical distribution of H2O2(blue circles) and E2:E3 (green triangles) at the 5 Go-Flo stations along the tra Upwelling. ution of H2O2(blue circles) and E2:E3 (green triangles) at the 5 Go-Flo stations along the transect in the Mauritanian open ocean sites in the Tropical North Atlantic not impacted by the ITCZ (Croot et al., 2004), though they were similar to observations near Cape Verde (Heller and Croot, 2010b). Most likely the lower surface concentrations observed here is reﬂecting strong consumption of H2O2 by the phytoplankton growing in the upwelling waters. On the shelf edge (stations 6– 7) there were slightly enhanced levels of H2O2 in the bottom waters possibly indicating a sedimentary source, coincident with elevated levels of C1 and C3 also seen at this station. As pore water measurements from sediments along the same transect, taken on the same expedition, indicated that surface sediments were always oxic and that only on the shelf itself did sediments accumulate H2S at depth (Dale et al., 2014). Thus, it is unlikely that these sediments are a source of H2O2 from the oxidation of Fe(II) released from the sediments, and it is more likely resulting from bacterial respiration of particulate carbon that has sunk to the sediments from the overlying productive waters. in the surface. This suggests that the productive waters in the upwelling region were a stronger source of C1, FDOMH to surface waters than photobleaching was a sink. At the oﬀshore stations, and on the shelf, C2 was low and almost constant (∼0.01 R.U.) throughout the water column with only a slight enrichment in surface waters close to the shelf break. The distribution of the tryptophan-like component, C3 (Figure 6), also showed some similarities to C2 with local maximum below the chlorophyll maxima and suggestive also of links to zooplankton or microbial activity. Although comparison with NH+ 4 (Figure 6), as a tracer of zooplankton activity, reveals that NH+ 4 concentrations are higher in the mixed layer and not coincident with the C3 maxima. However, as NH+ 4 is also taken up by phytoplankton/bacteria this approach may be too simplistic. Dissolved O2 concentrations were also weakly correlated with C2 (ρ = 0.36, n = 251, p < 0.001) and C3 (ρ = 0.45, n = 251, p < 0.001). Hydrography and Nutrients O− 2 Decay Rates Using SOTS-1 as Source Rates of O− 2 decomposition (Table 2; Table S4) as determined using SOTS-1 as a thermal source for O− 2 are shown in Figure 7 for seawater with DTPA (kDTPA) and unamended (kSW). Values Hydrography and Nutrients During the month of April, 2011 there was weak but persistent upwelling across the Mauritanian shelf with high concentrations of chlorophyll extending out into the Atlantic Ocean (Figure 2). Vertical proﬁles of chlorophyll ﬂuorescence (Figure 3) indicated highest surface concentrations in oﬀshore waters with lower levels inshore, indicating that satellite estimates of chlorophyll in these type 2 waters are currently overestimated, presumably by a combination of Saharan dust, suspended sediment, and CDOM. Transmission data (not shown) indicated that there was a considerable bottom nepheloid layer present across the shelf and shelf edge this was consistent with earlier microstructure measurements taken in the same region (Schafstall et al., 2010). The core of the OMZ of the ETNA was found in oﬀshore waters between 300 and 600 m depth, as observed previously (Stramma et al., 2009) and was still present at this depth upon encountering The vertical distribution of C1 and C2 at the Go-Flo stations are shown in Figure 5. Proﬁles of C1 were relatively constant throughout the water column (∼0.02 R.U.), the only noticeable feature is small positive excursions below the chlorophyll maxima (Figure 3) at the 3 stations oﬀshore and suggests a relationship to zooplankton grazing or microbial remineralization of sinking particles. At stations near the shelf break, C1 was slightly elevated in surface waters, in contrast with open ocean proﬁles (Heller et al., 2013) of FDOMH in open ocean tropical waters which typically exhibit a strong photobleaching eﬀect with a minimum August 2016 \| Volume 3 \| Article 132 7 FDOM/O− 2 Decay - Mauritanian Shelf Heller et al. FIGURE 3 \| Vertical distribution of chlorophyll a (black line)and a325 (green triangles) at the 5 Go-Flo stations along the transect in the Mauritanian Upwelling. FIGURE 3 \| Vertical distribution of chlorophyll a (black line)and a325 (green triangles) at the 5 Go-Flo stations along the transect in the Mauritanian Upwelling n of chlorophyll a (black line)and a325 (green triangles) at the 5 Go-Flo stations along the transect in the Mauritanian FIGURE 3 \| Vertical distribution of chlorophyll a (black line)and a325 (green triangles) at the 5 Go-Flo stations along th Upwelling. FIGURE 4 \| Vertical distribution of H2O2(blue circles) and E2:E3 (green triangles) at the 5 Go-Flo stations along the transect in the Mauritanian Upwelling. FIGURE 4 \| Vertical distribution of H2O2(blue circles) and E2:E3 (green triangles) at the 5 Go-Flo stations along the transect in the Mauritanian Upwelling. Sources of CDOM to the Mauritanian Upwelling Sources of CDOM to the Mauritanian Upwelling The main source of CDOM to our study region appears to be from new production by phytoplankton as evidenced by increases in a325 (Figure 3) coincident with the euphotic zone. However, there were also a potential source from the sediment as seen in the E2:E3 data (Figure 4) at the shelf break, which the increase in E2:E3 suggested an input of lower molecular weight CDOM. A high E2:E3 source from pore waters or sediments has not been described before to our knowledge and without pore water measurements to constrain these values we hesitate to assign them to any particular biogeochemical process. The E2:E3 values found in the present study were similar to that found in surface marine waters oﬀthe Portuguese coast (Santos et al., 2014), where a signiﬁcant negative correlation was found between E2:E3 and β- GlCase activity tentatively suggesting that decreases in E2:E3 are associated with increases in microbial activity. Photobleaching has been shown to increase E2:E3 (Helms et al., 2008) and while this process was clearly occurring in surface waters it was apparently not fast enough to change the phytoplankton or microbial signal observed. A recent study in the waters of the equatorial upwelling region of the Atlantic (Andrew et al., 2013) showed several lines of evidence that the CDOM found in surface waters there was derived from terrestrial components. They suggested that changes in the optical properties of CDOM associated with increases in AOU (Yamashita and Tanoue, 2008, 2009; Swan et al., 2009; Nelson et al., 2010; Yamashita et al., 2010) could be associated with redox changes in existing terrestrial material. A recent work (Aparicio et al., 2015) made a direct test of the hypothesis put forward by Andrew et al. (2013), by incubating microbial communities with a suite of organic compounds (glucose and acetate) and with or without humic matter. They found that new FDOMH was produced particularly when humic precursors were added, thus in accordance with the hypothesis. The results of Aparicio et al. (2015) also supported the hypothesis of Jørgensen et al. (2014) in which the less labile the precursor material is, the more humic ﬂuorescence is generated. of kDTPA and kSW were comparable to most other recent studies in Tropical waters (Table 2). H2O2 Distribution H2O2 concentrations (Figure 4) were as expected high in the sunlit surface waters with low background levels in the aphotic zone. Overall surface waters were low in H2O2 compared to other August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org 8 FDOM/O− 2 Decay - Mauritanian Shelf Heller et al. TABLE 1 \| PARAFAC analysis and identiﬁcation of ﬂuorophores. Component ex/em Fluorescence Characteristics ex/em Description and probable source C1 240/412 and 320/412 290– 310/370–410 Marine fulvic “M” peak (Coble, 1996)a 340/420 UV/Visible humic-like (Wedborg et al., 2007)b 315/418 Marine humic material “C2”(Murphy et al., 2010)b 335/400 Marine humic “C4” (Heller et al., 2013)b <270– 370/470 Humic like “C2” (Catalá et al., 2016)b, peak “M” (Catalá et al., 2015b)b C2 270/470 <260/400– 460 Terrestrial Humic Substance “A” peak (Coble, 1996)a Humic like (Dubnick et al., 2010)b Humic like “C1” (Jørgensen et al., 2011)b 250/475 Humic like “C2” (Heller et al., 2013)b 320/400 Humic like “C1” (Catalá et al., 2016)b, peak “A/C” (Catalá et al., 2015b) C3 280/330 275/340 Tryptophan-like peak “T” (Coble, 1996)a 280/328 Amino acids “C6”(Murphy et al., 2010)b 280/330 Tryptophan “C2” (Jørgensen et al., 2011)b 280/320 Tryptophan “C3” (Heller et al., 2013)b 290/340 Tryptophan-like “C3” (Catalá et al., 2016)b,Tryptophan-like (Catalá et al., 2015b) C2 350/470 330-350/420- 480 Terrestrial humic substance “C” peak (Coble, 1996)a Not observed 275/305 Tyrosine-like peak “B” (Coble, 1996)a 280/305 “BT” protein-like (Wedborg et al., 2007)b 280/310 Tyrosine “C5” (Jørgensen et al., 2011)b 270/310 Amino acid-like “C4” (Catalá et al., 2016)b, Tyrosine-like (Catalá et al., 2015b) aManual EEM interpretation. bPARAFAC analysis. CDOM Absorbance Along the Transect across the Mauritanian Upwelling CDOM Absorbance in EBUS There have been a few other studies of CDOM in EBUS (Kudela et al., 2006; Day and Faloona, 2009) and the North Atlantic (Kitidis et al., 2006; Nelson et al., 2007; Nelson and Siegel, 2013). In the Californian system, CDOM absorbance a325 was found to be lowest in recently upwelled waters (Day and Faloona, 2009) with the implication that it was derived from primary production and not from sediment resuspension. In a related study (Kudela et al., 2006), CDOM was found to exhibit seasonal patterns in the spectral slope (S350–600) related to both the strength of the upwelling and to the input of rivers to this region. DISCUSSION TABLE 1 \| PARAFAC analysis and identiﬁcation of ﬂuorophores. LE 1 \| PARAFAC analysis and identiﬁcation of ﬂuorophores. Sources of CDOM to the Mauritanian Upwelling FIGURE 6 \| NH+ 4 concentrations (blue circles) and protein-like FDOM component identiﬁed by PARAFAC, C3 (green triangles) at 5 stations along the transect in the Mauritanian Upwelling. E2:E3. These authors suggested that this inverse relationship between 81O2 and 8H2O2 was due to competitive formation of the 1O2 and H2O2 precursors. Sharpless and Blough (2014) further suggested that the inverse correlation of E2:E3 with 8H2O2 could be related to enhanced rates of CDOM radical formation due to higher levels of aromatic donors. that indicated that some of the lignin precursor compounds or breakdown products (including the Vanillyl, Syringyl, and Cinnamyl phenols) were exuded by diatom cultures under optimal growth and diﬀerent metal stress conditions (Rico et al., 2013; López et al., 2015; Rubino, 2015). Now it could be argued that the diatoms, or bacteria associated with them, were simply degrading existing lignin structures in seawater, however it is well-established that many marine organisms contain or synthesize polyphenols (Vreeland et al., 1998; Rico et al., 2013; Gómez et al., 2016) so there is a strong possibility that some of this material is exuded or released by grazing zooplankton or viral lysis. In the present study we were only able to measure a “snap shot” of the H2O2 concentration (Figure 4) and so have no production rates, though earlier work (M68-3) we performed in the same region over diel cycles suggested a strong gradient in H2O2 production from inshore to oﬀshore (unpublished data). Near surface values of E2:E3 (Figure 4) are relatively constant (∼6) perhaps indicating new production dominated over photobleaching (Helms et al., 2008) at this time. It was likely then that H2O2 concentrations and inventories in near surface waters were determined more by loss rates than production, as an earlier study oﬀshore of this region found higher H2O2 surface concentrations in low chlorophyll waters (Steigenberger and Croot, 2008), thus presumably the high phytoplankton and bacterial abundances in the near sure waters resulted in lower H2O2 due to the presence of cellular peroxidases. Potential for ROS Production from CDOM Recently there have been a number of studies that have examined the relationship between CDOM properties and ROS production in natural waters (Dalrymple et al., 2010; Peterson et al., 2012; Zhang et al., 2012; Powers and Miller, 2014). Dalrymple et al. Sources of CDOM to the Mauritanian Upwelling Over the entire transect the reaction between O− 2 and DOM was a major pathway ranging from 28 to 80% of the overall loss rate (kDTPA/kSW) with an average of 58 ± 13 (1 sd) %. This is slightly more than what was observed further to the south of the present study area (Wuttig et al., 2013a) and in strong contrast to the Southern Ocean where the organic pathway was found to be only minor (Heller and Croot, 2010c). There was no signiﬁcant correlation (ρ < 0.1, n = 36) between either kDTPA or kSW with the 3 components identiﬁed by PARAFAC or FDOMH. A signiﬁcant correlation was however found between kDTPA and S350–400 (ρ = 0.58, n = 36, p < 0.002), with a weaker correlation between kSW and S350–400 (ρ = 0.44, n = 36, p < 0.02). The hypothesis of Andrew et al. is partly based on evidence from ultra-resolution mass spectral data for the presence of terrestrial lignins in seawater (Opsahl and Benner, 1997; Kujawinski et al., 2009). However, recent data was published August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org 9 Heller et al. FDOM/O− 2 Decay - Mauritanian Shelf FIGURE 5 \| FDOM humic-like components identiﬁed by PARAFAC, C1(green triangles), and C2(blue circles) at 5 stations along the transect in the Mauritanian Upwelling. FIGURE 5 \| FDOM humic-like components identiﬁed by PARAFAC, C1(green triangles), and C2(blue circles) at 5 stations along the transect in the Mauritanian Upwelling. FIGURE 5 \| FDOM humic-like components identiﬁed by PARAFAC, C1(green triangles), and C2(blue circles) at 5 stations along the transect in the Mauritanian Upwelling. ike components identiﬁed by PARAFAC, C1(green triangles), and C2(blue circles) at 5 stations along the transect in the FIGURE 5 \| FDOM humic-like components identiﬁed by PARAFAC, C1(green triangles), and C2(blue circles) at 5 stations along the transect in the Mauritanian Upwelling. FIGURE 6 \| NH+ 4 concentrations (blue circles) and protein-like FDOM component identiﬁed by PARAFAC, C3 (green triangles) at 5 stations along the transect in the Mauritanian Upwelling. FIGURE 6 \| NH+ 4 concentrations (blue circles) and protein-like FDOM component identiﬁed by PARAFAC, C3 (green tr transect in the Mauritanian Upwelling. ns (blue circles) and protein-like FDOM component identiﬁed by PARAFAC, C3 (green triangles) at 5 stations along the pwelling. Sources of CDOM to the Mauritanian Upwelling (2010) in a laboratory study with freshwater humic substances observed that quantum yields for 1O2 (81O2)increased with increasing E2:E3 values, however quantum yields for H2O2 (8H2O2) decreased with increasing August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org 10 FDOM/O− 2 Decay - Mauritanian Shelf Heller et al. TABLE 2 \| Compilation of rates (with and without DTPA) for the decay of O− 2 in seawater during this work and from other studies. Study Location Station Depth kSW (s−1) kDTPA(s−1) This study (Table S4) Mauritanian shelf 0–1000 0.0003–0.0169 0.0006–0.0249 Wuttig et al., 2013a ETNA GoFlo 1 (CVOO) 19–600 0.025–0.043 0.010–0.030 GoFlo 2 46–391 0.021–0.086 0.016–0.36 GoFlo 3 20–400 0.029–0.065 0.016–0.089 GoFlo 4 20–400 0.015–0.048 0.011–0.028 GoFlo 5 20–400 0.013–0.031 0.006–0.017 GoFlo 6 20–400 0.009–0.063 0.011–0.051 Heller and Croot, 2010c Southern ocean 230–6 25–1000 0.014–0.041 236–5 25–2800 0.008–0.037 249–3 25–1000 0.009–0.021 Heller and Croot, 2010b ETNA 10 (CVOO) 10 0.023 0.013 Heller and Croot, 2011 ETNA 8 5 0.036 0.013 Roe et al., 2016 Station aloha 0.003–0.014 Roe et al., 2016 California current 0.006–0.017 Rose et al., 2010 Great barrier reef WQN157-184 0.08–0.31 TRICHO_1-3 0.07–0.43 Powers et al., 2015 Gulf of Alaska surface and deep 0.004–0.012 Hansard et al., 2011 GoA1-4 Gulf of Alaska GoA1-GoA4 10–50 0.0167 FIGURE 7 \| Vertical distribution of O− 2 decomposition rates kDTPA (green triangles) and kSW (blue circles) at 5 stations along the transect in the Mauritanian Upwelling. thout DTPA) for the decay of O− 2 in seawater during this work and from other studies. pilation of rates (with and without DTPA) for the decay of O− 2 in seawater during this work and from other studies. TABLE 2 \| Compilation of rates (with and without DTPA) for the decay of O− 2 in seawater during this work and from other studies. FIGURE 7 \| Vertical distribution of O− 2 decomposition rates kDTPA (green triangles) and kSW (blue circles) at 5 stations along the transect in the Mauritanian Upwelling. FIGURE 7 \| Vertical distribution of O− 2 decomposition rates kDTPA (green triangles) and kSW (blue circles) at 5 station Mauritanian Upwelling. ution of O− 2 decomposition rates kDTPA (green triangles) and kSW (blue circles) at 5 stations along the transect in the Recent Advances in Our Understanding of FDOM Recent Advances in Our Understanding of FDOM Two recent contributions to this ﬁeld have helped make signiﬁcant advances, ﬁrstly in a recent review article, Sharpless and Blough (2014) showed that ﬂuorescence spectra of natural water samples were not simply a superposition of individual chromophores and instead they suggested it could be explained using a physical model incorporating charge transfer interactions between electron donating and accepting chromophores within the CDOM. In this context then the identiﬁcation of speciﬁc individual chromophores from a seawater sample is highly unlikely but broad conclusions about the functional groups Frontiers in Marine Science \| www.frontiersin.org FDOM Distribution: Sources and Sinks in the Mauritanian Upwelling might be gathered. In this regard another recent advance was provided by Wünsch et al. (2015) who compared quantum yields and ﬂuorescence properties of chromophores identiﬁed as being potential components of CDOM and compared them to 3D EEM and PARAFAC data held in the OpenFluor database (Murphy et al., 2014). Production/Decomposition of FDOM by Phytoplankton, Zooplankton, and Bacteria Laboratory experiments with axenic phytoplankton indicates that many species can produce signiﬁcant concentrations of FDOM during both growth and senescence phases (Chari et al., 2013; Fukuzaki et al., 2014). Measured EEMs of axenic cultures of the diatom Ditylum brighwelli (Fukuzaki et al., 2014)were found to produce peaks (λex/λem = 350/450) similar to peak C, which had commonly been associated as a terrestrial humic. The raphidophyte Heterosigma akashiwo also produced FDOM (λex/λem = 370/450–470) similar to peak C but slightly red shifted from the diatom (Fukuzaki et al., 2014). In a further laboratory study (Romera-Castillo et al., 2011) grew phytoplankton under axenic conditions and also found that they exuded humic like substances (λex/λem = 310/392—similar to C1 in the current study). When bacteria were grown in the phytoplankton exudates, the ﬂuorescence from peak M decreased and new substances ﬂuoresced (λex/λem = 340/440—similar to peak C2 in our study). Peak T was seen to increase in the cultures during the exponential growth phase and decrease later. Phytoplankton cells may also release organic matter after viral lysis and this “viral shunt” is suggested to be a major source of DOM in aquatic systems (Wommack and Colwell, 2000). The impact on FDOM resulting from viral lysis of the marine phytoplankton Micromonas pusilla was recently studied by Lønborg et al. (2013), who found that protein-like FDOM (λex/λem = 280/320–Tryptophan like) and humic-like FDOM (λex/λem = 320/410 similar to FDOMH) was elevated 4.1 and 2.8 times, respectively in infected cultures. This pioneering study demonstrates that viral lysis must also be considered in the production of FDOM in seawater. In the context of the present study, and on the balance of the evidence from the literature, it appears that component C1 is most likely produced by phytoplankton, while C2 is derived from bacteria, with the possibility that elements of C1 are precursors for C2. However, this is a simplistic viewpoint as the relatively uniform vertical distribution of C1 at oﬀshore stations may be interpreted as implying that the bulk of this signal is recalcitrant DOM with more labile material being formed in the high production zones close to the shelf break. For component C3 there are a number of potential sources and sinks with grazing the most likely source. Humic material is both produced (Shimotori et al., 2009, 2012; Zhang et al., 2015) and consumed (Bussmann, 1999; Rocker et al., 2012) by bacteria in the ocean. PARAFAC Identiﬁcation of FDOM Components In the present work we identiﬁed 3 FDOM components (Table 1) that were related to the traditional M (C1), A and C (C2) and T (C3) peaks. Our PARAFAC results are similar to those found in other recent studies (Table 1). In particular, a series of paper published by Catalá et al. (2015a,b, 2016) examined the FDOM August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org 11 FDOM/O− 2 Decay - Mauritanian Shelf Heller et al. data gathered during the Spanish Malaspina circumnavigation of the globe. This work builds on an earlier study by Jørgensen et al. (2011) that used data collected during the Danish Galathea circumnavigation. In the global data set acquired by Catalá et al. they identiﬁed 4 FDOM species by PARAFAC (denoted here using the subscript M and their original designation), 2 humic like MC1 related to peaks A and C and MC2 associated with peak M) and 2 protein-like ﬂuorophores (MC3 Tryptophan −T, MC4 Tyrosine −B). They were able to estimate an overall turnover time in the deep ocean, for the diﬀerent peaks MC1 (A/C) 435 ± 41 years and MC2 (M) 610 ± 55 years and peak MC3 (T) 379 ± 103 years (Catalá et al., 2015b). Modeling of their data indicated a higher production rate of MC2 than MC1 (almost double) as a function of AOU. Comparison with the current work suggests that MC2 ∼C1 and MC1 ∼C2, with MC3 ∼C3 (Table 1). incubations in the eastern north Atlantic (Lønborg et al., 2015) results of dark incubation experiments where marine humic- like materials (λex/λem = 320/410 similar to FDOMH) were produced as a by-product of microbial DOM degradation. The study of Lønborg et al. (2015) also revealed that the protein-like ﬂuorescence (λex/λem = 280/320) can be used as a proxy for the dynamics of the labile dissolved organic nitrogen (DON) pool, opening up the potential for looking using FDOM to look at aspects of the nitrogen cycle. Copepods have been observed to exude a humic-like substance that ﬂuoresces at peak M (C1; Urban-Rich et al., 2006), however in the present study the distribution of this ﬂuorophore was relatively constant throughout the water column and showed no indication it was sourced from grazing (Figure 5). PARAFAC Identiﬁcation of FDOM Components The composition of the phytoplankton prey is also important to the FDOM formed via grazing (Urban-Rich et al., 2004), with a release of humic like material with a diet of either diatoms or dinoﬂagellates in the exponential growth phase, but feeding on senescent cells lead to an increase in protein like FDOM. Interestingly Urban-Rich et al. (2006) found a shift to lower wavelength humic-like material may reﬂect a unique zooplankton signal. However, in the present case it appears that PARAFAC is unable to resolve such small diﬀerences in spectral signals in order to determine these as separate individual components. Production/Decomposition of FDOM by Phytoplankton, Zooplankton, and Bacteria Laboratory studies have also shown that bacteria can simultaneously remove ﬂuorescence associated with FDOM peak M that had been exuded by phytoplankton, while producing DOM (λex/λem = 340/440) similar to FDOM peak C (Romera-Castillo et al., 2011). The generation of FDOMH by remineralization of particulate material resulting in the consumption of O2 in intermediate and deep waters is presumed to occur by microbial activity (Yamashita and Tanoue, 2008) and results in strong correlations between FDOMH and AOU and species which are similarly regenerated by remineralization processes (e.g., NO− 3 and PO3− 4 ). A strong correlation is found between FDOMH and AOU in most of the world’s ocean basins (Jørgensen et al., 2011; Catalá et al., 2016) with the exception of the North Atlantic (Jørgensen et al., 2011; Heller et al., 2013). In the equatorial Atlantic De La Fuente et al. (2014) found support for this relationship between AOU and FDOMH (deﬁned in their study as λex/λem = 340/440). In situ evidence for the formation of FDOMH comes from dark Marine The Fluorescence index (FI = λex,470/λex,520 at λem,370; Hansen et al., 2016) has been used to distinguish between DOM derived from terrestrial or microbial sources. In the present case there is almost no ﬂuvial input to this region (Cotrim Da Cunha et al., 2009), though terrestrial humic material may be associated with Saharan dust (Williams et al., 2007; Paris and Desboeufs, 2013) deposited in this region as has been previously suggested for the supply of organic matter to lakes in the Alps (Mladenov et al., 2011). In the present study we found values of FI = 2.6 ± 1.2, this is considerably higher than the typical range of reported values for FI of 1.2–1.8, however Hansen et al. (2016) found that leachates of the marine diatom Thalassiosira weissﬂogii had FI values up to 3.5 following biodegradation and lower values when this was combined with exposure to light. Earlier work at an oligotrophic site in the North Paciﬁc (Station Aloha), far from any freshwater inputs had lower values for FI ranging from 1.55 in the surface to 1.72 at 3500 m (Helms et al., 2013). Thus, it appears that FI when applied in a marine setting is not a good indicator of terrestrial input but could instead be an indicator or phytoplankton derived FDOM. Q + O− 2 →Q•−+ O2 (R1) QH2 + O− 2 →Q•−+ H2O2 (R2) Q•−+ Q•−2H+ −→QH2 + Q (R3) Q + O− 2 →Q•−+ O2 (R1) QH2 + O− 2 →Q•−+ H2O2 (R2) Q•−+ Q•−2H+ −→QH2 + Q (R3) (R3) where Q is the quinone, QH2 is the hydroquinone and Q•−is the semiquinone radical (Eyer, 1991; Roginsky et al., 1999). The semiquinone radical however can also generate superoxide by reactions with oxygen (Meisel, 1975). Q•−+ O2 →Q + O− 2 (R4) (R4) If no catalytic cycle is able to be established, then the reactant is consumed in a stoichiometric fashion and the reaction products will favor either O2 or H2O2 depending on whether it is a reducing or oxidizing reaction. Identiﬁcation of quinones as FDOM by 3D EEM is complicated as typically the hydroquinone has a high ﬂuorescence quantum yield than the quinone itself, with many of quinones having no apparent ﬂuorescence (Ma et al., 2010). Indeed it has been suggested that carbonyl compounds may play a more important role in the FDOM signal than quinones (Ma et al., 2010). Marine So while quinones are likely present in seawater there is currently no data on what concentrations they may be present in. In the present study, our PARAFAC analysis did not detect a distinct chromophore that aligned to peak C (C2 was aligned to A and C), which was initially regarded as being of terrestrial origin, though it has also been shown to form in seawater due to microbial action (Romera-Castillo et al., 2011). Another metric commonly used to assess terrestrial vs. marine FDOM is the ratio of Peak M to Peak C ﬂuorescence (M:C), which should correlate with the relative source strength of marine-derived FDOM (λex,310/λem,410) vs. terrestrial FDOM (λex,345/λex,445; Helms et al., 2013). Across the Mauritanian shelf we found M:C = 1.03 ± 0.19. Higher values (mean 2.09) have been observed in coastal Mediterranean surface waters (Para et al., 2010). In the context of the hypothesis by Andrew et al. (2013) that all marine humic material may be terrestrial in origin it is clear that there are no distinctly terrestrial only FDOM signals, as a marine source can also be found, and that testing of this hypothesis requires the application of other analytical techniques. y y It should be noted that the present work was conducted in a lab and in the absence of solar irradiation. During daylight in the ocean there are also reactants that could be photochemically generated, for instance oxidation of tryptophan (or other phenoxy species) to the semi oxidized Tryptophan radical occurs on reaction with Br− 2 , itself formed from OH reactions in seawater (Zaﬁriou et al., 1987). The tryptophan radical species reacts rapidly with O− 2 to form tryptophan hydroperoxide (Ehrenshaft et al., 2015), however it can also react with O2 to form O− 2 and so may be both a source and sink for O− 2 . Similarly, tyrosine and protein tyrosyl radicals react rapidly with O− 2 to form hydroperoxides (Möller et al., 2012; Das et al., 2014) and these reactions may be important under certain conditions (e.g., in the sea surface microlayer). Potential CDOM/FDOM Components as Sinks for O− 2 Potential CDOM/FDOM Components as Sinks for O− 2 A number of organic species (e.g., quinones, thiols) have been suggested previously as potential DOM sinks for O− 2 in seawater (Heller and Croot, 2010b). The correlation we found between kDTPA and S350−400(ρ = 0.58, n = 36, p < 0.002) is suggestive of a reaction between superoxide and aromatic moieties such as quinones, phenols/polyphenols, or humics which absorb over this range (Wünsch et al., 2015). Quinones have previously been identiﬁed as the most likely candidates as they react rapidly with O− 2 and can be involved in a catalytic cycle with regeneration of the original reactant and production of O2 and H2O2. CDOM Promoted O− 2 Decomposition Pathways O− 2 Decomposition Rates across the Mauritanian Upwelling Photobleaching and Photoformation of FDOM h l b d h k C h Photobleaching and Photoformation of FDOM It has previously been noted that EEM Peak C is the most photo- labile component of FDOM (Helms et al., 2013) as expected as it is presumably lost by direct photochemical reactions as its excitation spectrum stretches into the near UV and thus will be exposed to sunlight in near surface waters. Similarly peak B has also been identiﬁed as being prone to photobleaching (Helms et al., 2013). No component similar to peak B was identiﬁed using PARAFAC in the present work. Conversion between FDOM August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org Frontiers in Marine Science \| www.frontiersin.org 12 FDOM/O− 2 Decay - Mauritanian Shelf Heller et al. These values are similar to what has been observed in other recent studies of O− 2 loss rates (see Table 2). species is also possible, as a humic like material was produced from the photo irradiation of the protein tyrosine (peak B; Berto et al., 2016). Nitrate and nitrite may also act as a photosensitizers in the photo-transformation of phenol containing compounds to form FDOM (Calza et al., 2012). A further potential reaction that may result in changes to FDOM is from bromination of organic matter (Méndez-Díaz et al., 2014) due to the reaction of CDOM with Br− 2 formed by the reaction of Br−and OH radicals (Zaﬁriou et al., 1987). Polyphenols As Sources of O− 2 in Seawater Polyphenols As Sources of O2 in Seawater That some polyphenols (e.g., tannin, pyrogallol, or gallic acid) produce H2O2 in weak alkaline solutions from reactions between with O2 has been known for over 150 years (Schönbein, 1860). The reaction is thought to proceed via O− 2 as an intermediate and both thermal and photochemical pathways of H2O2 formation have been observed (Clapp et al., 1990). The presence of such compounds could result in an additional O− 2 thermal ﬂux in experiments utilizing SOTS-1, though at typical seawater temperatures and assuming nM concentrations of these polyphenols this ﬂux would be expected to be relatively insigniﬁcant. (1) Recent work has shown that polyphenols were released in response to metal stress by the diatom Phaeodactylum tricornutum (Rico et al., 2013; Santana-Casiano et al., 2014). Gallic acid was only detected in iron-enriched diatom cultures. (2) An earlier estuarine study (Maie et al., 2007) using size exclusion chromatography found that the “T-peak,” usually assigned to “Tryptophan-like” substances (Coble, 1996), typically of high molecule weight (Yamashita and Tanoue, 2003), could be separated into a high molecule weight protein signal and a lower molecular weight polyphenol signal. More recently an investigation of riverine humics (Pagano et al., 2012) showed that the polyphenol, tannic acid, gave a peak λex/λem∼270/340 nm, similar to the “T-peak.” Consideration of Other Sources of FDOM to the Study Region In our study region there are no major riverine sources (Cotrim Da Cunha et al., 2009) to the coastal and thus a terrestrial/ﬂuvial source for organic matter is considered unlikely. However, sandwiched between the upwelling region and the Saharan dust is the Banc d’Arguin, a shallow gulf with extensive tidal ﬂats covered with extensive seagrass beds, predominantly Zostera noltii and Cymodocea nodosa (Hemminga and Nieuwenhuize, 1991). Many species of seagrasses have been found to contain high concentrations of polyphenols (e.g., Gallic, Caﬀeic, and Ferulic acid; Vergeer and Develi, 1997), including Zostera noltii (Grignon-Dubois et al., 2012) and Cymodocea nodosa (Cariello et al., 1979). Decaying seagrass (Hemminga and Nieuwenhuize, 1991) could thus be a potential source of poly phenols and/or FDOM to our study area, though from the limited physical oceanography carried out over the Banc d’Arguin the evidence suggest there is little exchange between the warm salty inshore water and the oﬀshore upwelling (Peters, 1976; Loktionov, 1993; Carlier et al., 2015) and most of the seagrasses decay in situ (Hemminga and Nieuwenhuize, 1991). g (3) Analysis of the OpenFluor database (Murphy et al., 2014; Wünsch et al., 2015) indicated a number of polyphenol compounds as being similar to the excitation/emission spectra identiﬁed by PARAFAC in 3D EEM spectra of natural samples. (3) Analysis of the OpenFluor database (Murphy et al., 2014; Wünsch et al., 2015) indicated a number of polyphenol compounds as being similar to the excitation/emission spectra identiﬁed by PARAFAC in 3D EEM spectra of natural samples. In light of this, the T-peak appears to be related to the phenol content of Typtophan rather than the protein component. In the current work, PARAFAC component C3 was similar to the classical “T-peak,” however there was no apparent correlation between C3 and kDTPA (r = −0.19, n = 36). Though, it is recognized that there is a wide range of reaction rates with O− 2 for the diﬀerent phenolic compounds (Taubert et al., 2003) that may be present in this FDOM pool so it might be expected that there is very poor or no correlation at all between kDTPA and C3. As noted previously there is a diﬃculty in discriminating between similar phenol compounds using PARAFAC (Bosco et al., 2006). for maximal reaction rate constants with superoxide (Bors and Michel, 1999). 1998; Krachler et al., 2012) and that they can form iron humic complexes that stabilize riverine iron in seawater (Krachler et al., 2010, 2012, 2015). Indeed, CDOM optical properties have been used to predict lignin concentration in seawater (Fichot et al., 2016). Gallic acid, along with other polyphenols from peat lands, has also been suggested as a possible ligand for transporting Fe to the sea (Wu et al., 2016). However, several lines of direct and indirect evidence now point to a seawater source for some of these compounds: Frontiers in Marine Science \| www.frontiersin.org Polyphenols as Superoxide Sinks in Seawater Across our study area (Table S4), values of kDTPA varied from 0.0003 to 0.0169 s−1 (mean 0.0074 ± 0.0038 s−1) while kSW ranged from 0.0006 to 0.0241 s−1 (mean 0.0122 ± 0.0054 s−1). Previously it has been assumed that only source of polyphenols is from oxidation of terrestrial lignins (Opsahl and Benner, 1997, August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org 13 FDOM/O− 2 Decay - Mauritanian Shelf Heller et al. for maximal reaction rate constants with superoxide (Bors and Michel, 1999). ACKNOWLEDGMENTS We sincerely thank the oﬃcers and crew of the RV Maria S. Merian and the chief scientist, O. Pfannkuche, for their help at sea. Special thanks are also due to P. Streu, A. Bleyer (both GEOMAR), T. Kalvelage and M. Holtappels (both MPI) for their ship based and laboratory analysis. This work is a contribution of the Collaborative Research Centre 754 “Climate–Biogeochemistry Interactions in the Tropical Ocean” (http://www.sfb754.de), which is supported by the Deutsche Forschungsgemeinschaft (DFG). CDOM Red Fluorescence in OMZ Waters Overall studies have shown that compounds with pyrogallol or catechol moieties are the most rapid superoxide scavengers, and the gallate moiety was found to be the minimal essential structure August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org 14 FDOM/O− 2 Decay - Mauritanian Shelf Heller et al. FIGURE 8 \| Dissolved O2 (dotted line) and FDOM (λex/λem = 420/640) pigment “oxy” (green triangles) at 5 stations along the transect in the Mauritanian Upwelling. FIGURE 8 \| Dissolved O2 (dotted line) and FDOM (λex/λem = 420/640) pigment “oxy” (green triangles) at 5 stations along the transect in the Mauritanian Upwelling. (CR145/17-1). Research work at sea was funded through SFB754 (DFG), project B5 (PC). (CR145/17-1). Research work at sea was funded through SFB754 (DFG), project B5 (PC). wavelengths but there was no apparent correlation with oxygen concentrations (Figure 8) and the signal appeared to be related more to soluble pigments released in the photic zone presumably by zooplankton grazing (Kleppel, 1998). SUPPLEMENTARY MATERIAL MH’s participation was ﬁnancially supported by the German BMBF project SOPRAN III (FKZ 03F0611A and 03F0662A, PC co-PI) that forms part of the German contribution to SOLAS (Surface Ocean Lower Atmosphere Studies). Funding for the participation of KW was awarded to PC from the DFG The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmars. 2016.00132 CDOM Red Fluorescence in OMZ Waters CDOM Red Fluorescence in OMZ Waters Previously Röttgers and Koch (2012) had reported the presence of a distinct absorption shoulder in CDOM at 415–420 nm in the OMZ waters of the Atlantic that was partially correlated with AOU. On further examination using methanol extracts of CDOM they related the absorption peak at ∼415 nm to red ﬂuorescence at 650 nm. Röttgers and Koch suggested this peak may be bacterial in origin and most likely a non-chlorin, metal- free porphyrin, like degradation products of hemes, cytochromes, and chlorophyll c, etc. In an earlier work red ﬂuorescence (Ex/Em 420/660) had been found to correlate with dissolved O2 in the OMZ of the Arabian Sea (Breves and Reuter, 2000; Breves et al., 2003) with values ranging from 0 to 0.01 Raman units nm−1 (Ex/Em 270/300 Heuermann et al., 1995). Conversely while a small absorption line at 420 nm was also reported for CDOM data from the Equatorial Atlantic (Andrew et al., 2013), those authors did not observe any related ﬂuorescence at 640 nm and could not exclude it as a ﬁltration artifact. In our work we observed ﬂuorescence in the dissolved phase at these excitation/emission Several of the polyphenols are known sinks for O− 2 (Taubert et al., 2003; Terpinc and Abramovi´c, 2010) and while there is currently signiﬁcant interest in them as antioxidants there is surprisingly little agreement about their rate of reactivity with O− 2 as most studies focused on inhibition experiments. An example of this is Ferulic acid (FA), a cinnamyl phenol, and an oxidation product of lignin, that are particular abundant in grasses and many herbaceous tissues (Opsahl and Benner, 1998). FA reacts with O− 2 (Toda et al., 1991; Nasr Bouzaiene et al., 2015), to form a radical species which is resonance stabilized and can form the dimer curcumin and other products (Graf, 1992). FA was also one of the phenolic compounds excreted by diatoms in the study by Rico et al. (2013). Gallic acid (GA) reacts moderately fast with O− 2 (Table S5: kO2−= 5.4 × 106 M s−1 at 25◦C; Taubert et al., 2003). 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Atmospheres 112:D07302. doi: 10.1029/2005JD006702 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Wommack, K. E., and Colwell, R. R. (2000). Virioplankton: viruses in aquatic ecosystems. Microbiol. Mol. Biol. Rev. 64, 69–114. doi: 10.1128/MMBR.64.1.69- 114.2000 Copyright © 2016 Heller, Wuttig and Croot. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Wünsch, U. J., Murphy, K. R., and Stedmon, C. A. (2015). Fluorescence quantum yields of natural organic matter and organic compounds: implications for the ﬂuorescence-based interpretation of organic matter composition. Front. Mar. Sci. 2:98. doi: 10.3389/fmars.2015.00098 August 2016 \| Volume 3 \| Article 132 Frontiers in Marine Science \| www.frontiersin.org 19
https://openalex.org/W1991609863	https://europepmc.org/articles/pmc4060487?pdf=render	English	null	Prevalence of Corneal Astigmatism in Patients before Cataract Surgery in Northern China	Journal of ophthalmology	2,014	cc-by	5,112	Hindawi Publishing Corporation Journal of Ophthalmology Volume 2014, Article ID 536412, 7 pages http://dx.doi.org/10.1155/2014/536412 Hindawi Publishing Corporation Journal of Ophthalmology Volume 2014, Article ID 536412, 7 pages http://dx.doi.org/10.1155/2014/536412 Hindawi Publishing Corporation Journal of Ophthalmology Volume 2014, Article ID 536412, 7 pages http://dx.doi.org/10.1155/2014/536412 Correspondence should be addressed to Xin Tang; tangprofessor@aliyun.com Received 7 February 2014; Revised 26 April 2014; Accepted 19 May 2014; Published 3 June 2014 Received 7 February 2014; Revised 26 April 2014; Accepted 19 May 2014; Published 3 June 2014 Academic Editor: Suddhasil Mookherjee Academic Editor: Suddhasil Mookherjee Copyright © 2014 Xiaoyong Yuan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © 2014 Xiaoyong Yuan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Purpose. To analyze the prevalence and presentation patterns of corneal astigmatism in cataract surgery candidates in a teaching hospital in northern China. Methods. From May 1, 2012, to April 30, 2013, partial coherence interferometry (IOLMaster) measurements of all qualified cataract surgery candidates were retrospectively collected and analyzed. Results. The study evaluated 12,449 eyes from 6,908 patients with a mean age of 69.80±11.15 (SD) years. The corneal astigmatism was 0.5 diopters (D) or less in 20.76% of eyes, 1.0 D or more in 47.27% of eyes, 2.0 D or more in 13.16% of eyes, and 3.0 D or more in 3.75% of eyes. With-the-rule astigmatism was found in 30.36% of eyes, while against-the-rule was found in 52.41% of eyes. The percentage of against-the-rule astigmatism increased with age. Conclusion. Our study showed that almost one-half of preoperative eyes (47.27%) in northern China have a corneal astigmatism of 1.0 D or more, indicating that more surgical techniques or toric IOLs are needed to achieve better visual rehabilitation. Xiaoyong Yuan,1 Hui Song,1 Gang Peng,2 Xia Hua,1 and Xin Tang1 1 Tianjin Eye Hospital, Tianjin Key Laboratory of Ophthalmology and Vision Science, Clinical College of Ophthalm Tianjin Medical University, No. 4 Gansu Road, Heping District, Tianjin 300020, China Tianjin Medical University, No. 4 Gansu Road, Heping District, Tianjin 300020, China 2 Department of Bioinformatics and Computational Biology, University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA j y p g j 2 Department of Bioinformatics and Computational Biology, University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA 2 Department of Bioinformatics and Computational Biology, University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA Correspondence should be addressed to Xin Tang; tangprofessor@aliyun.com 1. Introduction reports have shown that IOLMaster can precisely measure preoperative corneal astigmatism and can predict the residual corneal astigmatism after cataract surgery [6].h Phacoemulsification is one of the most successfully and commonly performed cataract surgeries worldwide. With the development of modern surgical techniques and intraocular lenses (IOLs), patients expect and demand refractive error correction after cataract surgery. Advances in the calculation of IOL power have significantly reduced the incidence of spherical refractive errors, while residual astigmatism after surgery is a concern for both ophthalmologists and patients and can leave patients with symptomatic decreased visual function [1–5]. t The distribution and prevalence of corneal astigmatism in cataract patients of different countries have been previously reported [5, 7–10]. An estimated 13,780,000 cases of blindness have been caused by cataracts in China [11] and two groups from Guangzhou [8] and Shanghai [12] have reported the distribution of corneal astigmatism before cataract surgery in southern and central China. However, there are no similar reports for cataract patients in northern China. This study reviewed all of the cataract cases in one year in one of the largest eye hospitals in China to investigate the prevalence of corneal astigmatism in a large sample in northern China. The findings may aid hospitals and manufacturing companies in evaluating the requirement for the use of toric IOLs or other reported surgical methods. Partial coherence interferometry (IOLMaster, Carl Zeiss Meditec, Berlin, Germany) is widely used due to its superior performance in the measurement of ocular axial length. The IOLMaster analyzes six light reflections projected onto the anterior corneal surface within a 2.3 mm radius and can also be used as an automated keratometer (AK). Recent 2 Journal of Ophthalmology 2 Table 1: Patient demographics compared with 5 other published studies. Table 1: Patient demographics compared with 5 other published studies. Present Guan et al. [12] Chen et al. [8] Ferrer-Blasco et al. [7] Khan and Muhtaseb [5] De Bernardo et al. 1. Introduction [14] Eyes/patients 12449/6908 1430/827 4831/2849 4540/2415 1230/746 757/380 Age (y) Mean ± SD 69.80 ± 11.15 72.27 ± 11.59 70.56 ± 9.55 60.59 ± 9.87 75.54 ± 0.71 71.89 ± 10.19 Range 30, 97 16, 98 49, 95 32, 87 30, 104 33, 96 Male/female 3199/3709 359/468 1090/1759 768/1647 343/403 176/204 Corneal astigmatism (D) Mean ± SD 1.15 ± 0.84 1.07 ± 0.73 1.01 ± 0.69 0.90 ± 0.93 1.03 ± 0.73 1.02 ± 0.69 Range 0.0, 6.63 0.06, 5.52 0.05, 6.59 0.25, 6.75 0.0, 6.2 0.06, 4.57 K1 mean ± SD 43.93 ± 1.67 43.57 ± 1.56 43.76 ± 1.53 43.48 ± 1.61 43.43 ± 1.49 43.54 ± 1.43 K2 mean ± SD 45.08 ± 1.73 44.64 ± 1.65 44.76 ± 1.56 44.08 ± 1.59 44.46 ± 1.56 44.56 ± 1.52 Corneal astigmatism (%) ≤0.5 D 20.76 21.2∗ 23.14 58.8 24.47 23.38 ≥1.0 D 47.27 45.37 41.3 34.8 40.4 41.74 ≥2.0 D 13.16 10.33 8.22 9.26∗∗ 9.67 8.32 ≥3.0 D 3.75 2.22 3.52 5.61∗∗∗ 4.61 2.64 ∗Not including 0.5 D, ∗∗not including 2.0 D, and ∗∗∗not including 3.0 D. D = diopter, K1 = flat keratometry, and K2 = steep keratometry. 40 35 30 25 20 15 10 5 0 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 Corneal astigmatism (D) Frequency (%) 20.76 32.54 21.29 12.45 6.18 3.03 1.71 0.80 0.59 0.22 0.18 0.14 0.1 6.01 and above Figure 1: Distribution of corneal astigmatism in 0.5 D increments for all 12,449 eyes. 2. Patients and Methods Retrospective biometry data were collected for all patients who had routine cataract surgery at the Tianjin Eye Hospital between May 1, 2012, and April 30, 2013. Cataract patients with a history of ocular surgery, corneal disease, and inflam- mation and with an age younger than 30 years old and a dense cataract that did not allow IOLMaster measurement were excluded. Routine eye examinations were performed before operation, including visual acuity, refraction, tonometry, slit lamp evaluation, and dilated fundus examination. The study was approved by the Human Research Ethics Committee at the Tianjin Eye Hospital and all procedures adhered to the tenets of the Declaration of Helsinki. All patients provided written informed consent. Three experienced technicians collected the keratometric data for consecutive patients using IOLMaster version 5.3 and the mean of five measurements was used for the parameters.t Figure 1: Distribution of corneal astigmatism in 0.5 D increments for all 12,449 eyes. p Data were analyzed by the R software package ver- sion 2.15.2 R Core Team (R Foundation for Statistical Computing, Vienna, Austria, ISBN 3-900051-07-0, URL http://www.R-project.org/). The Kolmogorov-Smirnov test was used to evaluate the normal distribution of variables. The results showed that the data were normally distributed, except for the data regarding astigmatisms. One-way analysis of variance and the Kruskal-Wallis test were applied for the comparison of variance for normally and nonnormally distributed data among different age groups, respectively. The 𝑡-test was used to compare keratometry between the two groups and a Wilcoxon signed rank test was used to compare corneal astigmatism data. Spearman’s rank test was used to assess the relationship between age and astigmatism. A 𝑃 value less than 0.05 was considered statistically significant. ∗Not including 0.5 D, ∗∗not including 2.0 D, and ∗∗∗not including 3.0 D.l 3. Results This study was composed of 12,449 eyes from 6,908 patients. The patient demographics are shown in Table 1, which also shows a comparison of 5 other published papers. Figure 1 presents a histogram of the frequency distribution of corneal astigmatism. Among all of the patients, astigmatism of 0.51 to 1.00 D was the most common cylinder value (32.54%), followed by 1.01 to 1.50 D (21.29%) and 0.0 to 0.50 D (20.76%). In total, 3200 eyes (25.41%) exhibited a corneal astigmatism of 1.5 D or greater.l Table 2 presents the descriptive flat keratometry (K1) and steep keratometry (K2) in the 7 age groups. A gradually 3 Journal of Ophthalmology 3 Table 2: Descriptive statistics by age group. Age group (y) Astigmatism (D) K1 (D) mean ± SD K2 (D) mean ± SD Eyes (%) 30–40 1.33 ± 0.85 42.77 ± 2.23 44.10 ± 2.38 164 (1.32) 41–50 1.10 ± 1.10 43.51 ± 1.83 44.61 ± 1.92 571 (4.59) 51–60 0.99 ± 0.71 43.91 ± 1.61 44.90 ± 1.70 1869 (15.01) 61–70 1.05 ± 0.80 44.04 ± 1.63 45.10 ± 1.70 3226 (25.91) 71–80 1.20 ± 0.83 43.95 ± 1.65 45.14 ± 1.69 4517 (36.28) 81–90 1.34 ± 0.90 43.95 ± 1.68 45.28 ± 1.70 1993 (16.01) ≥91 1.39 ± 0.82 43.73 ± 1.65 45.12 ± 1.76 109 (0.88) 𝑃∗ <0.001 <0.001 <0.001 <0.001 D = diopter, K1 = flat keratometry, and K2 = steep keratometry. ∗Kruskal-Wallis test. Table 2: Descriptive statistics by age group. 10 8 6 4 2 0 30–40 41–50 51–60 61–70 71–80 81–90 91 and above Age groups (years) Corneal astigmatism (D) 10 8 6 4 2 0 30–40 41–50 51–60 61–70 71–80 81–90 91 and above Age groups (years) Corneal astigmatism (D) increased with age and the WTR astigmatism proportion decreased with age, except in the 30–40-year-old age group, which showed a slightly higher percentage of ATR astigma- tism (Figure 4). No significant difference was found between the right and left eyes in K1 (43.91 ± 1.76 versus 43.94 ± 1.66, 𝑡= 0.75, 𝑃= 0.45) or K2 (45.08±1.73 versus 45.06±1.72, 𝑡= 0.66, 𝑃= 0.51). A statistically significant difference was found between right and left eye corneal astigmatisms with the Wilcoxon signed rank test (1.17 ± 0.85 D versus 1.13 ± 0.82 D, statistic = 19840457, 𝑃= 0.02). 3. Results The K1 and K2 values in females were higher than those in males (K1: 44.11 ± 1.67 versus 43.71 ± 1.74, 𝑡= 13.20, 𝑃< 0.0001; K2: 45.30 ± 1.72 versus 44.82 ± 1.80, 𝑡= 15.34, 𝑃< 0.0001). The corneal astigmatism in females was significantly greater than that in males according to the Wilcoxon signed rank test (1.19 ± 0.87 versus. 1.11 ± 0.80, statistic = 18500144, 𝑃< 0.0001). Figure 2: Corneal cylinder in all 7 age groups. The bold lines in the boxes represent the median (50% percentile), the upper and lower limits of the box represent the first quartile (25% percentile) and third quartile (75% percentile), and the bars represent the minimum and maximum values. 4. Discussion The ATR astigmatism proportion ATR astigmatism was the predominant group, compris- ing 52.41% of the cases, and the prevalence increased with Journal of Ophthalmology 4 40 35 30 25 20 15 10 5 0 Frequency (%) 20.73 21.34 23.17 16.46 7.93 3.05 6.10 0.61 0.61 0.00 0.00 0.00 0.00 30–40years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (a) 40 35 30 25 20 15 10 5 0 Frequency (%) 25.22 30.12 19.61 12.61 5.43 3.85 2.10 0.53 0.35 0.00 0.00 0.00 0.18 41–50years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (b) 40 35 30 25 20 15 10 5 0 Frequency (%) 25.84 36.54 19.21 9.36 4.71 1.98 1.44 0.43 0.32 0.11 0.00 0.00 0.05 51–60years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (c) 40 35 30 25 20 15 10 5 0 Frequency (%) 24.33 35.37 19.44 10.51 4.93 2.14 1.61 0.59 0.34 0.28 0.25 0.09 0.12 61–70years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (d) 40 35 30 25 20 15 10 5 0 Frequency (%) 19.13 30.66 22.60 13.79 6.51 3.45 1.51 1.04 0.69 0.2 0.2 0.11 0.11 71–80years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (e) 40 35 30 25 20 15 10 5 0 Frequency (%) 13.20 30.56 23.28 14.65 8.68 4.21 2.16 1.00 1.05 0.40 0.25 0.45 0.10 81–90years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (f) 40 35 30 25 20 15 10 5 0 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 Corneal astigmatism (D) Frequency (%) 11.01 23.85 27.52 20.18 10.09 3.67 0.92 0.92 0.92 0.92 0.00 0.00 0.00 6.01 and above ≥91years (g) Figure 3: Frequency distribution of corneal astigmatism in 0.50 D steps for the 7 age groups. 4. Discussion This study determined the distribution of corneal astigma- tism in different age groups in northern China. Several stud- ies have investigated the prevalence of corneal astigmatism using IOLMaster [5, 7, 9, 12–14], which not only affords the measurement of corneal status but also enables the easy and reliable calculation of IOLs as well as postoperative refraction data. The IOLMaster database was accessed for all cataract candidates in an entire year. The results showed that the mean age was slightly younger than previously reported data [8, 12] and that the 71–80-year-old age group occupied 36.28% of all cases, followed by the 61–70-year-old age group (25.91%) and the 81–90-year-old age group (16.01%); these results were similar to those of Chen et al. report from Guangzhou [8] but differed from those of Khan and Muhtaseb’s report [5]. Khan et al. reported that the 71–80-year-old age group was the largest, followed by the 81–90- and 61–70-year-old age groups [5]. In terms of gender distribution, our study showed that the number of female patients was greater than that of males, which is consistent with other published studies [5, 7, 8, 12]. h d increasing keratometry value was observed with age, partic- ularly in K2. Most eyes in this cohort were between 71 and 80 years old, which represented more than one-third (36.28%) of all cases. Patients between 61 and 70 years old represented one-fourth (25.91%) of all cases. Figure 2 shows the corneal astigmatism values in each age group. Spearman’s rank correlation between age and astigmatism was 𝑟= 0.12 with 𝑃< 0.001. Figure 3 depicts the distributions of corneal astigmatism in the different age groups. With-the-rule (WTR, the steep meridian of the cornea being within 90 ± 30 degrees) corneal astigmatism was found in 3779 eyes (30.36%), against-the-rule (ATR, the steep meridian of the cornea being within 180 ± 30 degrees) corneal astigmatism was found in 6524 eyes (52.41%), and oblique (neither WTR nor ATR) corneal astigmatism was found in 2146 eyes (17.22%). 4. Discussion These findings are generally consistent with those of previous studies [7, 13, 15, 16].h The mean corneal astigmatism of this cohort was 1.15 ± 0.84 D (range from 0.0 to 6.84 D), which is slightly higher than that in other published studies [5, 7, 8, 12, 15]. The right (1.17 D) and left eyes (1.13 D) significantly differed, which is in contrast to the findings of Hoffmann and H¨utz’s study [15]. Interestingly, the corneal astigmatism in females (1.19 D) was significantly greater than that in males (1.11 D). p Corneal astigmatism changes significantly with age [15, 36–39]. Our study and the two previous Chinese investi- gations support this tendency [8, 12]. The mean values of K1, K2, corneal astigmatism, and other parameters in our study were slightly higher than those reported by some other studies [5, 7] but closely resemble those from reports from Shanghai [12] and Guangzhou [8], China. Possible reasons may be the inclusion of different racial and ethnic groups, different inclusion criteria, and different age distributions, among others. Our retrospective study was clinically based, which may lead to some selection bias. One advantage of our study is that we selected all of the cataract surgery candidates from an entire year, which presented more than one-half of cataract surgery cases in the same year in Tianjin, a city with 12,280,000 people http://en.wikipedia.org/wiki/Tianjin. i In our study, 20.76% of eyes had a corneal astigmatism of 0.5 D or less, which is lower than the results from other groups [5, 7, 8, 12]. A large proportion of eyes (47.27%) had a corneal astigmatism of 1.0 D or greater, which is higher than the results reported by the abovementioned groups. Additionally, 3.75% of eyes had 3.0 D of corneal astigmatism [8, 12], which is a greater prevalence than that reported by the other two Chinese studies (2.22%, 3.52%) but is lower than that reported by European studies (5.61%, 4.61%) [5, 7]. All age groups showed a similar distribution pattern of corneal astigmatism, except for the 30–40- and above 91-year-old age groups, which showed some variation in the astigmatism distribution. A previous study showed a similar distribution pattern [5, 7]. In conclusion, our study revealed the distribution of all cataract candidates in one year in a single hospital in northern China. A number of our cases (47.27%) exhibited a corneal astigmatism of 1.0 D or more. Corneal astigmatism increases with age. 4. Discussion The procedure chosen primarily depends on the precise measurements of preoperative corneal astigmatism. [19, 20], femtosecond laser-assisted astigmatic keratotomy [21, 22], and toric intraocular lens (IOL) implantation [23– 25]. The procedure chosen primarily depends on the precise measurements of preoperative corneal astigmatism. p p g A clear corneal incision may result in a surgically induced corneal astigmatism in patients with 0.5 D [26]. In our study, 53.30% of eyes had a corneal astigmatism of 1.0 D or less and received sufficient correction through the performance of on-axis phacoemulsification combined with monofocal IOL implantation [5]. Meanwhile, 23.41% of eyes exhibited more than 1.50 D of corneal astigmatism in our study, which is similar to the findings of Khan et al. (20.5%) [5] and Ferrer-Blasco et al. (22%) [7], although their studies required more manipulations or techniques to correct for better visual rehabilitation. Other techniques, such as manual or femtosecond laser-assisted arcuate keratotomy, were used to correct much worse corneal astigmatisms [27, 28]. Recently, limbal femtosecond laser-assisted intrastromal arcuate keratotomy has been used for corneal astigmatisms of 1.50 ± 0.47 D [22]. However, the results of this procedure are unpredictable or are associated with complications [29, 30]. Considering the high cost of femtosecond laser surgery, the majority of the population in China cannot afford such procedures. Figure 4: Percentages of WTR, ATR, and oblique corneal astigma- tisms in the 7 groups. p Toric IOLs have been used clinically since they were first described by Shimizu et al. [31], with encouraging results [32–35]. An analysis of the distribution of corneal astigmatism in a large cohort of cataract candidates will provide valuable information and benefits for manufacturers, ophthalmologists, and cataract patients. At present, toric IOLs can be used to correct corneal astigmatisms from 0.4 D to 8.4 D [7] during cataract surgery. In our case series, 1.51 D to 3.50 D represented 23.37% of all cases, most of which could be effectively corrected with toric IOLs. The higher cost of new IOLs may be another burden for patients and health insurance companies.i age, except for the 30–40-year-old age group, which showed a slightly higher percentage than the 41–50-year-old age group. Selection bias may account for this finding because the 30– 40-year-old age group represented only 1.32% of all cases. By contrast, the percentage of WTR astigmatism decreased with age. 4. Discussion 40 35 30 25 20 15 10 5 0 Frequency (%) 25.22 30.12 19.61 12.61 5.43 3.85 2.10 0.53 0.35 0.00 0.00 0.00 0.18 41–50years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (b) 40 35 30 25 20 15 10 5 0 Frequency (%) 20.73 21.34 23.17 16.46 7.93 3.05 6.10 0.61 0.61 0.00 0.00 0.00 0.00 30–40years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (a) 40 35 30 25 20 15 10 5 0 Frequency (%) 25.84 36.54 19.21 9.36 4.71 1.98 1.44 0.43 0.32 0.11 0.00 0.00 0.05 51–60years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (c) 40 35 30 25 20 15 10 5 0 Frequency (%) 24.33 35.37 19.44 10.51 4.93 2.14 1.61 0.59 0.34 0.28 0.25 0.09 0.12 61–70years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (d) 40 35 30 25 20 15 10 5 0 Frequency (%) 19.13 30.66 22.60 13.79 6.51 3.45 1.51 1.04 0.69 0.2 0.2 0.11 0.11 71–80years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (e) 40 35 30 25 20 15 10 5 0 Frequency (%) 13.20 30.56 23.28 14.65 8.68 4.21 2.16 1.00 1.05 0.40 0.25 0.45 0.10 81–90years 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 6.01 and above (f) (f) (e) (e) (f) 40 35 30 25 20 15 10 5 0 0.00–0.50 0.51–1.00 1.01–1.50 1.51–2.00 2.01–2.50 2.51–3.00 3.01–3.50 3.51–4.00 4.01–4.50 4.51–5.00 5.01–5.50 5.51–6.00 Corneal astigmatism (D) Frequency (%) 11.01 23.85 27.52 20.18 10.09 3.67 0.92 0.92 0.92 0.92 0.00 0.00 0.00 6.01 and above ≥91years (g) Figure 3: Frequency distribution of corneal astigmatism in 0 50 D steps for th (g) Figure 3: Frequency distribution of corneal astigmatism in 0.50 D steps for the 7 age groups. 5 Journal of Ophthalmology Journal of Ophthalmology 30–40 41–50 51–60 61–70 71–80 81–90 91 and above Age groups (years) 80 70 60 50 40 30 20 10 0 (%) With-the-rule Oblique Against-the-rule Figure 4: Percentages of WTR, ATR, and oblique corneal astigma- tisms in the 7 groups. 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https://openalex.org/W4395090339	https://www.researchsquare.com/article/rs-4068466/latest.pdf	English	null	Higher-order adaptive BFGS algorithm for nonconvex functions	Research Square (Research Square)	2,024	cc-by	6,213	Xiangli Li School of Mathematics and Computing Science, Guilin University of Electronic Technology Binglan Li School of Mathematics and Computing Science, Guilin University of Electronic Technology Zhiling Wang School of Mathematics and Computing Science Guilin University of Electronic Technology Higher-order adaptive BFGS algorithm for nonconvex functions Xiangli Li Xiangli Li Research Article Keywords: modi¦ed BFGS method, projection technique, global convergence Posted Date: March 15th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-4068466/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Keywords: modiﬁed BFGS method, projection technique, global convergence Higher-order adaptive BFGS algorithm for nonconvex functions Xiangli Li1,3, Binglan Li1,2 and Zhiling Wang1,2 1 School of Mathematics and Computing Science, Guilin University of Electronic Technology, 541004, Guangxi. 2 Guangxi Colleges and University Key Laboratory of Data Analysis and Computation 541004, Guangxi . 3 Center for Applied Mathmatics of Guangxi 541004 Guangxi 1 School of Mathematics and Computing Science, Guilin University of Electronic Technology, 541004, Guangxi. 2 Guangxi Colleges and University Key Laboratory of Data Analysis and Computation 541004, Guangxi . 3 1 School of Mathematics and Computing Science, Guilin University of Electronic Technology, 541004, Guangxi. 2 Guangxi Colleges and University Key Laboratory of Data Analysis and Computation 541004, Guangxi . 3 3 Center for Applied Mathmatics of Guangxi 541004, Guangxi . 3 Center for Applied Mathmatics of Guangxi 541004, Guangxi . Corresponding author(s). E-mail(s): lixiangli@guet.edu.cn; Contributing authors: binglanli0424@gmail.com; Abstract Quasi-Newton methods are commonly used to solve unconstrained optimization problems, among which BFGS method is one of the most common quasi-Newton method, which has stable and good performance.Whether BFGS method has global convergence or not under weak Wolfe-Powell (WWP) line search is a widely concerned issue in recent years. BFGS method can be easily proved to have global convergence for convex functions, but not for general nonconvex functions. There- fore we propose an modiﬁed BFGS algorithm with global convergence, and the following properties are shown in this algorithm:(i) a special descent condition is proposed to classify the iteration points into two categories; (ii) we construct a new paraboloid and the corresponding projection technique by utilizing the information of the third-order expansion of the function; (iii) for the points sat- isfying the special descent conditions, the original BFGS update formula is used; (iv) for points that do not satisfy the conditions, they are projected onto the proposed paraboloid.Our new improved BFGS algorithm has global convergence under WWP search and also has good numerical performance. 1 1 Introduction Consider the unconstrained nonlinear optimization problem min {ϕ(x) \| x ∈Rn} , (1.1) (1.1) where ϕ : Rn →R and ϕ(x) is twice continuously diﬀerentiable. where ϕ : Rn →R and ϕ(x) is twice continuously diﬀerentiable. The above unconstrained optimization model, which has a wide range of applica- tions on engineering ﬁelds, such as machine learning[1], artiﬁcial intelligence[2], image restoration and so on[3]. For (1.1), we often use the following (1.2)to solve it. xj+1 = xj + αjdj, (1.2) (1.2) where step size αj is generated by line search, and dj represents the search direction of the current point xj.In general we use inexact line search instead of exact line search to obtain αj.The following is a commonly used weak Wolfe-Powell (WWP) condition in inexact line searches[4]: where step size αj is generated by line search, and dj represents the search direction of the current point xj.In general we use inexact line search instead of exact line search to obtain αj.The following is a commonly used weak Wolfe-Powell (WWP) condition in inexact line searches[4]: ϕ (xj + αjdj) −ϕ (xj) ≤ζ1αjg (xj)T dj, (1.3) g (xj + αjdj)T dj ≥σg (xj)T dj, (1.4) (1.3) (1.4) where 0 < ζ1 < σ < 1. where 0 < ζ1 < σ < 1. where 0 < ζ1 < σ < 1. In this paper, a similar approach is taken to improve the BFGS algorithm for nonconvex functions, and the details of the property are as follows: • The suﬃcient descent condition similar to Yuan[15] is deﬁned as the boundary to separate iteration points. • A higher-order adaptive parabolic surface that is considered the projection surface is introduced. • The modiﬁed BFGS algorithm under the Wolfe line search also has the global convergence for nonconvex functions. • According to the numerical results, the proposed algorithms are more competitive than the similar algorithms. The next section describes the motivation for our modiﬁed BFGS algorithm and the speciﬁc algorithmic procedure. In section 3,we establish the global convergence of the algorithm and give a detailed proof process. In section 4, we perform numerical experiments and prove that the algorithm has good numerical experimental results by comparing it with the current popular quasi-Newton’s algorithm.The last section states the conclusion. where 0 < ζ1 < σ < 1. For the computation of the direction dj, the most common algorithms contain the conjugate gradient method and Newton’s method. Compared to the conjugate gradi- ent algorithm using only ﬁrst-order information of the function[5], Newton’s methods utilize the second-order information of the function through the computation of the Hessian matrix. The computation of the Hessian matrix can often be complex, leading many scholars[6–8] to propose quasi-Newton algorithms by simulating the computa- tion of Hessian matrix using a positive deﬁnite matrix Bj,and the direction dj is obtained by solving Bjdj = −gj. Among these, the most commonly proposed Newton algorithm is the Broyden-Fletcher-Goldfarb-Shanno (BFGS) method [9, 10], updates Bj via Bj+1 = Bj −BjsjsT j Bj sT j Bjsj + yjyT j sT j yj , (1.5) (1.5) where sj = xj+1 −xj and yj = gj+1 −gj, gjis the gradient functuon value. Although the BFGS method is the most eﬀective Newton algorithms, it does not have global convergence under the general Wolfe line search especially for nonconvex functions.The BFGS algorithm’s global convergence for convex problems under Wolfe line search was initially demonstrated by Powell[11]. In order to demonstrate that the BFGS approach does not converge for nonconvex functions under the Wolfe line search, Dai [10]has provided a six point counterexample. Even with the exact line search, Mascaren has [12] demonstrated that the convergence cannot be presented in the Broyden family methods. These two ﬁndings encourage us to look for other approaches to the BFGS method’s global convergence for nonconvex functions. Nowadays, a wide range of industries use projection technology [13]. In some bad circumstances, it can 2 also be used to acquire improved theoretical properties in a variety of optimization problems [14]. As a result,Yuan[15, 16] deﬁned a suﬃcient descent property condition as the boundary.They divided the points according to the boundary and projected the points that do not satisfy the conditions onto the corresponding paraboloid using the projection technique. In this paper, a similar approach is taken to improve the BFGS algorithm for nonconvex functions, and the details of the property are as follows: also be used to acquire improved theoretical properties in a variety of optimization problems [14]. As a result,Yuan[15, 16] deﬁned a suﬃcient descent property condition as the boundary.They divided the points according to the boundary and projected the points that do not satisfy the conditions onto the corresponding paraboloid using the projection technique. 2 Motivation and algorithm j j j j Recently, Yuan[16] added a new adaptive term ∥gj∥α to the special suﬃcient descent condition and projection surface, which further improves the performance of the algorithm. SD := n j \| dT j gj ≤−ραj ∥dj∥2 ∥gj∥α , j ≥0 o , (2.5) (2.5) where α ∈(∞, +∞) is a tuning parameter and ∥gj∥α can be viewed as an adaptive term. The deﬁnition of paraboloid is modiﬁed from (2.2) to (2.6) where α ∈(∞, +∞) is a tuning parameter and ∥gj∥α can be viewed as an adaptive term. The deﬁnition of paraboloid is modiﬁed from (2.2) to (2.6) n x ∈Rn \| µ ∥Vj −x∥2 ∥g(x)∥α + (Vj −x)T g (Vj) = 0 o . (2.6) (2.6) Case (c) : j ∈SD. Iteration proceeds as usual xj+1 = Vj. Case (c) : j ∈SD. Iteration proceeds as usual xj+1 = Vj. Case (c) : j ∈SD. Iteration proceeds as usual xj+1 = Vj. ∈SD. Iteration proceeds as usual xj+1 = Vj. Case (d) : j /∈SD. First, Yuan[16] projected the current point xj onto the surface(2.6), then the next iteration xj+1 is deﬁned below: xj+1 = xj + Pj ∥g (Vj) −g (xj)∥2 [g (Vj) −g (xj)] , (2.7) (2.7) where Pj = µ ∥Vj −xj∥2 ∥g (xj)∥α + (Vj −xj)T g (Vj) . (2.8) 2 3 (2.8) Similar to Yuan’s[15] idea of parabolic construction, µ ∥Vj −x∥2 + µ ∥Vj −x∥3 + (Vj −x)T g (Vj) can be viewed as the special combination of a ﬁrst-order expansion and a second-order expansion and a third-order expansion of a strongly convex function at the point Vj.On the basis of Yuan[15], we add third-order information to further characterize and inform the function, which is closer to the original function and makes the paraboloid more accurate.The new paraboloid is as follows: n x ∈Rn \| µ ∥Vj −x∥2 + µ ∥Vj −x∥3 + (Vj −x)T g (Vj) = 0 o . (2.9) (2.9) Case (i) : j ∈ID. Iteration proceeds as usual xj+1 = Vj. Case (i) : j ∈ID. Iteration proceeds as usual xj+1 = Vj. Case (ii) : j /∈ID. 2 Motivation and algorithm As we all known, sT j yj > 0 can insure the next iteration matrix Bj+1 of BFGS algorithm inherits the positive deﬁniteness of the previous matrix Bj, and it is one of the conditions for the BFGS algorithm to converge globally. This condition is well satisﬁed for convex functions, but not for general non-convex functions. Therefore, Yuan[15] introduces a projection technique to project the points that do not satisfy a particular descent condition onto the paraboloid. Yuan[15] deﬁnes a special suﬃcient descent condition(2.1)and projection surface (2.2): ID := n j \| dT j gj ≤−ραj ∥dj∥2 , j ≥0 o , (2.1) (2.1) and and n x ∈Rn \| µ ∥Vj −x∥2 + (Vj −x)T g (Vj) = 0 o , (2.2) (2.2) here ρ is a positive constant and step size αj is generated by line search. where ρ is a positive constant and step size αj is generated by line search. ositive constant and step size αj is generated by where ρ is a positive constant and step size αj is generated by line search. Case (a) : j ∈ID. Iteration proceeds as usual xj+1 = Vj. Case (b) : j /∈ID. First, Yuan[15] projected the current point xj onto the surface (2.2), then the next iteration xj+1 is deﬁned below: ρ j g y Case (a) : j ∈ID. Iteration proceeds as usual xj+1 = Vj. j Case (a) : j ∈ID. Iteration proceeds as usual xj+1 = Vj. j Case (a) : j ∈ID. Iteration proceeds as usual xj+1 = Vj. ( ) j p j+1 j Case (b) : j /∈ID. First, Yuan[15] projected the current point xj onto the surface (2.2), then the next iteration xj+1 is deﬁned below: Case (b) : j /∈ID. First, Yuan[15] projected the current point xj onto the surface (2.2), then the next iteration xj+1 is deﬁned below: xj+1 = xj + Pj dT j [g (Vj) −g (xj)]dj, (2.3) where Pj = µ ∥Vj −xj∥2 + (Vj −xj)T g (Vj) , (2.4) xj+1 = xj + Pj dT j [g (Vj) −g (xj)]dj, (2.3) (2.3) where Pj = µ ∥Vj −xj∥2 + (Vj −xj)T g (Vj) , (2.4) (2.4) 3 where Vj = xj + αjdj and parameter µ > 0. 2 Motivation and algorithm In summary, we propose a new higher-order adaptive paraboloid (2.15) instead of (2.9), and the updated iterative formula corresponding to x is changed from (2.10) to (2.16): n x ∈Rn \| µj ∥Vj −x∥2 + µj ∥Vj −x∥3 + (Vj −x)T g (Vj) = 0 o , (2.15) (2.15) xj+1 = xj + Pj ∥g (Vj) −g (xj)∥2 [g (Vj) −g (xj)] , (2.16) (2.16) where where Pj = µj ∥Vj −xj∥2 + µj ∥Vj −x∥3 + (Vj −xj)T g (Vj) . (2.17) (2.17) The speciﬁc algorithm ﬂow is shown in Algorithm 1. The speciﬁc algorithm ﬂow is shown in Algorithm 1. The speciﬁc algorithm ﬂow is shown in Algorithm 1. 2 Motivation and algorithm First, we project the current point xj onto the surface (2.9), then the next iteration xj+1 is deﬁned below: xj+1 = xj + Pj ∥g (Vj) −g (xj)∥2 [g (Vj) −g (xj)] , (2.10) (2.10) where Pj = µ ∥Vj −xj∥2 + µ ∥Vj −x∥3 + (Vj −xj)T g (Vj) . (2.11) (2.11) In (2.2), µ is a ﬁxed scalar, which cannot accurately describe the function infor- mation at the current iteration point. As mentioned by Yuan, the paraboloid (2.2) can be considered as the ﬁrst and second order expansions of a strongly convex func- tion inVj, where µ can be regarded as the eigenvalue of the Hessian matrix. From this point of view, the paraboloid can be portrayed more accurately if we can allow µ to be updated adaptively as k . If we use the eigenvalues of the Hessian matrix directly, it requires a larger amount of computation and longer computation time, so we simulate 4 the eigenvalues of the Hessian matrix by using the special diagonal Hessian matrix updating method[17]. the eigenvalues of the Hessian matrix by using the special diagonal Hessian matrix updating method[17]. ˜µj =      1 1+wj s(max) j 2 if sT j sj > sT j yj sT j yj sT j sj otherwise , (2.12) (2.12) where where wj = sT j sj −sT j yj Pn i=1 s(i) j 4 , (2.13) (2.13) s(i) j denotes the ith component of sj,s(max) j denotes the max component of sj. s(i) j denotes the ith component of sj,s(max) j denotes the max component of sj. To ensure that the proposed new algorithm is globally convergent, we furt boundedly restrict µ: s( ) j denotes the ith component of sj,s( ) j denotes the max component of sj. To ensure that the proposed new algorithm is globally convergent, we further boundedly restrict µ:  µj =      η if ˜µj > η ˜µj if 4σρ ⩽˜µj ⩽η 4σρ if ˜µj < 4σρ , (2.14) (2.14) where η is a large enough constant bigger than 4σρ. 3 Global convergence of the algorithm This section aims to analyze the global convergence and convergence rate of Algorithm 1. First, the required assumptions similar to [4] are given as follows: Assumption 3.1. The level set S0 = {x ∈Rn \| ϕ(x) ≤ϕ (x0)} is bounded, and ϕ(x) is lower bounded by ϕ (x∗) ∈R. And the same order between its gradient and the variables holds, i.e., ∥g( ˙x) −g(¨x)∥= O(∥˙x −¨x∥), ∀˙x, ¨x ∈Rn. (3.1) (3.1) Assumption 3.2. When j /∈SD, there will always be at least one step-size αj that satisﬁes the following condition: ϕ (xj+1) −ϕ (xj) ≤ζ1αjg (xj)T dj. (3.2) (3.2) 5 Algorithm 1 Higher-oder adaptive projection technique-BFGS algorithm for non- convex unconstrained optimization problems (HAPT-BFGS) Algorithm 1 Higher-oder adaptive projection technique-BFGS algorithm for non- convex unconstrained optimization problems (HAPT-BFGS) step 1 : Choose an initial point x0 ∈Rn , an initial symmetric and positive deﬁnite matrix B0 ∈Rn×n. Given the necessary parameters ρ > 0, ζ1 ∈ 0, 1 2 , σ ∈(ζ1, 1) , ε ∈ 0, 1 2 , let j := 0. , 2 , j step 2 : if ∥gj∥⩽ε , the algorithm stops. , 2 , j step 2 : if ∥gj∥⩽ε , the algorithm stops. ∥j∥ step 3 : Get the direction dj by solving the linear equation j step 3 : Get the direction dj by solving the linear equation (2.18) Bjdj = −gj. (2.18) step 4 : Determine a step size αj > 0 that satisﬁes the WWP condition (1.4) and (1.5). j j j j step 6 : If j ∈ID, let xj+1 := Vj, sj := xj+1 −xj and yj := g (xj+1) −g (xj), then skip to Step 9. j j j j step 6 : If j ∈ID, let xj+1 := Vj, sj := xj+1 −xj and yj := g (xj+1) −g (xj), then skip to Step 9. step 7 : Else j /∈ID, deﬁne the next iteration xj+1 as shown in (2.16)(2.17), sj := xj+1 −xj and yj := g (Vj) −g (xj). step 7 : Else j /∈ID, deﬁne the next iteration xj+1 as shown in (2.16)(2.17), sj := xj+1 −xj and yj := g (Vj) −g (xj). j+ j yj g ( j) g ( j) step 8 : Determine Bj+1 by the ordinary BFGS formula(1.5) Bj+1 = Bj −BjsjsT j Bj sT j Bjsj + yjyT j sT j yj . 3 Global convergence of the algorithm (2.19) (2.19) step 9 : Let j := j + 1 and, return to Step 3 step 9 : Let j := j + 1 and, return to Step 3 Remark 1. From Assumption 3.1 and Vj is bounded on the level set S0 we can infer Remark 1. From Assumption 3.1 and Vj is bounded on the level set S0 we can infer ∥αjdj∥⩽M, (3.3) (3.3) Mis a large enough constant. Mis a large enough constant. lemma 1. Suppose that the sequence {xj, dj, αj, gj} is generated by Algorithm 1 and Assumptions 3.1 and 3.2 hold. Then, the following inequality sT j yj ≥λ ∥αjdj∥2 , (3.4) (3.4) holds, where λ is a positive constant. holds, where λ is a positive constant. Proof. The lemma will be completed by the following two cases. Case (i): xj ∈ID, by the Wolfe line search (1.4), and the deﬁnition of ID, we get sT j yj = sT j [g (xj + αjdj) −gj] = sT j [g (xj+1) −gj] ≥−(1 −σ)gT j sj = αj(1 −σ) −gT j dj ≥(1 −σ)ρ ∥αjdj∥2 . (3.5) (3.5) Case (ii): xj /∈ID, by Step 7 of Algorithm 1 and the deﬁnition of ID, we have Case (ii): xj /∈ID, by Step 7 of Algorithm 1 and the deﬁnition of ID, we have sT j yj = Pj ∥yj∥2 yT j yj = Pj. (3.6) (3.6) 6 By the deﬁnition of Pj Pj = µj ∥αjdj∥2 + µj ∥αjdj∥3 + g (xj + αj)T αjdj ⩾µj ∥αjdj∥2 + µj ∥αjdj∥3 + αjσgT j dj ⩾µj ∥αjdj∥2 + µj ∥αjdj∥3 −σρ ∥αjdj∥2 = (µj −σρ) ∥αjdj∥2 + µj ∥αjdj∥3 ⩾(µj −σρ) ∥αjdj∥2 ⩾(4σρ −σρ) ∥αjdj∥2 = 3σρ ∥αjdj∥2 , (3.7) the ﬁrst inequality use the wolfe line search (1.4), the second inequality use the deﬁnition xj /∈ID, the last inequality utilizes (2.14).Therefore, by setting λ = min{(1 −σ)ρ, 3σρ}, we obtain(3.4). The proof is complete. lemma 2. The sequence Bk is generated by (1.5) and Assumptions 3.2 hold. Then, the matrix Bk designed by(1.5) is positive deﬁnite for all k. Proof. To get this lemma, it is suﬃcient to have the condition sT j yj > 0 . By (3.4), we can immediately complete the proof. Remark 2. The above lemma and the relationship (2.18) prove that dT j gj < 0 (3.8) (3.8) holds for all k ⩾0. Therefore, by (1.4), we conclude that the sequence ϕk is descent. lemma 3. [15] Suppose that the sequence {xj, dj, αj, gj} is generated by Algorithm 1 and Assumptions 3.1 and 3.2 hold. Then, the following inequality ∞ X k=0 gT j dj 2 ∥dj∥2 < ∞ (3.9) (3.9) holds. holds. Remark 1. From Assumption 3.1 and Vj is bounded on the level set S0 we can infer lemma 4. Suppose that the conditions of Lemma 1 hold. Then, there exist two constants b1 ≥b2 > 0 with any integer t > 0 such that holds. lemma 4. Suppose that the conditions of Lemma 1 hold. Then, there exist two constants b1 ≥b2 > 0 with any integer t > 0 such that ∥Bjsj∥≤b1 ∥sj∥, (3.10) (3.10) and and sT j Bjsj ≥b2 ∥sj∥2 , (3.11) (3.11) sj Bjsj ≥b2 ∥sj∥, (3.11) hold for at least ⌊t/2⌋values of k ∈{1, 2, . . . , t}. j hold for at least ⌊t/2⌋values of k ∈{1, 2, . . . , t}. hold for at least ⌊t/2⌋values of k ∈{1, 2, . . . , t}. hold for at least ⌊t/2⌋values of k ∈{1, 2, . . . , t}. Proof. We prove this lemma using the following two cases. 7 Case (i):j ∈ID. By the deﬁnition of ID and (3.4),we obtain sT j yj ∥sj∥2 ⩾λ ∥αjdj∥2 ∥αjdj∥2 = λ. (3.12) (3.12) By Assumption 3.1 and (3.4), we have By Assumption 3.1 and (3.4), we have By Assumption 3.1 and (3.4), we have ∥yj∥2 sT j yj ⩽L2 p ∥αjdj∥2 λ ∥αjdj∥2 = L2 p λ . (3.13) (3.13) Case (ii):j /∈ID.By Step 7 of Algorithm 1 and (2.16),we can have Case (ii):j /∈ID.By Step 7 of Algorithm 1 and (2.16),we can have ∥sj∥2 = Pj2 ∥yj∥2 , (3.14) sT j yj = Pj. (3.15) Therefore, we get sT j yj ∥sj∥2 = Pj P 2 j ∥yj∥2 = ∥yj∥2 Pj . (3.16) Due to j /∈ID and (3.8) we have ∥sj∥2 = Pj2 ∥yj∥2 , (3.14) sT j yj = Pj. (3.15) ∥sj∥2 = Pj2 ∥yj∥2 , (3.14) (3.14) (3.15) Therefore, we get Therefore, we get sT j yj ∥sj∥2 = Pj P 2 j ∥yj∥2 = ∥yj∥2 Pj . (3.16) e have ∥sj∥ Due to j /∈ID and (3.8) we have αjgT j dj = −αjgT j dj ≤ρ ∥αjdj∥2 . Remark 1. From Assumption 3.1 and Vj is bounded on the level set S0 we can infer (3.17) (3.17) Using the deﬁnition of Pj, we obtain Using the deﬁnition of Pj, we obtain Using the deﬁnition of Pj, we obtain Using the deﬁnition of Pj, we obtain Pj = ∥Pj∥= g (xj + αjdj)T αjdj + µj ∥αjdj∥2 + µj ∥αjαj∥3 ⩽ (g (xj + αjdj) −g (xj))T αjdj + g (xj)T αjdj + µj ∥αjdj∥2 + µj ∥αjdj∥3 ⩽Lp ∥αjdj∥2 + ρ ∥αjdj∥2 + µj ∥αjdj∥2 + µj ∥αjdj∥3 Pj = ∥Pj∥= g (xj + αjdj)T αjdj + µj ∥αjdj∥2 + µj ∥αjαj∥3 ⩽ (g (xj + αjdj) −g (xj))T αjdj + g (xj)T αjdj + µj ∥αjdj∥2 + µj ∥αjdj∥3 ⩽Lp ∥αjdj∥2 + ρ ∥αjdj∥2 + µj ∥αjdj∥2 + µj ∥αjdj∥3 = (Lp + ρ + µj + µj ∥αjdj∥) ∥αjdj∥2 ⩽(Lp + ρ + µj + µjM) ∥αjdj∥2 ⩽(Lp + ρ + η + ηM) ∥αjdj∥2 . (3.18) (3.18) The ﬁrst equation holds naturally due to (3.7), the ﬁrst inequality is caused by trigonometric inequalities. Using (3.17), it is natural to obtain the second inequality, the last inequality can be obtained using Remark 1 and (2.14). By Assumption 2 and (3.18), we obtained sT j yj ∥sj∥2 = ∥yj∥2 pj ⩾ L0 ∥αjdj∥2 (Lp + ρ + η + ηM) ∥αjdj∥2 = L0 (Lp + ρ + η + ηM). (3.19) (3.19) 8 ∥yj∥2 sT j yj = ∥yj∥2 Pj ⩽L2 p ∥αjdj∥2 λ ∥αjdj∥2 = L2 p λ . (3.20) (3.20) Above all, we let δ1 = min n L0 (Lp+ρ+η+ηM), λ o and δ2 = L2 p λ , we can always have δ1 = min n L0 (Lp+ρ+η+ηM), λ o and δ2 = L2 p λ , we can always have sT j yj ∥sj∥2 ⩾δ1, ∥yj∥2 sT j yj ⩽δ2. (3.21) (3.21) Using a method similar to the proof of Theorem 2.1 in [18], it is not diﬃcult to complete the proof. Theorem 1. Suppose that the conditions of the above lemmas are true. Then we have lim k→∞inf ∥gk∥= 0. (3.22) (3.22) Proof. The proof can be found in Theorem 5.1 in [15] Proof. The proof can be found in Theorem 5.1 in [15] Proof. The proof can be found in Theorem 5.1 in [15] 4 Numerical Experiments For example, in the ﬁrst row of the table, we list the total number of iterations, the total number of 9 Table 1: The test problems Table 1: The test problems No. Test problem No. Test problem 1 Extended Freudenstein and Roth Function 35 ARWHEAD Function (CUTE) 2 Extended Trigonometric Function 36 NONDIA Function (CUTE) 3 Extended Rosenbrock Function 37 HIMMELBG Function (CUTE) 4 Extended White and Holst Function 38 DQDRTIC Function (CUTE) 5 TRIDIA Function (CUTE) 39 EG2 Function (CUTE) 6 Extended Penalty Function 40 DIXMAANA Function (CUTE) 7 Perturbed Quadratic Function 41 DIXMAANB Function (CUTE) 8 Raydan 1 Function 42 DIXMAANC Function (CUTE) 9 Raydan 2 Function 43 Extended Tridiagonal-2 Function 10 Diagonal 1 Function 44 Partial Perturbed Quadratic Function 11 ARGLINB Function (CUTE) 45 Broyden Tridiagonal Function 12 Diagonal 3 Function 46 Almost Perturbed Quadratic Function 13 Hager Function 47 Tridiagonal Perturbed Quadratic Function 14 Generalized Tridiagonal-1 Function 48 EDENSCH Function (CUTE) 15 Extended Tridiagonal-1 Function 49 VARDIM Function (CUTE) 16 Extended Three Exponential Terms Function 50 STAIRCASE S1 Function 17 Generalized Tridiagonal-2 Function 51 LIARWHD Function (CUTE) 18 Diagonal 4 Function 52 DIAGONAL 6 Function 19 BDQRTIC Function (CUTE) 53 FLETCHCR Function (CUTE) 20 Extended Himmelblau Function 54 ENGVAL1 Function (CUTE) 21 Generalized PSC1 Function 55 DENSCHNA Function (CUTE) 22 Extended PSC1 Function 56 DENSCHNC Function (CUTE) 23 Extended Powell Function 57 DENSCHNB Function (CUTE) 24 Extended Block Diagonal BD1 Function 58 DENSCHNF Function(CUTE) 25 Extended Maratos Function 59 SINQUAD Function (CUTE) 26 Extended CliﬀFunction 60 DIAGONAL 9 Function 27 Quadratic Diagonal Perturbed Function 61 Extended Block-Diagonal BD2 Function 28 Extended Wood Function 62 Generalized quartic GQ1 Function 29 Extended Hiebert Function 63 Diagonal 7 Function 30 Quadratic Function QF1 Function 64 Diagonal 8 Function 31 Extended Quadratic Penalty QP1 Function 65 Full Hessian Function 32 Extended Quadratic Penalty QP2 Function 66 SINCOS Function 33 A Quadratic Function QF2 Function 67 Generalized quartic GQ2 Function 34 Extended EP1 Function 68 EXTROSNB Function (CUTE) functions and gradients used, and the total iteration time for each of the four algo- rithms for each of the 68 functions tested in 500 dimensions. Speciﬁcally, HAPTBFGS method iterates a total of 7789 times for the 68 functions, uses functions and gradients a total of 146895 times, and has a total running time of 53.256, all of which are smaller than the other algorithms. 4 Numerical Experiments In this section, we perform experiments on the proposed algorithms and other nor- mal algorithms under the Wolfe line search. We show the numerically experimental test results for sixty-eight problems from an unconstrained optimization test functions collection which presented by Andrei[19], as seen in Table 1. To test and compare the numerical performance of Algorithm 1, we show some well performed algorithms [15, 16] related to technique proposed by Yuan, called OPTBFGS and PTBFGS respec- tively. To show the eﬀect of Algorithm 1 with high-order adaptive projection technique, we name Algorithm 1 as HAPTBFGS, and when µ takes a constant of 4σρ, we name the algorithm HPTBFGS. If \|ϕ (xj)\| > 10−5 holds,set Ter1 = \|ϕ(xj)−ϕ(xj+1)\| \|ϕ(xj)\| ; other- wise, let Ter1 = \|ϕ (xj) −ϕ (xj+1)\|. If the condition ∥gj∥⩽10−6 (or Ter1 < 10−5 ) is true or the total number of iteration loops exceeds 2000, this program terminates. We give the parameter and initialization settings in Algorithm 1: ζ1 = 0.2, σ = 0.8, ρ = 0.7, η = 1000,B0 = I; Each problem has diﬀerent initialization settings, and the initialization settings for x0 are provided by Andrei[19]. This algorithm code is writ- ten on Matlab R2022a software. The program runs on a PC with an Intel (R) Core (TM) i7-6700HQ CPU at 2.60GHz of RAM, and the operating system is Windows 11. We performed numerical experiments on these sixty-eight test functions in 500, 1000, and 1500 dimensions respectively, and the overall experimental results are shown in Table 2. Some notations need to be clariﬁed before analysis: Dim: the dimension of the functions. Dim: the dimension of the functions. Ni: the total iteration numbers of algorithm iterations. Ni: the total iteration numbers of algorithm iterations. Nfg: the total number of functions and gradient computations during the iteration. Nfg: the total number of functions and gradient computations during the iteration. Time: the total time required to complete the algorithm. Nfg: the total number of functions and gradient computations during the iteratio Nfg: the total number of functions and gradient computati Time: the total time required to complete the algorithm. As can be seen from Table 2, we have all compared these four algorithms separately from each dimension and the minimum values are indicated in bold. 4 Numerical Experiments The algorithm presented in the paper is competitive with the other algorithms in Ni, Nfg, and time on the tested problems. The proﬁles of these algorithms are compared and analyzed by the tool of Dolan and Mor`e[20]. The per- formances of algorithms OPTBFGS, PTBFGS, HPTBFGS, HAPTBFGS, in terms of Nf,Ng, time and Ni are shown in Figs. 1, 2, 3 and 4 respectively, where Nf,and Ng denote the norm of the functions, norm of the gradients respectively. As can be seen from Figs 1 - 4, when we add higher order terms, the performance of the algorithm can be improved both in terms of the norm of the function value, the norm of the gradient, the number of iterations, and the running time, as the curve is closer to 1, representing that the proposed algorithm can solve more problems. By 10 Table 2: The total numerical results Table 2: The total numerical results Algorithm Dim Ni Nfg Time OPTBFGS 500 8892 169883 68.062 PTBFGS 500 8410 153650 57.914 HPTBFGS 500 8448 174674 57.609 HAPTBFGS 500 7789 146895 53.256 OPTBFGS 1000 9969 228997 284.753 PTBFGS 1000 9184 194582 264.438 HPTBFGS 1000 9358 258818 269.269 HAPTBFGS 1000 8608 243281 245.431 OPTBFGS 1500 9155 195291 659.057 PTBFGS 1500 9360 208548 671.093 HPTBFGS 1500 5409 129816 381.101 HAPTBFGS 1500 5989 165800 423.991 Figs 1 - 4, it is found that the algorithm performance is further improved signiﬁcantly when the parameters µ can be updated adaptively. This shows that higher order terms and adaptive updating of parameters µ are both essential to improve the performance of the proposed algorithm. Figs 1 - 4, it is found that the algorithm performance is further improved signiﬁcantly when the parameters µ can be updated adaptively. This shows that higher order terms and adaptive updating of parameters µ are both essential to improve the performance of the proposed algorithm. Figs 1 - 4, it is found that the algorithm performance is further improved signiﬁcantly when the parameters µ can be updated adaptively. This shows that higher order terms and adaptive updating of parameters µ are both essential to improve the performance of the proposed algorithm. Fig. 1: Performance proﬁles of the HAPTBFGS and other algorithms in terms of Nf Fig. 1: Performance proﬁles of the HAPTBFGS and other algorithms in terms of Nf 5.1 Ethical Approval This article does not contain any studies with human participants or animals performed by any of the authors. 5.4 Funding This work is supported by National Natural Science Foundation of China (11961010,61967004). 5 Conclusions For the BFGS algorithm that does not have global convergence for nonconvex functions, we construct a new paraboloid by utilizing the information of the third-order Taylor expansion of the function, so that for the points that do not satisfy the special 11 Fig. 2: Performance proﬁles of the HAPTBFGS and other algorithms in terms of Ng Fig. 2: Performance proﬁles of the HAPTBFGS and other algorithms in terms of Ng Fig. 3: Performance proﬁles of the HAPTBFGS and other algorithms in terms of time suﬃcient descent condition, we project them onto the paraboloid. Further, for the constant µ in the paraboloid, we take an adaptive update of µ . Numerical experiments also demonstrate that the new paraboloid and the new projection technique can greatly improve the performance of the algorithm.As a future work, the following points are Fig. 3: Performance proﬁles of the HAPTBFGS and other algorithms in terms of time suﬃcient descent condition, we project them onto the paraboloid. Further, for the constant µ in the paraboloid, we take an adaptive update of µ . Numerical experiments also demonstrate that the new paraboloid and the new projection technique can greatly improve the performance of the algorithm.As a future work, the following points are considered: (i) a new paraboloid and the corresponding projection technique can be proposed; (ii) take a better adaptive update for the parameter µ. suﬃcient descent condition, we project them onto the paraboloid. Further, for the constant µ in the paraboloid, we take an adaptive update of µ . Numerical experiments also demonstrate that the new paraboloid and the new projection technique can greatly improve the performance of the algorithm.As a future work, the following points are considered: (i) a new paraboloid and the corresponding projection technique can be proposed; (ii) take a better adaptive update for the parameter µ. suﬃcient descent condition, we project them onto the paraboloid. Further, for the constant µ in the paraboloid, we take an adaptive update of µ . Numerical experiments also demonstrate that the new paraboloid and the new projection technique can greatly improve the performance of the algorithm.As a future work, the following points are considered: (i) a new paraboloid and the corresponding projection technique can be proposed; (ii) take a better adaptive update for the parameter µ. 12 Fig. 4: Performance proﬁles of the HAPTBFGS and other algorithms in terms of Ni Fig. 5.3 Competing interests The authors declare no competing interests 5.2 Availability of supporting data The data that support the ﬁndings of this study are available on request from the corresponding author, upon reasonable request. 5 Conclusions 4: Performance proﬁles of the HAPTBFGS and other algorithms in terms of Ni 5.5 Authors’ contributions Xiangli li: conceptualization, methodology, supervision. Binglan Li: data cura- tion, writing—original draft preparation, writing—reviewing and editing, software, validation, visualization. Zhiling Wang: writing—review and editing. 5.6 Acknowledgments The authors are grateful to the editor and anonymous reviewers for their suggestions 13 References [1] Fu, Z., Wu, X., Guan, C., Sun, X., Ren, K.: Toward eﬃcient multi-keyword fuzzy search over encrypted outsourced data with accuracy improvement. IEEE Transactions on Information Forensics and Security 11(12), 2706–2716 (2016) [2] Pan, Z., Zhang, Y., Kwong, S.: Eﬃcient motion and disparity estimation opti- mization for low complexity multiview video coding. IEEE Transactions on Broadcasting 61(2), 166–176 (2015) [3] Xia, Z., Wang, X., Sun, X., Liu, Q., Xiong, N.: Steganalysis of lsb matching using diﬀerences between nonadjacent pixels. Multimedia Tools and Applications 75, 1947–1962 (2016) [4] Nocedal, J., Wright, S.J.: Numerical Optimization. Springer, Boston (1999) [5] Yuan, G., Wei, Z., Li, G.: A modiﬁed polak–ribi`ere–polyak conjugate gradi- ent algorithm for nonsmooth convex programs. 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https://openalex.org/W4291295525	https://www.researchsquare.com/article/rs-314550/latest.pdf	English	null	Coherent optical communications using coherence-cloned Kerr soliton microcombs	arXiv (Cornell University)	2,021	cc-by	11,176	Coherent optical communications using coherence- cloned Kerr soliton microcombs Coherent optical communications using coherence cloned Kerr soliton microcombs Yong Geng University of Electronic Science and Technology of China Heng Zhou (  zhouheng@uestc.edu.cn ) University of Electronic Science and Technology of China Xinjie Han University of Electronic Science and Technology of China Wenwen Cui University of Electronic Science and Technology of China Qiang Zhang University of Electronic Science and Technology of China Boyuan Liu University of Electronic Science and Technology of China Deng Guang-Wei Qiang Zhou University Of Electronic Science And Technology Of China https://orcid.org/0000-0001-7099-1995 Kun Qiu University of Electronic Science and Technology of China Article Coherent optical communications using coherence cloned Kerr soliton microcombs Yong Geng University of Electronic Science and Technology of China Heng Zhou (  zhouheng@uestc.edu.cn ) University of Electronic Science and Technology of China Xinjie Han University of Electronic Science and Technology of China Wenwen Cui University of Electronic Science and Technology of China Qiang Zhang University of Electronic Science and Technology of China Boyuan Liu University of Electronic Science and Technology of China Deng Guang-Wei Qiang Zhou University Of Electronic Science And Technology Of China https://orcid.org/0000-0001-7099-1995 Kun Qiu University of Electronic Science and Technology of China Article Keywords: Dissipative Kerr Soliton Microcomb, Multi-wavelength Laser Source, Fiber Optical Communications, Pump Laster Conveying, Two-point Locking Posted Date: April 2nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-314550/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Nature Communications on February 28t 2022. See the published version at https://doi.org/10.1038/s41467-022-28712-y. Coherent optical communications using coherence-cloned Kerr soliton 1 microcombs 2 Yong Geng1,3, Heng Zhou ∗1,3, Xinjie Han1, Wenwen Cui1, Qiang Zhang1, Boyuan Liu1, 3 Guangwei Deng2, Qiang Zhou2, and Kun Qiu1 4 1Key Lab of Optical Fiber Sensing and Communication Networks, University of Electronic 5 Science and Technology of China, Chengdu 611731, China 6 2Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology 7 of China, Chengdu 611731, China 8 3These authors contributed equally: Yong Geng, Heng Zhou 3These authors contributed equally: Yong Geng, Heng Zhou ∗zhouheng@uestc.edu.cn Article Posted Date: April 2nd, 2021 Version of Record: A version of this preprint was published at Nature Communications on February 28th, 2022. See the published version at https://doi.org/10.1038/s41467-022-28712-y. Abstract Dissipative Kerr soliton microcomb has been recognized as a promising on-chip multi- wavelength laser source for ﬁber optical communications, as its comb lines possess fre- quency and phase stability far beyond the independent lasers. In the scenarios of coherent optical transmission and interconnect, a highly beneﬁcial but rarely explored target is to re-generate a Kerr soliton microcomb at the receiver side as local oscillators that conserve the frequency and phase property of the incoming data carriers, so that to enable coher- ent detection with minimized optical and electrical compensations. Here, by using the techniques of pump laser conveying and two-point locking, we implement re-generation of a Kerr soliton microcomb that faithfully clones the frequency and phase coherence of another microcomb sent from 50 km away. Moreover, leveraging the coherence-cloned soliton microcombs as carriers and local oscillators, we demonstrate terabit coherent data interconnect, wherein traditional digital processes for frequency oﬀset estimation is totally dispensed with, and carrier phase estimation is substantially simpliﬁed via slowed-down phase estimation rate per channel and joint phase estimation among multi- ple channels. Our work reveals that, in addition to providing a multitude of laser tones, regulating the frequency and phase of Kerr soliton microcombs among transmitters and receivers can signiﬁcantly improve optical coherent communication in terms of perfor- mance, power consumption, and simplicity. 1 1 Introduction 30 Wavelength division multiplexing (WDM) optical coherent transmission greatly enhances the ca- 31 pacity and spectral eﬃciency of ﬁber communication by modulating information onto both the 32 amplitudes and phases of a multitude of laser carriers at the transmitter, and demodulating infor- 33 mation at the receiver through coherently mixing the data signals with matched local oscillators 34 (LO) [1]. Frequency and phase coherence between the carrier and LO lasers thus play a crucial role 35 in determining the performance of coherent data receiving. To date, most commercial systems still 36 use independent carrier and LO lasers, which have weak mutual coherence (Fig. 1a left) and entail 37 large guard band and power-hungry digital signal processing (DSP) to gauge their frequency and 38 phase uncertainties [2]. Optical frequency comb consisting of a large quantity of even spaced and 39 phase locked laser tones can provide spectral stability orders of magnitude higher than individual 40 lasers (Fig. 1a right) [3], thus being considered as a promising laser source for coherent WDM 41 transmission and interconnect. Their strengths to carry massive parallel data channels have already 42 been demonstrated in various of optical frequency comb platforms, including electro-optical (EO) 43 modulating comb [4–6], nonlinear broadened comb [7, 8], mode-locked ﬁber laser comb [9], semi- 44 conductor gain-switched laser comb [10], and dissipative Kerr soliton (DKS) microcomb [11–16]. 45 Therein, DKS microcomb generated in nonlinear optical microcavity has evoked special interests 46 thanks to its unique features including large frequency spacing [11, 12], ultra-broadband spectrum 47 [17], high stability [18], excellent SWaP (size, weight and power) factors and compatibility for chip 48 integration [19–21]. It had been reported that chip-scale DKS microcombs can simultaneously 49 provide more than 100 laser tones to transmit coherent data signals with line rate up to 55 Tbit 50 s−1 [12]. 51 On the other hand, to employ DKS microcombs in coherent communication networks, it is of 52 great importance to re-generate the LO microcomb at the receiver side that inherits the frequency 53 2 and phase coherence of the transmitted data carrier comb [22–24]. In fact, the generation dynamics 54 and physical characteristics of DKS microcomb make it an ideal platform to realize coherence- 55 cloned comb re-generation among transmitters and receivers [11, 17, 23, 24]. Introduction 30 First, DKS microcomb 56 is commonly generated by a single continuous-wave pump laser, which directly set the central 57 frequency fc of the whole comb spectrum [11]. Second, the mode spacing fspc of a DKS microcomb 58 is preset by the cavity geometry and can be ﬁnely adjusted by conﬁguring either the intracavity 59 pump power or the pump-cavity frequency detuning δ (i.e., fundamentally the nonlinear phase- 60 matching) [23, 25]. Third, in soliton mode-locked state, the phases of all the DKS microcomb lines 61 φm(m = ±1, 2, 3, ...) uniformly align to the phase of the pump laser φ0 [11, 26]. That is to say, all 62 the spectral parameters (fc, fspc and φm) of a DKS microcomb can be precisely manipulated by 63 controlling the pump laser parameters. Relying on these eﬀects, here we demonstrate coherence- 64 cloned DKS microcomb re-generation by relaying the pump laser between a pair of transmitter 65 and receiver separated by 50 km, with the assistant of a pilot tone to achieve further mode spacing 66 stabilization and phase noise suppression between the original and re-generated microcombs, based 67 upon the mechanisms of two-point locking and optical frequency division. We illustrate that 68 the re-generated receiver microcomb achieves excellent frequency and phase consistency with the 69 transmitter microcomb, enabling high performance and energy-saving coherent data transmission 70 with substantially simpliﬁed processing for the carrier-LO frequency oﬀsets and phase drifts. 71 Results 72 Coherence-cloned re-generation of DKS microcomb. Our experiments utilize two silicon 73 nitride micro-ring cavities with similar free spectral range (FSR) of ∼100 GHz [27]. A low-noise 74 ﬁber laser with wavelength λpump ∼1550.0 nm is used as the pump laser CTx(0) to produce a 75 DKS microcomb CTx(m)(m = ±1, 2, 3, ...) in the transmitter microcavity, via the technique of 76 3 auxiliary laser heating (ALH) (see Methods) [28–30]. ALH is adopted in order to suppress the 77 thermal nonlinearity of microcavity resonances and allow the pump laser to stably access single 78 soliton state in the red-detuning regime. Afterwards, the transmitter microcomb CTx including 79 the pump laser CTx(0) is sent through a 50 km standard-single-mode-ﬁber (SSMF) to the re- 80 ceiver, where the conveyed pump laser CTx(0) is used to re-generate another DKS microcomb 81 CRx(m)(m = ±1, 2, 3, ...) in the receiver microcavity, also using ALH method [29]. Optical spectra 82 of the transmitter microcomb CTx and receiver microcomb CRx are shown in Fig. 1b. Once gen- 83 erated, the microcombs can operate for weeks maintained by simple stabilization techniques [27]. 84 At this stage, both microcombs have identical central wavelength λpump, and the comb line phases 85 φRx m and φTx m , (m = ±1, 2, 3, ...) all align approximately to the corresponding pump laser phase φRx 0 86 and φRx 0 respectively [26]. However, due to the distinct soliton repetition rates (f Tx spc ∼100.53 GHz, 87 f Rx spc ∼100.58 GHz) and their uncorrelated jitters caused by the ﬂuctuations of the two independent 88 microcavities, the frequency and phase coherence between comb lines within CTx and CRx are still 89 weak [25, 31], exhibiting inter-comb beat note spectrum with full-width-half-maximum (FWHM) 90 linewidth > 3 kHz (e.g., m = 1), as shown in Fig. 1c. Therefore, at this stage CRx is not yet a 91 coherence-cloned copy of CTx. 92 Then, we implement phase locking of the 17th receiver comb line CRx(17) to the arrived 17th 93 transmitter comb line CTx(17) (see Methods), and narrow their beat note FWHM linewidth down 94 to ∼5 Hz. Results 72 By doing this, CTx and CRx are two-point locked [32] by the shared pump laser CTx(0), 95 and the 17th comb modes CTx(17) and CRx(17), thus the inter-comb frequency and phase noise 96 of those in-between comb lines CTx(m) and CTx(m) (m = ±1, 2, 3, ...16) can be substantially sup- 97 pressed obeying the law of optical frequency division (OFD) [33–35]. As shown in Fig. 1c, after 98 two-point locking, the linewidths of inter-comb beat notes (m = 1, 5, 10) signiﬁcantly decrease and 99 their noise backgrounds drop approximately as the function of 1/(m−17)2 [32, 34]. Minor close-in 100 (< 3.0 kHz) noises are observed in the beat note spectra due to dispersion incurred temporal walk 101 4 oﬀamong diﬀerent comb lines as CTx transmitted through the 50 km SSFM (see Methods), which, 102 however, have no inﬂuence to the performance of coherent data detection, as will be discussed 103 below. Moreover, Fig. 1d shows the Allan deviation of the beat note frequency ∆fCL(m) between 104 CTx(m) and CRx(m) (m = 1, 5, 17), it is seen that two-point locking improves the stability of ∆fCL 105 by about 4 orders of magnitude (at 1 s gate time) comparing with the situation without two-point 106 locking. Of note, here we choose m = 17 as the locked comb mode due to the bandwidth limitation 107 (< 1.0 GHz) of our phase comparator. According to OFD theory, the locked comb mode index 108 should be further increased by adopting either faster phase comparator or smaller discrepancy 109 between f Tx spc and f Rx spc, so that to obtain larger division factor and stronger coherence enhancement 110 between CRx and CTx. Nevertheless, the low noise beat notes and stable Allan deviations shown in 111 Fig. 1c-d indicate that the mutual coherence between the DKS microcombs CTx and CRx is already 112 high. Next, we will show how the highly coherent microcombs facilitate coherent data transmission. 113 Coherent data interconnect using coherence-cloned microcombs. Fig. 2 115 the experimental setup of optical data interconnect using the coherence-cloned DKS microcombs 116 as carriers and LOs [28]. At the transmitter, 20 comb lines CTx(m = ±2... ± 11) are selected 117 and sent into an IQ modulator (Fig. 2c), where 21 Gbaud/s single polarization 16-QAM data 118 are encoded to all the comb lines. The modulated data channels together with the pump laser 119 CTx(0) and pilot tone CTx(17) are combined and sent to the receiver through 50 km SSMF. At 120 the receiver, microcomb CRx is re-generated and two-point locked to CTx following the process 121 described above, and then used as LOs for coherent date receiving (Fig. 2c). Of note, as CTx(0) 122 and CTx(17) propagate through the 50 km ﬁber link together with the high speed data channels, a 123 matter of concern is that the data signals may impose linewidth broadening to them via nonlinear 124 cross-phase modulation (XPM) and degrade their spectral purity as the pump laser and reference 125 pilot. Nevertheless, as shown in Fig. 2d-2e, after co-propagating with data channels, the beat 126 5 5 note between CTx(17) and CRx(17) remains almost identical with the case without co-propagating 127 data, indicating that XPM only induces negligible linewidth distortion to CTx(17) and CTx(0) in 128 our experiment. The underlying mechanism is because ﬁber dispersion induces spatiotemporal 129 walk-oﬀamong signals at diﬀerent wavelength channels along the transmission link [36, 37], there- 130 fore, XPM imposed to CTx(17) and CTx(0) from diﬀerent data channels are smoothed out as a 131 quasi-constant phase envelop without high frequency component (see Supplementary Information 132 for numerical analysis). 133 Fig. 2b shows the performance of coherent data receiving enabled by CTx and CRx, it is seen 134 that excellent signal-to-noise ratio (SNR) and bit-error rate (BER) are achieved for all the 20 135 channels, with a total bit rate of 1.68 Tbit s−1. More importantly, thanks to the high coherence 136 between CTx and CRx, DSP-based electrical frequency oﬀset estimation (FOE) and carrier phase 137 estimation (CPE) between carriers and LOs are substantially simpliﬁed during coherent data 138 retrieval [1, 2, 38, 39]. First, after two-point locking, the frequency oﬀset ∆fCL(17) between 139 CRx(17) and CTx(17) has been locked to the reference clock fREF = 941.101000 MHz, with tiny 140 residual frequency jitter < 1 Hz (at 100 ms gate time, see Fig. Coherent data interconnect using coherence-cloned microcombs. Fig. 2 115 1d), so the FOE between CTx(m) and 141 CRx(m) (m = ±1, 2, ...16) can be precalculated using the simple relation ∆fCL(m) = m · fref/17. 142 To validate this scheme, we conduct coherent data demodulation using the precalculated ∆fCL(m) 143 as the FOE result for each channel, and resolve the actual frequency oﬀset error by extracting 144 the ﬁrst-order derivative of time from the data phase evolution (see Fig. 2f) [37]. As summarized 145 in Fig. 2g, the discrepancies between the precalculated ∆fCL(m) and actual frequency oﬀsets 146 are within ±500 Hz for all the 20 data channels. In comparison, when CRx and CTx are not 147 two-point locked, the errors of precalculated ∆fCL(m) become ∼3 orders of magnitude bigger. 148 Hypothetically, if traditional DSP algorithms for FOE (e.g., 4th power spectrum peak search) are 149 used to achieve such accuracy of ±500 Hz, it would entail unacceptably heavy DSP operations 150 and super-long training sequence [38]. Thus, by virtue of the cloned frequency stability between 151 6 CTx and CRx, one can save substantial DSP power and complexity while simultaneously achieving 152 FOE accuracy that is orders of magnitude higher than relying on conventional digital methods. 153 Second, besides FOE, to retrieve data information from coherently modulated signal, random 154 phase drift rooted in the residual frequency oﬀset and intrinsic phase noise between the carrier 155 and LO needs to be traced using CPE algorithms [1, 38]. Essentially, the processing rate and 156 corresponding power consumption of CPE depend on the phase coherence between the carrier and 157 LO tones [39], or in other words, the lowest CPE rate should be properly chosen to minimize power 158 consumption (considering that typical CPE algorithms such as Viterbi&Viterbi phase estimation 159 and blind-phase search are usually power hungry), while making sure that stochastic phase drift 160 within the interval between two CPE operations causes acceptable data bit error [38, 39]. It has 161 been shown above that the phase coherence between CRx and CTx is greatly enhanced by two- 162 point locking and OFD, so it is expected that the CPE rate and related power budget can be 163 reduced when using them as carriers and LOs. As shown in Fig. Coherent data interconnect using coherence-cloned microcombs. Fig. 2 115 3b, the data channels carried 164 and demodulated by CRx and CTx exhibit extremely stable phase evolution (see Methods for phase 165 retrieval scheme), with the phase ﬂuctuation much smaller than generated by unlocked microcombs. 166 Larger indexed data channels show slightly bigger phase ﬂuctuations as the corresponding comb 167 lines experienced smaller frequency division factors, but are still conﬁned in a small range (e.g., 168 < ±0.2 rad). Then we gradually slow down the CPE rate (i.e., increasing the number of skipped 169 data blocks after one CPE) while continuously recording the BER, and evaluate the lowest CPE 170 rate allowed for diﬀerent data channels. As shown in Fig. 3c, if we set the 7% hard forward- 171 error-correction (FEC) threshold 3.8e-3 as the target BER, two-point locked CRx and CTx enable 172 1 order of magnitude lower CPE rate than unlocked microcombs, and 3 orders of magnitude lower 173 CPE rate than independent carrier and LO lasers. Particularly, for those lower indexed data 174 channels (e.g., m < 5), only one CPE block (32 symbols) is suﬃcient to warrant satisfying BER 175 of the whole data frame (400,000 symbols), implying substantial power saving of the relevant DSP 176 CTx and CRx, one can save substantial DSP power and complexity while simultaneously achieving 152 FOE accuracy that is orders of magnitude higher than relying on conventional digital methods. 153 Larger indexed data channels show slightly bigger phase ﬂuctuations as the corresponding comb 167 lines experienced smaller frequency division factors, but are still conﬁned in a small range (e.g., 168 < ±0.2 rad). Then we gradually slow down the CPE rate (i.e., increasing the number of skipped 169 data blocks after one CPE) while continuously recording the BER, and evaluate the lowest CPE 170 rate allowed for diﬀerent data channels. As shown in Fig. 3c, if we set the 7% hard forward- 171 error-correction (FEC) threshold 3.8e-3 as the target BER, two-point locked CRx and CTx enable 172 1 order of magnitude lower CPE rate than unlocked microcombs, and 3 orders of magnitude lower 173 CPE rate than independent carrier and LO lasers. Particularly, for those lower indexed data 174 channels (e.g., m < 5), only one CPE block (32 symbols) is suﬃcient to warrant satisfying BER 175 of the whole data frame (400,000 symbols), implying substantial power saving of the relevant DSP 176 7 module. Coherent data interconnect using coherence-cloned microcombs. Fig. 2 115 Practically, such stable phase evolutions between coherence-cloned CRx and CTx can even 177 be tracked by adaptive equalizer module without conducting CPE, and possibly to bring about 178 further simpliﬁcation to the coherent receiver structure. Detailed module and circuit design of 179 coherent receiver that fully copes with coherence-cloned microcombs is beyond the scope of the 180 current work, but will be an important topic as Kerr microcomb moving fast towards utility. 181 module. Practically, such stable phase evolutions between coherence-cloned CRx and CTx can even 177 be tracked by adaptive equalizer module without conducting CPE, and possibly to bring about 178 further simpliﬁcation to the coherent receiver structure. Detailed module and circuit design of 179 coherent receiver that fully copes with coherence-cloned microcombs is beyond the scope of the 180 current work, but will be an important topic as Kerr microcomb moving fast towards utility. 181 Furthermore, Fig. 3e shows the data phase evolutions of diﬀerent channels that are simulta- 182 neously demodulated in two coherent receivers, it is observed that strong phase correlations exist 183 among channels. Such phenomenon can be interpreted by Eq. 10 in the Methods section. It 184 shows that fast phase ﬂuctuations between two-point locked microcombs mainly result from the 185 term m · ∆φ/17 (∆φ is the residual phase noise of the phase lock loop), which linearly scales up 186 with m and means that we can use the CPE result of one channel to predict the phase of other 187 channels in a master-slave fashion, as sketched in Fig. 3d [5, 37]. Fig. 3f shows the measured 188 data receiving performance when master-slave CPE is conducted among channel 1 to channel 10, 189 excellent BER is achieved when the phase of higher indexed data channel (e.g., channel 10) is 190 used to detect lower indexed data channels (e.g., channel 1 to 9). For example, when the CPE 191 result of channel 10 is used for channel 1, only minor BER penalty is observed in comparison with 192 the result of independent CPE. Moreover, comparing with recent demonstrated master-slave CPE 193 using uncorrelated carrier comb and LO comb [37], coherence-cloned DKS combs possess much 194 longer mutual coherent time, so they should be less sensitive to phase de-coherence caused by ﬁber 195 dispersion, and can potentially support longer transmission distance. 196 , p p g y Furthermore, Fig. Coherent data interconnect using coherence-cloned microcombs. Fig. 2 115 3e shows the data phase evolutions of diﬀerent channels that are simulta- 182 neously demodulated in two coherent receivers, it is observed that strong phase correlations exist 183 among channels. Such phenomenon can be interpreted by Eq. 10 in the Methods section. It 184 shows that fast phase ﬂuctuations between two-point locked microcombs mainly result from the 185 term m · ∆φ/17 (∆φ is the residual phase noise of the phase lock loop), which linearly scales up 186 with m and means that we can use the CPE result of one channel to predict the phase of other 187 channels in a master-slave fashion, as sketched in Fig. 3d [5, 37]. Fig. 3f shows the measured 188 data receiving performance when master-slave CPE is conducted among channel 1 to channel 10, 189 excellent BER is achieved when the phase of higher indexed data channel (e.g., channel 10) is 190 used to detect lower indexed data channels (e.g., channel 1 to 9). For example, when the CPE 191 result of channel 10 is used for channel 1, only minor BER penalty is observed in comparison with 192 the result of independent CPE. Moreover, comparing with recent demonstrated master-slave CPE 193 using uncorrelated carrier comb and LO comb [37], coherence-cloned DKS combs possess much 194 longer mutual coherent time, so they should be less sensitive to phase de-coherence caused by ﬁber 195 dispersion, and can potentially support longer transmission distance. 196 Discussion 197 Synthesizing the results in Fig. 3c and 3e, we can choose a desired trade-oﬀbetween BER perfor- 198 mance and CPE simplicity according to speciﬁc system requirements. For example, if our system 199 8 8 has a target BER of 3.8e-3, we can run CPE every other 1001 data block (i.e., skip 1000 blocks 200 after each CPE) for channel 10 and use the CPE result to detect channel 9 to channel 1. So, only 201 13 CPE operations (⌈12, 500 ÷ 1000⌉) are needed for channel 10 and the total decoded symbol 202 number sums up to 4,000,000. In comparison, if independent carriers and LOs are used, 12,500 203 CPE operations (12, 500 ÷ 10 × 10 channels) are needed to reach the 3.8e-3 BER (i.e., skipping 10 204 data blocks after each CPE, see Fig. 3c) within 4,000,000 symbols. According to such evaluation, 205 10 data channels carried and detected by coherence-cloned microcombs (i = 1000, j = 9) bring 206 about more than 3 orders of magnitude less pilot symbols and related CPE operations comparing 207 with same data capacity implemented by individual laser carriers and LOs (i = 10, j = 0). Such 208 prominent source saving can further scale up when the number of data channel increases. 209 In summary, we demonstrated coherence-cloned re-generation of DKS microcombs over long 210 distance and used them as the transmitter carriers and receiver LOs for terabit coherent data 211 interconnect. Enabled by two-point locking and OFD, excellent frequency and phase coherence 212 are achieved between the original and re-generated microcombs, which are leveraged to implement 213 totally saving of FOE and substantially reducing of CPE in coherent data detection. In our 214 experiment, simple point-to-point interconnect is demonstrated for which the pump laser and pilot 215 tone can be conveyed from transmitter to the receiver. Indeed, such scheme would become diﬃcult 216 for networks with multiple nodes and sophisticated topology [13]. However, instead of conveying 217 pilot tones, two-point locking among transmitter and receiver microcombs can also be implemented 218 using local optical frequency standard such as atomic gas cell or ultra-stable optical cavities [40]. Discussion 197 219 As long as the mutual stability among microcombs at diﬀerent network nodes is suﬃciently high, 220 the above demonstrated beneﬁts regarding FOE and CPE for coherent data receiving can be 221 obtained, oﬀering a potential solution to cope with the impending energy crisis that vexes the 222 optical transmission industry. 223 has a target BER of 3.8e-3, we can run CPE every other 1001 data block (i.e., skip 1000 blocks 200 after each CPE) for channel 10 and use the CPE result to detect channel 9 to channel 1. So, only 201 13 CPE operations (⌈12, 500 ÷ 1000⌉) are needed for channel 10 and the total decoded symbol 202 number sums up to 4,000,000. In comparison, if independent carriers and LOs are used, 12,500 203 CPE operations (12, 500 ÷ 10 × 10 channels) are needed to reach the 3.8e-3 BER (i.e., skipping 10 204 data blocks after each CPE, see Fig. 3c) within 4,000,000 symbols. According to such evaluation, 205 10 data channels carried and detected by coherence-cloned microcombs (i = 1000, j = 9) bring 206 about more than 3 orders of magnitude less pilot symbols and related CPE operations comparing 207 with same data capacity implemented by individual laser carriers and LOs (i = 10, j = 0). Such 208 prominent source saving can further scale up when the number of data channel increases. 209 In summary, we demonstrated coherence-cloned re-generation of DKS microcombs over long 210 distance and used them as the transmitter carriers and receiver LOs for terabit coherent data 211 interconnect. Enabled by two-point locking and OFD, excellent frequency and phase coherence 212 are achieved between the original and re-generated microcombs, which are leveraged to implement 213 totally saving of FOE and substantially reducing of CPE in coherent data detection. In our 214 experiment, simple point-to-point interconnect is demonstrated for which the pump laser and pilot 215 tone can be conveyed from transmitter to the receiver. Indeed, such scheme would become diﬃcult 216 for networks with multiple nodes and sophisticated topology [13]. However, instead of conveying 217 pilot tones, two-point locking among transmitter and receiver microcombs can also be implemented 218 using local optical frequency standard such as atomic gas cell or ultra-stable optical cavities [40]. Discussion 197 219 As long as the mutual stability among microcombs at diﬀerent network nodes is suﬃciently high, 220 the above demonstrated beneﬁts regarding FOE and CPE for coherent data receiving can be 221 obtained, oﬀering a potential solution to cope with the impending energy crisis that vexes the 222 optical transmission industry. 223 9 9 Acknowledgments The authors thank Professor Chee Wei Wong for helpful comments and sug- 224 gestions on this work, and VLC Photonics S. L. and LiGenTec SA for device fabrication. This work 225 is supported by National Key Research and Development Program of China (2019YFB2203103, 226 2018YFA0307400); National Natural Science Foundation of China (62001086, 61705033, 61775025); 227 The 111 project (B14039). 228 Methods 229 Generation and locking of CTx and CRx. DKS microcomb CTx is ﬁrst generated in the 230 transmitter microcavity, using the auxiliary laser heating method [29, 30]. Particularly, an auxiliary 231 laser is tuned into the blue-detuning regime of a cavity mode (∼1536.2 nm), and subsequently 232 a pump laser is tuned into another cavity mode (∼1549.9 nm). By properly setting the power 233 and detuning of the pump and auxiliary laser, the heat ﬂow caused by them can be balanced out 234 allowing the pump laser to stably scan into the red-detuning regime and access single soliton state 235 [29]. Moreover, using the same pump laser sent from the transmitter to the receiver trough 50 km 236 SSMF, DKS microcomb CRx is similarly generated in the receiver microcavity, by using another 237 auxiliary laser (∼1536.2 nm) to simultaneously control the pump detuning and maintain cavity 238 thermal stability [27]. 239 To achieve two-point locking, CTx(17) and CRx(17) are ﬁltered out and sent into a fast photodi- 240 ode in which their beating frequency ∆fCL(17) is detected. Then, ∆fCL(17) and a reference clock 241 fREF = 941.101000 MHz is sent into a phase comparator to generate the error signal and feedback 242 control the power of the auxiliary laser for generating CRx. Particularly, the auxiliary laser power 243 controls the pump detuning in the receiver microcavity via thermal resonance shift and in turn 244 adjust the repetition rate of CRx [25, 27], so that to lock CRx(17) to CTx(17). The bandwidth of 245 our phase lock loop is about 100 kHz, set by the amplitude modulation frequency limitation of the 246 10 adopted auxiliary laser module. 247 Coherence analysis between CTx and CRx. After 50 km ﬁber transmission, the phase of the 248 pump laser when it arrives at the receiver side is[36]: 249 Coherence analysis between CTx and CRx. After 50 km ﬁber transmission, the phase of the 248 pump laser when it arrives at the receiver side is[36]: 249 φTx(0) = φTx(0) int + φTx(0) ﬀ + φTx(0) nl (1) (1) φTx(0) int is the intrinsic phase noise (i.e., linewidth) of the pump laser, φTx(0) ﬀ denotes the phase noise 250 caused by the random ﬂuctuation of 50 km SSMF, φTx(0) nl denotes the nonlinear phase modulation 251 acquired during ﬁber transmission. Note that all the phase terms in Eq. 1 are time-varying. Methods 229 252 Similarly, the phase of the mth transmitter comb line CTx(m) when it arrives at the receiver side 253 is: 254 φTx(0) int is the intrinsic phase noise (i.e., linewidth) of the pump laser, φTx(0) ﬀ denotes the phase noise 250 caused by the random ﬂuctuation of 50 km SSMF, φTx(0) nl denotes the nonlinear phase modulation 251 acquired during ﬁber transmission. Note that all the phase terms in Eq. 1 are time-varying. 252 Similarly, the phase of the mth transmitter comb line CTx(m) when it arrives at the receiver side 253 is: 254 φTx(m) = φTx(m) int + φTx(m) ﬀ + φTx(m) nl + φTx(m) rep (2) φTx(m) = φTx(m) int + φTx(m) ﬀ + φTx(m) nl + φTx(m) rep (2) (2) Here φTx(m) rep = 2π · m · ∆f Tx rep · t is the phase noise caused by the ﬂuctuation of soliton repetition 255 rate ∆f Tx rep of CTx. 256 Here φTx(m) rep = 2π · m · ∆f Tx rep · t is the phase noise caused by the ﬂuctuation of soliton repetition 255 rate ∆f Tx rep of CTx. 256 At the receiver site, the phase of the mth line of the re-generated DKS microcomb CRx is: 257 At the receiver site, the phase of the mth line of the re-generated DKS microcomb CRx is: 257 At the receiver site, the phase of the mth line of the re-generated DKS microcomb CRx is: 257 φRx(m) = φRx(m) int + φRx(m) rep (3) φRx(m) = φRx(m) int + φRx(m) rep (3) φRx(m) = φRx(m) int + φRx(m) rep (3) Here φRx(m) rep = 2π · m · ∆f Rx rep · t, with ∆f Rx rep the soliton repetition rate jitter of CRx. For CRx we 258 neglect the random length ﬂuctuation and nonlinear phase modulation in those short ﬁber patch 259 cord within the receiver. 260 Here φRx(m) rep = 2π · m · ∆f Rx rep · t, with ∆f Rx rep the soliton repetition rate jitter of CRx. For CRx we 258 neglect the random length ﬂuctuation and nonlinear phase modulation in those short ﬁber patch 259 cord within the receiver. Methods 229 Since in our experiment the nonlinear XPM among diﬀerent channels 271 are small within the 50 km SSFM (see Supplementary Information), we can neglect the nonlinear 272 phase modulation term φTx(m) nl m = ±1, 2, 3, ... and obtain: 273 φTx(m) −φRx(m) = (φTx(m) ﬀ −φTx(0) ﬀ ) −m · (φTx(17) ﬀ −φTx(0) ﬀ )/17 + m · ∆φ/17 (10) (10) In the presence of ﬁber dispersion, diﬀerent comb modes temporally walk oﬀamong each other, 274 so the instantaneous phases of diﬀerent comb lines at the output of the SSFM are diﬀerent: 275 φTx(m) ﬀ ̸= φTx(n) ﬀ , m ̸= n. This results in the non-zero ﬁrst and second term to the right-hand-side 276 of Eq. 10. Considering that ﬁber ﬂuctuations are low frequency, we speculate that the ﬁrst and 277 second term in Eq. 10 explain the minor low-frequency noise (< 3.0 kHz) observed in the beat 278 note spectra shown in Fig. 1c. However, in our coherent detection experiment the electrical CPE 279 rate is generally larger than 3.0 kHz, so such slow phase drifts caused by ﬁber ﬂuctuations hardly 280 inﬂuence CPE and data receiving. The last term in Eq. 10 is linearly depends on comb mode 281 index m, based on which master-slave CPE is conducted. 282 In the presence of ﬁber dispersion, diﬀerent comb modes temporally walk oﬀamong each other, 274 so the instantaneous phases of diﬀerent comb lines at the output of the SSFM are diﬀerent: 275 φTx(m) ﬀ ̸= φTx(n) ﬀ , m ̸= n. This results in the non-zero ﬁrst and second term to the right-hand-side 276 of Eq. 10. Considering that ﬁber ﬂuctuations are low frequency, we speculate that the ﬁrst and 277 second term in Eq. 10 explain the minor low-frequency noise (< 3.0 kHz) observed in the beat 278 note spectra shown in Fig. 1c. However, in our coherent detection experiment the electrical CPE 279 rate is generally larger than 3.0 kHz, so such slow phase drifts caused by ﬁber ﬂuctuations hardly 280 inﬂuence CPE and data receiving. The last term in Eq. 10 is linearly depends on comb mode 281 index m, based on which master-slave CPE is conducted. 282 Coherent data modulation and receiving using CTx and CRx. At the transmitter, 20 DKS 283 comb lines are ﬁltered out by a C-band programmable wavelength selective switch (WSS) and used 284 as data carriers. Methods 229 260 11 As CRx and CTx are both in the state of DKS mode locking, we can assume that the intrinsic 261 phase of each comb line is aligned to the phase of corresponding pump laser: 262 φTx(m) int = φTx(0) int , φRx(m) int = φTx(0) (4) φTx(m) int = φTx(0) int , φRx(m) int = φTx(0) (4) (4) Also, the phase variation caused by soliton repetition rate change for CRx(m) and CTx(m) has the 263 relationship: 264 Also, the phase variation caused by soliton repetition rate change for CRx(m) and CTx(m) has the 263 relationship: 264 φTx(m) rep = m × φTx(1) rep , φRx(m) rep = m × φRx(1) rep (5) φTx(m) rep = m × φTx(1) rep , φRx(m) rep = m × φRx(1) rep (5) φTx(m) rep = m × φTx(1) rep , φRx(m) rep = m × φRx(1) rep (5) Locking CRx(17) to CTx(17) leads to: φTx(17) −φRx(17) = ∆φ, ∆φ is the residual technical noise of 265 Locking CRx(17) to CTx(17) leads to: φTx(17) −φRx(17) = ∆φ, ∆φ is the residual technical noise of 265 Locking CRx(17) to CTx(17) leads to: φTx(17) −φRx(17) = ∆φ, ∆φ is the residual technical noise of 265 the adopted phase lock loop, and we obtain: 266 Locking CRx(17) to CTx(17) leads to: φTx(17) −φRx(17) = ∆φ, ∆φ is the residual technical noise of 265 the adopted phase lock loop, and we obtain: 266 ( ) ( ) the adopted phase lock loop, and we obtain: 266 the adopted phase lock loop, and we obtain: 266 the adopted phase lock loop, and we obtain: 266 the adopted phase lock loop, and we obtain: 266 267 φTx(17) int + φTx(17) ﬀ + φTx(17) nl + φTx(17) rep = φRx(17) int + φRx(17) rep + ∆φ (6) φTx(17) int + φTx(17) ﬀ + φTx(17) nl + φTx(17) rep = φRx(17) int + φRx(17) rep + ∆φ (6) (6) Using Eq(1-6), we can calculate the phase of the mth inter-comb beat note: 268 φTx(m) −φRx(m) = ∆φ(m) p −∆φ(m) l + m · ∆φ/17 (7) (7) ∆φ(m) p = (φTx(m) ﬀ + φTx(m) nl −φTx(0) ﬀ −φTx(0) nl ) (8) (8) 12 ∆φ(m) l = m · (φTx(17) ﬀ + φTx(17) nl −φTx(0) ﬀ −φTx(0) nl )/17 (9) (9) ∆φ(m) p and ∆φ(m) l are produced due to that, when CRx is generated the pump laser has been 269 attached with extra phase noise φTx(0) ﬀ + φTx(0) nl comparing with the original pump laser linewidth 270 φTx(0) int when CTx is generated. Methods 229 Each of the 20 comb lines is boosted to about 0 dBm using a low-noise Er-doped 285 ﬁber ampliﬁer (EDFA) while maintaining >40 dB optical carrier-to-noise-ratio (OCNR)(Fig. 2c). 286 13 13 An IQ modulator is used to encode single-polarization 16-QAM data onto all the 20 comb lines, 287 driven by an electrical arbitrary waveform generator (eAWG) to generate the 16-QAM waveform 288 with rectangle pulse shaping. After modulation, all data channels are ampliﬁed by another EDFA 289 to generate -10.0 dBm launched power for each channel. 290 An IQ modulator is used to encode single-polarization 16-QAM data onto all the 20 comb lines, 287 driven by an electrical arbitrary waveform generator (eAWG) to generate the 16-QAM waveform 288 with rectangle pulse shaping. After modulation, all data channels are ampliﬁed by another EDFA 289 to generate -10.0 dBm launched power for each channel. 290 The 20 data channels together with the pump laser CTx(0) and pilot tone CTx(17) are trans- 291 mitted through 50 km SSMF to the receiver. At the receiver, CRx is re-generated as LOs for 292 coherent data detection. Each data channel and the corresponding LO is selected by another WSS 293 and fed into a coherent receiver. The detected electrical signal of each channel is recorded by 294 a real-time digital phosphor oscilloscope (DPO) and then processed oﬄine. Multiple algorithms 295 are used to achieve optimal data retrieval, including IQ imbalance compensation based on Gram- 296 Schmidt orthogonalization, chromatic dispersion compensation, Volterra channel equalization, and 297 pilot-aided CPE. Particularly, CPE is implemented by comparing the phase of 1 data block (32 298 symbols) of the received data sequence with the originally sent pilot symbols. 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Frequency-stabilized links for coherent wdm ﬁber interconnects in the datacenter. 413 Journal of Lightwave Technology, 38(13):3376–3386, 2020. 414 17 17 Figure 1: Coherence-cloned re-generation of DKS microcomb. a Left: for conventional WDM system based on independent lasers, guard intervals are necessary to tolerate the random frequency drifts among adjacent channels at the transmitter side, while at the receiver side power-hungry DSP must be implemented to recovery and compensate the random frequency and phase drifts between the carriers and LOs. Right: optical frequency comb has much better spectral stability than independent lasers, thus holds great potentials to improve spectral eﬃciency by eliminating guard intervals and reduce the DSP complexity of coherent transmission. b Upper: schematics of coherence-cloned DKS microcomb re-generation and two-point locking. Lower: optical spectra of the original transmitter comb CTx and re-generated receiver comb CRx. c Inter-comb beat note spectra between CTx(m) and CRx(m), m = 1, 5, 10, 17. It is seen that after two-point locking by the conveyed pump laser and pilot-tone, the beat note linewidths are substantially narrowed down implying that coherence between CTx and CRx are signiﬁcantly enhanced. d Allan deviations of the inter-comb beat note frequency ∆fCL(m), m = 1, 5, 17, which conﬁrm the eﬃcacy of frequency stability enhancement by two-point locking. The fundamental repetition rate oﬀset between CTx and CRx is ∆fCL(1) = 55.358882 MHz. Figure 1: Coherence-cloned re-generation of DKS microcomb. References 304 a Left: for conventional WDM system based on independent lasers, guard intervals are necessary to tolerate the random frequency drifts among adjacent channels at the transmitter side, while at the receiver side power-hungry DSP must be implemented to recovery and compensate the random frequency and phase drifts between the carriers and LOs. Right: optical frequency comb has much better spectral stability than independent lasers, thus holds great potentials to improve spectral eﬃciency by eliminating guard intervals and reduce the DSP complexity of coherent transmission. b Upper: schematics of coherence-cloned DKS microcomb re-generation and two-point locking. Lower: optical spectra of the original transmitter comb CTx and re-generated receiver comb CRx. c Inter-comb beat note spectra between CTx(m) and CRx(m), m = 1, 5, 10, 17. It is seen that after two-point locking by the conveyed pump laser and pilot-tone, the beat note linewidths are substantially narrowed down implying that coherence between CTx and CRx are signiﬁcantly enhanced. d Allan deviations of the inter-comb beat note frequency ∆fCL(m), m = 1, 5, 17, which conﬁrm the eﬃcacy of frequency stability enhancement by two-point locking. The fundamental repetition rate oﬀset between CTx and CRx is ∆fCL(1) = 55.358882 MHz. 18 Figure 2: Optical interconnect using coherence-cloned DKS microcombs as carriers and LOs. a Setup of the optical coherent data interconnect experiment. At the transmitter, microcomb CTx is generated in a silicon nitride micro-ring cavity based on the ALH method and used as multi-wavelength laser carriers. A programmable de-multiplexer (DEMUX) is adopted to select 20 comb lines from CTx and sent them to a high speed IQ modulator, where 21 Gbaud 16-QAM data is encoded to all the comb lines, forming 20 data channels with total bitrate 1.68 Tbit s−1. The pump laser CTx(0), pilot-tone CTx(17) and 20 data channels are then combined together in a multiplexer (MUX) and sent to the receiver through 50 km SSMF. At the receiver, microcomb CRx is re-generated and two-point locked to CTx and used as LOs for coherent data receiving. PID: proportional integral derivative; PD: photodiode; OBPF: optical bandpass ﬁlter. b SNR and BER measurements for all the 20 data channels. c Optical spectra of the carrier and LO comb lines, showing high OCNR > 40 dB for each line. d-e Comparison of beat note spectra between CTx(17) and CRx(17) before (d) and after (e) two-point locking. References 304 The results indicate that nonlinear XPM from the high speed data imposes negligible linewidth distortion to the pilot-tone CTx(17), therefore does not impact the performance of microcomb re-generation and coherent data receiving (see supplementary for more discussions). f The resolve phase evolution of channel 1 when FOE is directly calculated via m · fREF/17. The dotted lines show the residual FOE error extracted from the ﬁrst-order derivative of time from the actual data phase evolution, showing a 17.1 Hz (11.5 kHz) FOE error with two-point locking (without two-point locking). g Summarized FOE errors with and without two-point locking for all the 20 data channels. Figure 2: Optical interconnect using coherence-cloned DKS microcombs as carriers and LOs. a Setup of the optical coherent data interconnect experiment. At the transmitter, microcomb CTx is generated in a silicon nitride micro-ring cavity based on the ALH method and used as multi-wavelength laser carriers. A programmable de-multiplexer (DEMUX) is adopted to select 20 comb lines from CTx and sent them to a high speed IQ modulator, where 21 Gbaud 16-QAM data is encoded to all the comb lines, forming 20 data channels with total bitrate 1.68 Tbit s−1. The pump laser CTx(0), pilot-tone CTx(17) and 20 data channels are then combined together in a multiplexer (MUX) and sent to the receiver through 50 km SSMF. At the receiver, microcomb CRx is re-generated and two-point locked to CTx and used as LOs for coherent data receiving. PID: proportional integral derivative; PD: photodiode; OBPF: optical bandpass ﬁlter. b SNR and BER measurements for all the 20 data channels. c Optical spectra of the carrier and LO comb lines, showing high OCNR > 40 dB for each line. d-e Comparison of beat note spectra between CTx(17) and CRx(17) before (d) and after (e) two-point locking. The results indicate that nonlinear XPM from the high speed data imposes negligible linewidth distortion to the pilot-tone CTx(17), therefore does not impact the performance of microcomb re-generation and coherent data receiving (see supplementary for more discussions). f The resolve phase evolution of channel 1 when FOE is directly calculated via m · fREF/17. The dotted lines show the residual FOE error extracted from the ﬁrst-order derivative of time from the actual data phase evolution, showing a 17.1 Hz (11.5 kHz) FOE error with two-point locking (without two-point locking). References 304 g Summarized FOE errors with and without two-point locking for all the 20 data channels. 19 Figure 3: Carrier phase estimation facilitated by coherence-cloned DKS microcombs. a Scheme for CPE rate conﬁguration. CPE is conducted once every (i + 1) data blocks, namely, the CPE results of block 1 is used for the following i data blocks. b CPE results for channel 1, 5 and 10. It is seen that two-point locking between CTx and CRx signiﬁcantly enhance the phase stability between carriers and LOs. For this measurement the data format is 12.5 Gbaud 16-QAM, each panel illustrates a time window of 32 us containing 400,000 symbols. c Measured BER as a function of CPE rate. The block size for pilot-based CPE is 32 symbols. It is obvious that coherence-cloned microcombs allow much slower CPE rate to reach the target BER 3.8e-3 comparing with unlocked microcombs and independent carriers and LOs. d Scheme for master-slave joint CPE among multiple data channels. The carrier phase is estimated from the master channel (CH M) and then applied to j slave channels (CH S). e Retrieved data phases by individual CPE and master-slave CPE based on Eq. 10. Upper: channel 1 as slave and channel 2 as master; Middle: channel 1 as slave and channel 5 as master; Lower: channel 1 as slave and channel 10 as master. f Summarized BER performance of individual CPE and master-slave CPE of multiple data channels. For master-slave CPE measurement, channel 10 is used as the master channel and channel 1 to 9 are processed as slave channels. The inset shows the constellation maps and BER for channel 1 retrieved via individual and master-salve CPE Figure 3: Carrier phase estimation facilitated by coherence-cloned DKS microcombs. a Scheme for CPE rate conﬁguration. CPE is conducted once every (i + 1) data blocks, namely, the CPE results of block 1 is used for the following i data blocks. b CPE results for channel 1, 5 and 10. It is seen that two-point locking between CTx and CRx signiﬁcantly enhance the phase stability between carriers and LOs. For this measurement the data format is 12.5 Gbaud 16-QAM, each panel illustrates a time window of 32 us containing 400,000 symbols. c Measured BER as a function of CPE rate. The block size for pilot-based CPE is 32 symbols. References 304 It is obvious that coherence-cloned microcombs allow much slower CPE rate to reach the target BER 3.8e-3 comparing with unlocked microcombs and independent carriers and LOs. d Scheme for master-slave joint CPE among multiple data channels. The carrier phase is estimated from the master channel (CH M) and then applied to j slave channels (CH S). e Retrieved data phases by individual CPE and master-slave CPE based on Eq. 10. Upper: channel 1 as slave and channel 2 as master; Middle: channel 1 as slave and channel 5 as master; Lower: channel 1 as slave and channel 10 as master. f Summarized BER performance of individual CPE and master-slave CPE of multiple data channels. For master-slave CPE measurement, channel 10 is used as the master channel and channel 1 to 9 are processed as slave channels. The inset shows the constellation maps and BER for channel 1 retrieved via individual and master-salve CPE. 20 Figures Figures Figure 1 Coherence-cloned re-generation of DKS microcomb. a Left: for conventional WDM system based on independent lasers, guard intervals are necessary to tolerate the random frequency drifts among adja channels at the transmitter side, while at the receiver side power-hungry DSP must be implemented to Figure 1 Figure 1 Coherence-cloned re-generation of DKS microcomb. a Left: for conventional WDM system based on independent lasers, guard intervals are necessary to tolerate the random frequency drifts among adjacent channels at the transmitter side, while at the receiver side power-hungry DSP must be implemented to recovery and compensate the random frequency and phase drifts between the carriers and LOs. Right: optical frequency comb has much better spectral stability than independent lasers, thus holds great potentials to improve spectral eﬃciency by eliminating guard intervals and reduce the DSP complexity of coherent transmission. b Upper: schematics of coherence-cloned DKS microcomb re-generation and two- point locking. Lower: optical spectra of the original transmitter comb CTx and re-generated receiver comb CRx. c Inter-comb beat note spectra between CTx(m) and CRx(m), m = 1, 5, 10, 17. It is seen that after two- point locking by the conveyed pump laser and pilot-tone, the beat note linewidths are substantially narrowed down implying that coherence between CTx and CRx are signiﬁcantly enhanced. d Allan deviations of the inter-comb beat note frequency ∆fCL(m), m = 1, 5, 17, which conﬁrm the eﬃcacy of frequency stability enhancement by two-point locking. The fundamental repetition rate oﬀset between CTx and CRx is ∆fCL(1) = 55.358882 MHz. Figure 2 Optical interconnect using coherence-cloned DKS microcombs as carriers and LOs. a Setup of the optical coherent data interconnect experiment. At the transmitter, microcomb CTx is generated in a silicon nitride micro-ring cavity based on the ALH method and used as multi-wavelength laser carriers. A programmable de-multiplexer (DEMUX) is adopted to select 20 comb lines from CTx and sent them to a high speed IQ modulator, where 21 Gbaud 16-QAM data is encoded to all the comb lines, forming 20 data channels with Figure 2 Optical interconnect using coherence-cloned DKS microcombs as carriers and LOs. a Setup of the optical coherent data interconnect experiment. At the transmitter, microcomb CTx is generated in a silicon nitride micro-ring cavity based on the ALH method and used as multi-wavelength laser carriers. A programmable de-multiplexer (DEMUX) is adopted to select 20 comb lines from CTx and sent them to a high speed IQ modulator, where 21 Gbaud 16-QAM data is encoded to all the comb lines, forming 20 data channels with total bitrate 1.68 Tbit s−1. The pump laser CTx(0), pilot-tone CTx(17) and 20 data channels are then combined together in a multiplexer (MUX) and sent to the receiver through 50 km SSMF. At the receiver, microcomb CRx is re-generated and two-point locked to CTx and used as LOs for coherent data receiving. PID: proportional integral derivative; PD: photodiode; OBPF: optical bandpass ﬁlter. b SNR and BER measurements for all the 20 data channels. c Optical spectra of the carrier and LO comb lines, showing high OCNR > 40 dB for each line. d-e Comparison of beat note spectra between CTx(17) and CRx(17) before (d) and after (e) two-point locking. The results indicate that nonlinear XPM from the high speed data imposes negligible linewidth distortion to the pilot-tone CTx(17), therefore does not impact the performance of microcomb re-generation and coherent data receiving (see supplementary for more discussions). f The resolve phase evolution of channel 1 when FOE is directly calculated via m · fREF/17. The dotted lines show the residual FOE error extracted from the ﬁrst-order derivative of time from the actual data phase evolution, showing a 17.1 Hz (11.5 kHz) FOE error with two-point locking (without two- point locking). g Summarized FOE errors with and without two-point locking for all the 20 data channels. Figure 3 Figure 3 Carrier phase estimation facilitated by coherence-cloned DKS microcombs. a Scheme for CPE rate conﬁguration. CPE is conducted once every (i + 1) data blocks, namely, the CPE results of block 1 is used for the following i data blocks. b CPE results for channel 1, 5 and 10. It is seen that two-point locking between CTx and CRx signiﬁcantly enhance the phase stability between carriers and LOs. For this measurement the data format is 12.5 Gbaud 16-QAM, each panel illustrates a time window of 32 us containing 400,000 symbols. c Measured BER as a function of CPE rate. Figure 2 The block size for pilot-based CPE is 32 symbols. It is obvious that coherence-cloned microcombs allow much slower CPE rate to reach the target BER 3.8e-3 comparing with unlocked microcombs and independent carriers and LOs. d Scheme for master-slave joint CPE among multiple data channels. The carrier phase is estimated from the master channel (CH M) and then applied to j slave channels (CH S). e Retrieved data phases by individual CPE Figure 3 Figure 3 Carrier phase estimation facilitated by coherence-cloned DKS microcombs. a Scheme for CPE rate conﬁguration. CPE is conducted once every (i + 1) data blocks, namely, the CPE results of block 1 is used for the following i data blocks. b CPE results for channel 1, 5 and 10. It is seen that two-point locking between CTx and CRx signiﬁcantly enhance the phase stability between carriers and LOs. For this measurement the data format is 12.5 Gbaud 16-QAM, each panel illustrates a time window of 32 us containing 400,000 symbols. c Measured BER as a function of CPE rate. The block size for pilot-based CPE is 32 symbols. It is obvious that coherence-cloned microcombs allow much slower CPE rate to reach the target BER 3.8e-3 comparing with unlocked microcombs and independent carriers and LOs. d Scheme for master-slave joint CPE among multiple data channels. The carrier phase is estimated from the master channel (CH M) and then applied to j slave channels (CH S). e Retrieved data phases by individual CPE Carrier phase estimation facilitated by coherence-cloned DKS microcombs. a Scheme for CPE rate conﬁguration. CPE is conducted once every (i + 1) data blocks, namely, the CPE results of block 1 is used for the following i data blocks. b CPE results for channel 1, 5 and 10. It is seen that two-point locking between CTx and CRx signiﬁcantly enhance the phase stability between carriers and LOs. For this measurement the data format is 12.5 Gbaud 16-QAM, each panel illustrates a time window of 32 us containing 400,000 symbols. c Measured BER as a function of CPE rate. The block size for pilot-based CPE is 32 symbols. It is obvious that coherence-cloned microcombs allow much slower CPE rate to reach the target BER 3.8e-3 comparing with unlocked microcombs and independent carriers and LOs. d Scheme for master-slave joint CPE among multiple data channels. The carrier phase is estimated from the master channel (CH M) and then applied to j slave channels (CH S). e Retrieved data phases by individual CPE and master-slave CPE based on Eq. 10. Upper: channel 1 as slave and channel 2 as master; Middle: channel 1 as slave and channel 5 as master; Lower: channel 1 as slave and channel 10 as master. f Summarized BER performance of individual CPE and master-slave CPE of multiple data channels. Figure 3 For master-slave CPE measurement, channel 10 is used as the master channel and channel 1 to 9 are processed as slave channels. The inset shows the constellation maps and BER for channel 1 retrieved via individual and master-salve CPE. Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. DKSCommunicationssupplementary.pdf
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JUILLET CHEF DES TRAVAUX DE MICROGRAPHIE A L'ÉCOLE DE L'UNIVERSITÉ DE SUPÉRIEURE DE PHARMACIE MONTPELLIER ire THÈSE. — RECHERCHES ANATOMIQUES, EMBRYOLOGIQUES, HISTOLOGIQUES ET, COMPARATIVES SUR LE POUMON Due THÈSE. — PROPOSITIONS DONNÉES PAR LA FACULTÉ. DES OISEAUX. Soutenues J ( Le la Commission d'examen MM. Y. DErAGE: Président. MICHEL Examinateurs. MOLLIARD PARIS ÉDITION DES ARCHIVES DE Z00LOGIE EXPÉRIMENTALE LIBRAIRIE A. SCHULZ 8, Place de la 4912 Sorbonne, 38 Ci AN meÈ av \| Ÿ PE DT 1 28 4 8 VUE ue A LA FACULTÉ DES SCIENCES DE L'UNIVERSITÉ DE PARIS POUR LE GRADE OBTENIR DE DOCTEUR ES SCIENCES NATURELLES PAR M. A JUILLET CHEF 1 DES THÈSE. TRAVAUX — DE MICROGRAPHIE A L'ÉCOLE DE L’UNIVERSITÉ DE RECHERCHES TOLOGIQUES DES One THÈSE. — Soutenues le SUPÉRIEURE PHARMACIE EMBRYOLOGIQUES, ANATOMIQUES, ET DE MONTPELLIER COMPARATIVES SUR LE OISEAUX. PROPOSITIONS — DONNÉES PAR LA FACULTÉ. / ‘an TT de, . devant la Commission MM. Y. DErAGE: \ MOLLIARD =—— d'examen Président. MICHEL -— À Examinateurs. — PARIS ÉDITION DES ARCHIVES DE Z00LOGIE EXPÉRIMENTALE LIBRAIRIE A. SCHULZ 8, Place de la 1912 Sorbonne, 3 HIS- POUMON FACULTÉ DEN SCIENCES DE L'ONNERNITÉ DE PARIS LE 2 MM. Doteneer cie Doyen honoraire Professeur.... P. APPELL, G. DARBOUX, ........ Projesseurs honoraires... .... — CHNOEE J, RIBAN. ETPPMANNE eur ee cc LOUE BOUSSINESQ. .......:..e Physique mathémat. et calcul des probabilités. IBICARD rene tee CE Analyse supérieure et algèbre supérieure. HÉXPOINCARÉ SR \| VAN DETAGEE. Reese Astronomie mathématique et mécanique céleste. Zoologie, anatomie, physiologie comparée. IMCCMBONNEERE Botanique. ER eme NS EE Ce uoe Mécanique MÉTAIN ee Géographie physique. neo neets physique -et expérimentale. GOURSAT re Calcul différentiel CHATINS. 65m see sine HAGGERN LR ee Histologie. Chimie organique. JOANNIS Rem Chimie (Enseignement P. C. N.). -e RARetre ee Mie WAGLERANTES Eee \PMANDOVER CE ie PAINDEVÉRT AU EE ne HOUSSAVE ce crc 7 cote © eue cree H. LE CHATELIER....... GABRIEL BERTRAND..... Mme PCURIE.CAUREERMEE À Physiologie. IRŒONTÉRES RC tr TANETIS ose Physique, Cotote ses vaio c Physique. DASTRE PNOJESSEUTS ER Mécanique rationnelle. Géométrie supérieure. Se et calcul intégral. Physique. Minéralogie. : Astronomie : physique. Mathématiques générales. Géologie. Zoologie. Chimie. Chimie biologique. Physique générale. ee ce Zoologie, Evolution CICHABRIÉ Se GAURPBAINE CT eee Chimie appliquée. Chimie. BOBREL = see eee Théorie des fonctions, MARCHIS EEE Ce Aviation. GPRUVOTS EC Rec Zoologie, JEANVPERRIN.-.....---.. NS re Re LE CR INR Re Le CE des êtres organisés. anatomie, physiologie comparée. Chimie Physique. Application de l'analyse à la géométrie. Calcul différentiel et calcul intégral. \| SPUISEUXÉ EEE Projesseurs-adjoints ..... TT Physique. Botanique. MICHEL AZ Re sleeee coute Minéralogie. HÉROUARD ee Zoologie. Pr Léon BERTRAND ........ Rémy PERRIER MOBDIARDE COTON. D CCTÉLORTE ee Ce Mécanique et astronomie. DEDUCIE PEER ECC CE MATRUCHOT EEE Eee ........ EEETerre rte D. TOMBECK. : Géologie. Zoologie (Enseignement Physiologie Physique. végétale. P. C. X... A Monsieur le Professeur de la Faculté de médecine L. VIALLETON de Montpellier. Hommage de respectueuse gratitude. A MES MAIÏITRES de l'École supérieure de Pharmacie de Montpellier ARCHIVES DE ZOOLOGIE 5° Série, Tome EXPÉRIMENTALE IX, p. 207 à 371, pl. XV ET GÉNÉRALE à XX. 20 Janvier 1912. RECHERCHES ANATOMIQUEN, EMBRYOLOGIQUES, HINTOLOGIQUES ET COMPARATIVEN SUR LE POUMON DES OISEAUX" PAR A. JUILLET, Chef des travaux de à l’École supérieure de Pharmacie micrographie de l’Université de Montpellier. TABLE DES MATIÈRES ÉSÉRODUOIONEE En denis ea ETS RO RTO IEEE Aie es ele ces etes ocie ol eie se cine soc sise onie diaie o c\oicio aie o die 2 eco nee + 207 ataie eine ele ele)stolela calsieelalelerais cietas se 8 cialelo aiclore @i0r0ore sine ciele 84e cd e 210 216 RIRE OR ES EEE cel s miteisie cie se © oise else see + 210 © a oies o saisie o ecole se. 8e 2 oislele e e.s 0e o sie CHAP. I. — RAPPORTS DES POUMONS : Rapports des poumons avec le squelette (p. 226). — Forme des poumons (p. 228). — Diaphragme et plèvre (p. 231). — Rapports de la face ventrale des poumons C2 RÉSUMÉ CHAP. II. — PRINCIPALES DESCHOUONNAES CHAP. III. — IV. — NL. VOIES LM ne AÉRIENNES ETOSMTONCS IDIONCHIQUES RAPPORT Rolesphysiologique CHAP. A DES SACS AÉRIENS des ibronChes DÉVELOPPEMENT celle 246 INTRA-PULMONAIRES....,......... a nes ee lee sn A 249 LM ser eee tee en ce serletstele siecle els oise ele eteiele ET DES BRONCHES................4.. TÉCUITENLES.. DES POUMONS ee ET DES ses 254 269 ses ose oi" es «+ 1e 287 SACS...............suee 295 CHAPELLE PARENCHYMEZ PULMONAIRE... rise» cn vies etele sin else eo/ee CAP AVE PAIN ATOMIE COMPARE sec see sie seine ele eee closes cesse se Forme du poumon (p. 338). — Bronches (p. 339)— Rapports de la face ventrale avec les sacs aériens (p. 340). — Comparaison de la stucture du poumon des Oiseaux Avec Celui des autres AMNIOLCS. 0-2 eee see = = == nee lee sieis se = 1e«1e CONCHUSIONSIENNSE LE Re dentaire molle stle cecesssolsisielnie ele ele alese se,efon oo cie clolsis cine sie NDS TBLIOGRAPHIQUEME Eee seance nsiieisle sielslniels ele ee oc se scie slele see © eines ee RIMPHICATIONSSDES PIANCHES 2. ess sie e an » simie ee 6e nee e sole ete soie else ele a 01e 01e e1#1e1eLe 318 336 396 361 366 369 INTRODUCTION Le titre de ce travail a besoin d’être expliqué; il précise d’abord à quelle partie de l’appareil respiratoire il a été consacré et indique que, contrairement à tant d’autres travaux (1) Travail du laboratoire d’histologie de la Faculté de Médecine de Montpellier, M. L. Vialleton professeur, ARCH. DE ZOOL. EXP. ET GÉN. — 5° SÉRIE. — T. IX. — (I). 14 208 A. JUILLET qui embrassent toute l’étendue de cet appareil, il s’adresse au poumon seul ; mais en même temps, il ne se borne point à la structure de cet organe et envisage son anatomie tout entière, y compris ses rapports avec le squelette et les autres viscères. C’est ce que veut exprimer le mot « anatomiques » destiné en même temps à bien montrer toute l’importance qu’il faut attacher à l’étude macroscopique du poumon et de ses connexions. Pour avoir négligé cette étude macroscopique l’on est encore très mal fixé sur la constitution et sur le rôle du diaphragme pulmonaire des Oiseaux et l’on a décrit la face ventrale du poumon d’une manière absolument schématique. A la suite de cette étude anatomique nouvelle, il était indispensable de bien relier entre elles les données macroscopiques que l’on possédait déjà sur la structure du poumon, car, faute d’avoir fait ce rapprochement, bien des détails de la structure restaient incompréhensibles, et en tous cas ne pouvaient être comparés aisément avec ceux du poumon des autres animaux. Le meilleur moyen de bien faire comprendre la structure du poumon des Oiseaux était évidemment d’en faire une étude complète, c’est-à-dire à la fois ‘anatomique, histologique et embryologique, afin de bien coordonner entre elles les notions de divers ordres que l’on possèdait sur le poumon et de suivre la structure depuis les dispositions macroscopiques aisément visibles à l’œil nu jusqu'aux plus fins détails, perceptibles seulement à l’aide de forts grossissements. Cette étude devait être corroborée par celle du développement qui permet de suivre graduellement la complication de l’ébauche pulmonaire et d'assister à la création des différentes parties observées chez l'adulte. Pour une semblable étude, une espèce s’offrait avant toutes les autres à cause de la facilité avec laquelle on peut s’en pro- curer les embryons aux différents moments de l’évolution: c’est le Poulet domestique. Les embryons ont été recueillis à tous les stades et surtout pendant les stades tardifs — qui POUMON DES OISEAUX 209 n'avaient fait jusqu'ici l’objet d’aucune recherche — c’està-dire à partir du huitième jour de l’incubation jusqu’à l’éclosion et pendant les premiers jours qui suivent cette dernière. Mais si le Poulet à été le principal objet de mes recherches, je ne me suis pas borné à cette espèce, et j’ai étudié, chez l'adulte, vingt-quatre espèces appartenant à huit ordres. Elles m'ont montré des dispositions un peu différentes de celles rencontrées chez le Poulet et m'ont permis de prendre une idée de la variation structurale du poumon dans un certain nombre de types. Enfin, j'ai comparé le poumon des Oiseaux avec celui des autres Amniotes et particulièrement avec celui des Mammifères en faisant ressortir les corrélations étroites qui lient la structure du poumon au reste de l’organisation avienne et qui caractérisent d’une manière si frappante l’organe respiratoire des Oiseaux. Ce travail m’a été inspiré par M. le professeur Vialleton qui a bien voulu mettre à ma disposition les ressources de son laboratoire pendant toute la durée de mes recherches et en écrivant son nom en tête de cette étude, je sens quel est le faible hommage de ma reconnaissance à sa bienveillance éclairée. Ce travail n’est que le résultat de son remarquable enseignement et les encouragements, les conseils que ne cessa de me prodiguer mon Maître porteront un jour, je lespère, des fruits dignes des méthodes scientifiques que je me suis efforcé d'acquérir dans son laboratoire. Je croirais manquer à mon devoir en ne remerciant pas 1ci M. Pontal, étudiant en médecine et M. F. Delanoë, chef des travaux d’histologie à la Faculté de Médecine de Montpellier, dont j'ai pu apprécier tant de fois la charmante courtoisie et qui sacrifièrent si généreusement leurs loisirs au travail de leur ami. 210 À. JUILLET Historique Les travaux sur l’appareil respiratoire des Oiseaux sont excessivement nombreux et si l’on se reporte à la liste donnée p. 366, on voit qu'il en existe plus d’une centaine; mais ces travaux se rapportent d’une manière très inégale au sujet que nous traitons. Beaucoup s'occupent avant tout des sacs aériens, et un petit nombre seulement ont trait au poumon proprement dit envisagé sous les différents aspects où nous voulons l’examiner. Il nous paraît inutile de refaire un historique complet des mémoires consacrés à l’appareil respiratoire, d'autant que cet historique a été fort bien fait pour ce qui regarde le dixhuitième siècle et la première partie du dix-neuvième par SAPppEy (1847), et que l’on peut aussi trouver dans OPPEL (1905) un exposé détaillé des travaux postérieurs à celui de anatomiste français dont nous venons de parler. Mais nous voudrions faire ressortir les principales étapes de nos connaissances sur l’anatomie du poumon afin de bien préciser les questions qui restent à résoudre et de mettre en relief l’œuvre des principaux chercheurs qui se sont occupés de cette étude. Sans parler des données anciennes d’ARISTOTE et de l’empereur FRÉDÉRIC qui avait remarqué l’adhérence des poumons aux côtes et d’autres détails de leur anatomie, nous prendrons comme point de départ la magistrale étude de SAPPEY qui renferme, ainsi qu'on la vu plus haut, un excellent historique. Combi- nant les résultats obtenus par ses devanciers et ses propres découvertes, SAPPEY donne une bonne description des grosses bronches intra-pulmonaires qu'il divise en « diaphragmatiques » et « costales » suivant qu’elles sont placées sur la face ventrale ou sur la face dorsale du poumon. Il montre leur mode de ramification pennée et leur situation superficielle, il découvre aussi quelques anastomoses réunissant les rameaux issus de ces grosses bronches, mais ne va pas plus loin dans l’étude de la structure intime. Il remarque que si l’on remplit POUMON DES OISEAUX 211 d’une masse solide les grosses bronches sans atteindre celles qui en naissent, ces dernières qui communiquent entre elles par les anastomoses précitées, n’étendent point leur domaine et leurs communications sur une surface un peu étendue, mais qu’elles fournissent seulement un territoire restreint du poumon, qu’il compare à celui d’un lobe ou même d’un lobule pulmonaire. Il ne s’est donc point rendu compte de la facilité du passage de l’air dans les différentes parties du poumon et s’est mépris sur la fine structure de ce dernier, en admettant dans le poumon des Oiseaux des territoires lobulaires qui n’y existent point. D'ailleurs il n’a guère cherché les fins détails de structure du parenchyme pulmonaire, et toute son attention, comme celle de ses prédécesseurs du reste, s’est portée essentiellement sur les sacs aériens. Il à certainement donné de ces sacs la meilleure des descriptions qui puisse en être faite si l’on met à part la mention de leur origine embryologique. Les auteurs qui ont suivi n'ont pas ajouté grand’-chose à sa description. Pour le poumon proprement dit, il faut signaler aussi qu'il à fort bien vu l’absence de plèvre et ses données sur ce sujet ont été pleinement confirmées par les recherches embryologiques de BERTELLI (1905) qui ont mis fin aux longues discussions résultant d’une compréhension restée imparfaite, faute de la connaissance du développement. Peu de temps après les recherches de SAPPEY, RAINEY (1849) fit une étude histologique du poumon des Oiseaux et donna la première figure de la structure du parenchyme. Bien que schématique, cette figure représente assez exactement l'enveloppe prismatique que le parenchyme forme autour de la lumière des bronches. Les détails histologiques sont toutefois très imparfaits : c’est ainsi qu’il affirme l’absence d’epithélium sur les travées du parenchyme, idée qui se fera encore jour dans certains travaux récents. La présence de cet épithélium fut au contraire affirmée dix ans plus tard par WILLrams (1859) qui a donné une étude d'ensemble du poumon. EBERTH (1863) apporta quelques contributions à l’histologie du poumon, 212 A. JUILLET et en 1871 F. E. SCHULZE donna une description histologique du poumon des Oiseaux avec deux figures schématiques, l’une des voies aériennes dans le parenchyme, l’autre des vaisseaux. Comme ce sont les meilleures figures se rapportant à l’anatomie microscopique du poumon, elles ont été reproduites jusque dans les ouvrages récents comme celui d’OrPEz (1905). Le même auteur a récemment donné une étude importante du poumon de l’Autruche, on en reparlera plus loin. En 1875, parut un admirable travail, aussi complet qu’il pouvait l’être à ce moment, et qui embrasse tous les points essentiels de la structure du poumon. C’est le travail de CamPANA (1875.) CAMPANA étudia spécialement le Poulet, mais il ne négligea aucune des parties de l’appareil respiratoire : il en fit une monographie véritablement magistrale, qui n’eut point le succès et le retentissement qu’elle méritait, car bien des résultats qu’elle rénferme ont été méconnus jusqu'ici, alors même qu’ils étaient l’expression exacte de la vérité. CAMPANA injecta à l’aide de mélanges céro-résineux l’arbre bronchique du Poulet et put ainsi suivre les bronches dans tout leur parcours. Il montra que la bronche souche donne d’abord les gros troncs superficiels signalés par ses prédécesseurs, puis que ces troncs se divisent en rameaux plus fins qui vont s’anastomoser avec les rameaux analogues venus des bronches de la face opposée du poumon et forment avec ces derniers des circuits spiroïdes qui parcourent toute l'épaisseur du poumon en traversant le parenchyme pulmonaire. Il fit voir aussi que tout autour de ces circuits bronchiques, le parenchyme pulmonaire se dispose en fines travées anastomosées, entre lesquelles l’air circule de toutes parts, et cette donnée est plus exacte que celle de SCHUIZE figurant autour de la lumière des gros conduits aériens des tubes branchés et communicants entre eux mais terminés en cul-de-sac. En un mot, il donna une description du poumon de Poulet à laquelle on pourrait souscrire aujourd’hui sans rien changer d’essentiel et en y ajoutant seulement certains faits tirés de l’embryologie qui lexpliquent te 2P ee POUMON DES OISEAUX 213 et la complètent. Les détails de l’œuvre de CAMPANA seront discutés au fur et à mesu re qu’ils se présenteront à propos de nos recherches, et d’ailleurs il serait inutile de les indiquer dès maintenant, parce qu’ils ne pourraient être compris d’une manière satisfaisante sans entrer dans des développements qui feraient double emploi avec la suite. Il suffit d’insister encore une fois sur l’importance de son travail. De 1875 à 1890, peu de travaux d’ensemble furent faits sur le poumon des Oiseaux. HUXLEY (1882) donna une description et une nomenclature nouvelle des bronches. Il appelle «mésobronche » la bronche souche, centobronches » les bronches ventrales ou diaphragmatiques, « ectobronches » les bronches costales et désigne sous le nom de « parabronches » les bronches issues de ces troncs principaux. Cette nomenclature très commode par l’unité des termes proposés, constitue un très réel progrès pour l’exposé de la structure pulmonaire et pour la description des parties. Mais en dehors de cette acquisition didactique, le travail de HUXLEY n’apporte aucune contribution nouvelle à nos connaissances qui resteront encore longtemps après lui à un état assez peu satisfaisant, étant donné loubli des descriptions de CAMPANA. Aussi la monographie de GaAnow (1890) donnée dans le règne animal de BRONN ne renferme aucune bonne figure du poumon. De nouveaux travaux, ceux de Rocré (1891), de BIrGNoN ont paru sur les sacs aériens, mais ils ne s'occupent point de la structure pulmonaire. IL en est de même de ceux de BERTELLI (1905) qui ont toutefois une grande valeur parce qu’ils donnent des détails très intéressants sur le développement des sacs aériens et sur leur rôle dans le cloisonnement de la cavité viscérale. Grâce à ces travaux, les rapports du poumon avec les viscères et avec les parois costales sont, d’une manière générale, exactement com- pris et les discussions interminables soulevées à propos de l'existence des plèvres sont enfin tranchées par l’embryologie d’une manière satisfaisante. Pour quelques détails, il est vrai, 214 A. JUILLET nous différerons de l’opinion de BERTELLI, mais il n’en est pas moins vrai que, pour ce qui regarde les rapports des poumons, son travail à une importance capitale et occupe un rang des plus honorables parmi ceux qui ont fixé nos connaissances à ce sujet. Avec les travaux embryologiques de BERTELLI nous devons rappeler ceux qui ont été consacrés par divers auteurs au développement du poumon des Oiseaux. Ces travaux, dont certains remontent à une date fort reculée (VON BAER, RATHKE) sont fort nombreux comme on se \|l’explique aisément, par le fait que l'embryon de Poulet a été longtemps l’objet d’étude favori des embryologistes, mais ils ne portent que sur les premiers ‘stades du développement et par conséquent relèvent plutôt de l’embryologie générale que de celle des organes que nous avons en vue. Nous ne ferons donc qu’énumérer ceux de VON BAER (1828), RATHKE (1828), REMAK (1855), SELENKA (1866), GÔTTE (1867), H1s(1868), Senssez (1877), FIscHELIS (1885), KASTSCHENKO (1887), WEBER ET BuviGnrER (1903) pour insister un peu plus sur ceux de ZUMSTEIN (1891-92), de MILLER (1893), de FANNyY Moser (1902). ZUMSTEIN à énuméré la formation des sacs ; FANNY Moser a poussé plus loin que ses prédécesseurs l’étude du développement du poumon et a étudié des poumons de huit jours. MILLER a essayé de représenter les rapports entre la structure des poumons des Oiseaux et ceux des autres Vertébrés. SUPINO (1899) a donné quelques renseignements histologiques sur les poumons des Oiseaux. OPPEL (1905) dans son ouvrage, surtout bibliographique, a donné une étude histologique très complète de la structure de l’appareil respiratoire, étude il est vrai appuyée sur des recherches personnelles et sur des critiques qui lui donnent une valeur importante. Mais il s’en est tenu surtout, comme lindique d’ailleurs son titre, à l’histologie, de sorte que celui qui chercherait à se faire une idée exacte et complète du poumon des Oiseaux d’après son traité, serait certainement déçu et ne trouverait pas au milieu POUMON DES OISEAUX 215 de la masse énorme de documents bibliographiques qu’il renferme, le fil conducteur capable de lui faire comprendre la structure du poumon. Un dernier travail doit être signalé maintenant qui semble répondre très exactement au but que se propose le nôtre, c’est celui de G. FISCHER (1905) qui a étudié à la fois anatomiquement et histologiquement l’arbre bronchique des Oiseaux, et donne des moulages du poumon d’une trentaine d’espèces. A première vue, ce travail paraît bien combler toutes les lacunes qui existaient jusqu'alors dans nos connaissances et semblerait devoir décourager toute autre recherche dans le même domaine. Mais en y regardant de plus près, on s’aperçoit aisément qu’il est loin de répondre à tous les desiderata et qu’il est en réalité bien au-dessous de celui de CAMPANA, qu’il a méconnu comme tant d’autres. En effet, les anastomoses des parabronches sont représentées d’une manière tout à fait schématique et, chose plus grave, les rapports des sacs aériens avec les bronches intrapulmonaires sont tout à fait négligés, de sorte que lun des traits les plus caractéristiques de la structure du poumon des Oiseaux a totalement échappé à l’auteur. Il n’a pu voir notamment la disposition différente des orifices bronchiques dans les différentes espèces qu’il étudie ; par conséquent les nombreux moulages qu’il donne (et qui pour la plupart, étant vus par la surface dorsale, ne laissent apercevoir aucun des traits essentiels de la structure), n’ont que la valeur d’une image tout à fait superficielle des bronches. Dans un travail récent, F. E. ScHuzze (1908) a donné uno description du poumon de l’Autruche, à laquelle il à déjà été fait allusion. Enfin je dois signaler diverses notes que j'ai publiées au cours de cette année dans les comptes rendus de l’Académie des Sciences et dans ceux de la Société de Biologie, sur divers points de la structure pulmonaire et sur lesquelles je reviendrai. 216 A. JUILLET Méthodes Nous indiquerons ici toutes les méthodes employées au cours de ce travail pour l’étude purement anatomique et l’étude embryologique ou histologique. Tout d’abord, il est indispensable de se servir d’un matériel très frais, non seulement pour l'étude histologique où il est inutile d’en faire ressortir la nécessité, mais encore pour l’étude anatomique parce que diverses circonstances interviennent qui peuvent rendre absolument inutilisables des animaux morts depuis quelque temps déjà, et fréquemment aussi des animaux venant d’être tués à la chasse. Le matériel de choix est donc donné par les Oiseaux de basse-cour et par ceux que l’on peut se procurer vivants chez les marchands. On peut aussi utiliser d’autres pièces rares obtenues autrement, mais cela ne pourra jamais se faire que pour une étude partielle d’un point quelconque de la structure, et non pour une étude d’ensemble de toutes les questions qui se soulèvent à propos de la structure pulmonaire. Pour tuer les animaux le procédé le plus simple et le meilleur est le suivant : on fait tenir l’oiseau par un aide, on découvre rapidement la trachée à la partie antérieure du cou, on l’isole et on fait la section du cou en dessous de la trachée ; de cette façon l’animal est saigné et meurt rapidement. La saignée est utile surtout pour les recherches histologiques en dégageant le poumon d’une certaine quantité du sang qu’il renferme, quantité telle, que lorsque la saignée préalable n’a pas été faite les globules rouges masquent presque totalement la fine structure pulmonaire. Après la saignée, le poumon n’est certes pas dépourvu de sang, mais il en contient moins et l’observation histologique est beaucoup plus facile. La saignée ordinairement opérée par les marchands de volailles et qui consiste soit dans une section totale du cou, soit dans une section des artères linguales, à l’inconvénient d'entraîner toujours le passage d’une POUMON DES OISEAUX 217 certaine quantité de sang dans la trachée et même dans les bronches intrapulmonaires, ce qui peut gêner l’observation histologique et rendre les injections par la trachée souvent très difficiles, Pour l’étude anatomique, la dissection sur le frais est absolument insuffisante et ne donne que des résultats très incomplets. Les parois des sacs aériens qu’il importe de conserver pour les bien délimiter, sont sur le frais beaucoup trop molles et trop rétractiles pour conserver leur forme et leur disposition après qu'elles ont été partiellement sectionnées. Elles s’affaissent donc ou se déchirent de manière à ne plus laisser reconnaître leur situation ou leurs rapports. D’autre part, le sang contenu dans les oreillettes et dans les grosses veines est encore assez abondant pour gêner beaucoup la dissection et exiger des tamponnements avec l’ouate hydrophile qui déforment plus ou moins les parties. Par conséquent, pour faire une bonne dissection de l’appareil pulmonaire, donnant des résultats précis, relativement faciles à observer et permettant de conserver des pièces, il faut procéder de la manière suivante. Sur un oiseau tué comme il vient d’être dit et absolument intact d'autre part, on ouvre la trachée, on y met une canule de calibre convenable et on injecte lentement sous une faible pression de l’alcool à 80° qui pénètre dans le poumon, parcourt tout l’arbre bronchique dont les rameaux, continus entre eux lui laissent un passage facile et s’écoule dans les sacs aériens qu’il distend. Le remplissage de ces sacs est indiqué à l’extérieur par le gonflement du ventre et un léger écartement des ailes. On maintient alors la pression pendant quelque temps en se gardant bien de l’augmenter pour ne pas entraîner de ruptures, puis on lie la trachée, on enlève la canule et l’on plonge l’animal entier dans de l’alcool à 80°. On peut d’ailleurs, lorsqu'on veut faire une simple dissection, sans recherche histologique, remplacer l’alcool bon goût, par de l'alcool à brûler qui est meilleur marché et donne d’aussi bons résultats. Pour ces injections, j'ai utilisé appareil à pression continue L2 218 A. JUILLET indiqué dans la Technique histologique de VIALLETON (1909) et qui consiste en une soufflerie de thermo-cautère produisant dans un flacon rempli d'alcool une pression susceptible d’être réglée à volonté et que l’on peut évaluer avec un manomètre à mercure. Les pressions extrêmes employées évoluaient entre 8 et 10 centimètres de mercure. Au bout de vingt-quatre heures les parties sont devenues suffisamment résistantes pour que l’on puisse ne point craindre leur affaissement si l’on vient à ouvrir un sac aérien. On peut encore enlever les parties inutiles de l’oiseau pour éviter l’emploi d’une trop grande quantité d’alcool ou la pénétration difficile de ce liquide dans l’intérieur du corps et par suite une mauvaise conservation. On enlèvera donc par désarticulation les jambes et les ailes, on coupera le cou à sa partie moyenne et on ouvrira avec soin la paroi abdominale pour permettre à l'alcool de pénétrer aisément entre les différents viscères qu’il faudra d’ailleurs laisser en place jusqu’au moment de la dissection définitive. On peut aussi écarter légèrement avec le doigt quelques anses intestinales pour faciliter la pénétration de l’alcool ; mais il faut avoir grand soin de ne déchirer aucun des mésos qui cloisonnent la cavité viscérale. J’ai essayé de remplacer l'alcool par le formol, mais les résultats obtenus sont beaucoup moins bons. Le formol gonfle légerement le tissu conjonctif et ne lui donne point autant de résistance que l’alcool : il doit donc être rejeté dans ce cas. Comme je me suis servi pour la préparation des pièces histologiques d’un procédé de fixation sur place, par injection intra-pulmonaire faite dans les mêmes conditions que celle qui vient d’être dite, je l’indiquerai ici avec ses différentes modalités. 10 INJECTION DE NITRATE D'ARGENT. — Dans un oiseau préparé comme il a été dit, on injecte une solution de nitrate d'argent en se servant de l’appareil à pression continue. On maintientla pression pendant quelques minutes, puis on enfonce une canule piquante dans les sacs diaphragmatiques posté- POUMON DES OISEAUX 219 rieurs où abdominaux : le nitrate d'argent s'échappe par la canule. On fait alors passer à sa place un courant d’eau distillée puis un courant d’alcool et sans attendre davantage on coupe l’animal en arrière de la dernière côte : on enlève le thorax, les ailes, les jambes et le cou, et on plonge la pièce dans l’alcool où elle achève de se durcir. IL est inutile ici de laisser l’animal entier, puisqu'on n’a pas à faire de recherches anatomiques exigeant la conservation des viscères. On procéderait de même avec le liquide de Renaut (mélange picro-osmio-ar- gentique) mais sans faire de lavage à l’eau. 20 FIXATION AU LIQUIDE DE ZENKER. — [L'animal étant préparé pour l’injection, on pousse du liquide de Zenker dans la trachée et on lie cette dernière. S'il s’agit d’un petit oiseau, on le plonge en entier dans ce fixateur en se contentant d’enlever les jambes et les ailes. Douze heures après, on porte dans l’eau comme d’habitude, on passe dans l’alcool iodé puis dans les alcools successifs et on dégage le poumon que l’on conserve seul. 30 FIXATION AU LIQUIDE DE FLEMMING. — [’injection est faite avec du liquide de Flemming : puis l'animal, s’ilest petit, étant rapidement débarrassé des ailes et des pattes, sectionné en arrière de la dernière côte, est plongé dans le fixateur précité. On l’y laisse pendant plusieurs jours de façon à obtenir la décalcification du squelette ; puis on lave à l’eau, on déshydrate par l'alcool et on peut faire des coupes totales des poumons en place. MÉTHODE DES ALLIAGES FUSIBLES. — (Cette méthode donne pour le poumon des Oiseaux des résultats vraiment merveilleux. En effet, les bronches communiquant toutes entre elles et d’autre part les sacs aériens fournissant une issue facile à l'air contenu dans le poumon, les alliages fusibles injectés dans cet organe remplissent complètement les bronches et donnent des moulages tout à fait complets qui ont en outre l’avantage de pouvoir être conservés indéfiniment et étudiés à loisir, 220 A. JUILLET Différents alliages fusibles sont signalés par les auteurs. Un des plus connus est celui de Darcet dont la composition est la suivante en poids : Bisübhte ANNEE ER MERE Ur POMPES ERNEST 1p Pain enr 2 RNA RETAN 22 PROS 1p Cet alliage est fusible à 939 ; mais comme il faut forcément pour l’injecter, l’élever à une température supérieure d’au moins 30 à 40° au-dessus du point de fusion, il n’est pas très pratique. J’ai pensé l’améliorer par une addition de mercure, ainsi que différents auteurs l’ont nroposé. On peut ainsi en prenant : 2 p. de l’alliage initial 1 p. de mercure obtenir une masse fusible à 539 et qui par conséquent n’a pas besoin d’être élevée à une très haute température pour l’injection. J’ai employé avec succès cette masse ; mais, à cause des inconvénients que je vais rapporter, je l’ai bientôt délaissée pour une autre dont il sera question plus loin. D’abord et surtout, cette masse a une tendance à se dissocier en ses deux composants, mercure et alliage de Darcet, entre 30 et 400 ; de plus elle se ramollit suffisamment à ces températures pour que les moulages se déforment sous leur propre poids. C’est là un inconvénient très fâcheux d'autant plus que dans le traitement destiné à débarrasser les moulages de leurs parties molles, ces derniers doivent être portés à une température de 45 à 50°. D'autre part les moulages se brisent aisément même sous la pression des doigts surtout lorsqu'il s’agit de petits animaux. Enfin un dernier inconvénient, et du reste de beaucoup le moins important, réside en ce que les masses métalliques obtenues à l’aide de ce mélange et qui ont d’abord un éclat assez vif sont très rapidement ternies par l’air et se désagrègent. Le mélange auquel je me suis arrêté après divers tâtonnements et qui m'a donné les résultats les meilleurs est l’alliage de Wood dont la composition en poids est la suivante : nd be se ra POUMON DES OISEAUX Bismuth "ordinaire! &. /:.:1.....:.. Plomb'envbaguettes : 2:........... Etain en baguettes ............... Cadmium en grenaiïlle............. 221 Tip: 2p 2 p. 2 p. Cvt alliage est fusible vers 709. Pour l’employer on cominence par faire fondre le bismuth réduit en petits fragments, puis tenant les baguettes de plomb avec une pince en fer on projette sur elles la flamme d’un chalumeau de façon à les échauffer rapidement et à faire tomber le plomb fondu dans le bismuth en fusion. On procède de même avec l’étain. Le cadmium est alors versé dans la masse où il fond très rapidement. On brasse vigoureusement l’alliage avec une spatule en fer et on le maintient sur le feu à la température de 1209 constatée fréquemment avec un thermomètre ordinaire gradué à 2000 et bien desséché au préalable. Pendant ce temps, on prépare un oiseau comme il a été dit pour les injections ordinaires. Suivant la taille de l’oiseau, la canule mise dans la trachée est, soit une canule métallique d’un millimètre de diamètre au minimum, soit pour les oiseaux de plus forte taille, une canule plus volumineuse, les plus grosses étant naturellement les meilleures à la condition cependant qu’elles glissent librement dans la trachée sansla distendre. La partie libre de la canule doit dépasser longuement la trachée pour pouvoir être chauffée avec une flamme au moment de l’injection. Celle-ci est faite au moyen de l’appareil représenté dans la figure 1 et qui consiste tout simplement en un entonnoir de cuivre d’une capacité d'environ 80 centimètres cubes sur lequel s'adaptent à frottement dur des tubes de 18 à 20 centimètres de long également en cuivre, de calibres divers et dont l’extrémité inférieure peut entrer parfaitement dans la canule trachéenne ou dans la trachée elle-même. L’entonnoir est supporté par un anneau glissant le long d’une tige verticale et formant lui-même rampe à gaz, avec une série de trous de façon à chauffer tout le pourtour de l’entonnoir. L'appareil en place et l’oiseau destiné à être injecté étant DD AVJUILLET couché sur le dos près de la masse en fusion dans une cuvette r ; \| En Fig. I. — Appareil pour injections métalliques. — Ca, canule ; Cy, couronne à gaz; En, entonnoir ; G, gaz. à photographie, pour éviter l’écoulement du métal en cas de rupture on chauffe l’entonnoir avec la rampe à gaz et on promène le long du tube la flamme d’un tampon imbibé d’alcool POUMON DES OISEAUX 223 pour l’échauffer. La température de l’alliage étant alors de 1109, on le verse dans l’entonnoir de façon à remplir,ce dernier que l’on continue de chauffer selon le besoin ainsi que le tube conducteur. L’injection se produit d’elle-même comme le montrent bien vite le gonflement du ventre et l’écartement des ailes. Lorsque ces phénomènes se sont produits, on cesse de verser la masse et on laisse refroidir sans s’occuper de l’excès de masse restant dans l’entonnoir et dans le conduit, sa fusi- bilité permettant de l'enlever facilement après l’opération. Il est bon de ne pas toucher à l’oiseau pendant tout le temps nécessaire au refroidissement, c’est-à-dire une vingtaine de minutes. On porte ensuite la cuvette contenant l’oiseau sous un courant d’eau pour achever le refroidissement. Dès que ce résultat est obtenu, on commence la dissection de l’animal : on enlève les aïles, les pattes et l’on attaque avec précaution les viscères comprimés et diversement refoulés par les sacs aériens distendus. Ces derniers sont très inégalement injectés par la masse à injection et on ne trouve peut-être pas deux cas où le résultat soit semblable, l’un ou l’autre des sacs étant absolument rempli tandis que d’autres ne le sont que partiellement ; mais ce résultat qui serait très fâcheux si on étudiait les sacs aériens, à moins d'importance dans le cas qui nous occupe et il est en quelque sorte la rançon d’une bonne injec- tion pulmonaire : l'air refoulé dans certains sacs, servant de tampon et favorisant la circulation de la masse fluide dans les voies aériennes qui communiquent toutes entre elles. Au cours de la dissection de la pièce injectée, les sacs aériens se rompent d'habitude au niveau de leur pédicule et permettent ainsi de dégager aisément le poumon. $i quelques-uns d’entre eux ne se détachent pas spontanément, on peut toujours les enlever en coupant leur pédicule avec une scie fine d’horloger. Pendant la dissection, il importe de manier doucement la pièce pour que la rupture des gros sacs (abdominaaux et diaphragmatiques postérieurs) se fasse avec ménagement et sans entraîner de délabrement. Il est AROH, DE ZOOL, EXP, ET GÊN, — 5° SÉRIE, — T, IX, — (III), 15 224 À. JUILLET bon de débarrasser le plus possible le poumon des viscères qui l’entourent (cœur et foie), mais il faut éviter d’essayer d'extraire du moulage la colonne vertébrale et les côtes auxquelles le poumon adhère si étroitement qu’on ne saurait les séparer sans danger pour le moment. La pièce ainsi préparée et comprenant le poumon, les petits sacs aériens (diaphragmatiques antérieurs, interclaviculaire et cervicaux) et la colonne vertébrale avec la portion dorsale du thorax est maintenant portée dans une liqueur acide destinée à décalcifier le squelette. Cette liqueur est préparée avec : Acide chlorhydrique pur 30 centimètres cubes ANNE Re A0 — L'action de cette solution, pourvu qu’elle ne soit pas prolongée au delà d’un quart d'heure, n’attaque pas lalliage et suffit pour permettre d'enlever impunément les os qu’elle ramollit suffisamment et pour pousser assez loin le dégagement du moulage pulmonaire. Il ne reste plus, pour obtenir ce dernier à son état parfait, qu'à digérer les parties molles enveloppant le moulage et en particulier le parenchyme pulmonaire. On arrive très facilement à ce résultat en portant les pièces dans le mélange suivant: Pepsine amylacée titre 20... 0,20 centigranrmes RaUSAÉE CREER RER ES 120 grammes Acide chlorhydrique dilué à 30 9 ...... 3 grammes. La pièce est mise dans un cristallisoir avec la quantité de liquide suffisante pour la recouvrir et le tout est porté dans une étuve à 509, IT est très important de ne pas faire reposer directement le cristallisoir sur le fond même de l’étuve qui peut avoir une température supérieure à 500: pour cela, on interpose un corps peu conducteur comme une boîte en carton. Au bout de quarante-huit heures la digestion est achevée ou, en tout cas, suffisamment avancée pour que l’on puisse nettoyer complètement le moulage métallique en le lavant sous un faible filet d’eau et en le brossant très légèrement avec une brosse à dents savonneuse. On rince ensuite dans l’eau courante pour POUMON DES OISEAUX 225 di sl enlever tous les débris et pour faire disparaître les traces de savon qui pourraient oxyder ultérieurement les moulages. Les restes de squelette qui n’avaient pas été enlevés après la première décalcification sont maintenant faciles à extraire et la conservation de la pièce est définitive. J’ai pu constater d'autre part que l’attaque de l’alliage par le liquide acide employé pour la digestion était nulle : en effet la liqueur acide, après un contact de plusieurs jours à l’étuve avec de petits fragments de la masse à injection, ne présentait aucune réaction sensible des composants de l’alliage. La méthode de Wood donne des résultats vraiment remarquables, son emploi est très facile sur des animaux pris vivants et préparés comme il a été dit. Cependant comme je le faisais remarquer déjà au commencement de ce chapitre, les oiseaux tués à la chasse, ayant presque toujours leurs sacs aériens déchirés et leurs poumons envahis par le sang, sont impropres à ces injections. Il paraît nécessaire d’autre part pour le succès de cette méthode que le corps de l’animal soit encore chaud, car plusieurs essais faits sur des animaux tués depuis quelques heures ne m'ont point donné de résultat. 11 est probable qu’une fois le refroidissement du corps produit, la masse se prend même dans les gros troncs pulmonaires et arrête la pénétration de l'injection. Mais dans les conditions sus indiquées, elle doit être préférée à toute autre, VIALLETON et A. JUILLET (1911). Ceci fait surtout allusion à la méthode des injections à la photoxyline et à la celloïdine dont se servait G. FISCHER (1905). Cette méthode, très longue, donne des résultats incon- testablement inférieurs. Les pièces doivent être conservées dans des mélanges d’alcool, de glycérine et d’eau distillée et sont moins aisément maniables. Elles se prêtent moins bien aussi à la photographie que les moules métalliques. J’ai essayé également les injections au collodion, mais je n'ai pas poursuivi leur étude devant les résultats infiniment supé- 226 A. JUILLET rieurs que me donnait l’alliage de Wood. J’ai cru devoir néanmoins signaler la méthode employée par G. FISCHER, parce qu’elle est applicable à des pièces de collection conservées dans l’alcool. CHAPITRE RAPPORTS Rapports des PREMIER DES poumons POUMONS avec le squelette Le poumon des Oiseaux occupe la portion dorsale de la cage thoracique : il s'étend très peu du côté ventral de cette dernière et son bord latéral, qui descend le plus bas, n’atteint jamais l'articulation reliant la partie vertébrale avec la partie sternale des côtes. La forme particulière de cette portion de la cage thoracique exige quelques détails. En effet le corps des vertebres thoraciques offre sur sa face ventrale une crête sagittale plus ou moins saillante, et qui atteint sa plus grande longueur sur les vertèbres moyennes. Cette crête est formée par les apophyses ventrales des deuxième, troisième et quatrième dorsales qui, chez le Poulet, sont réunies entre elles en une lame osseuse qui s’interpose entre les deux poumons. Cette lame interpulmonaire est percée d’un trou entre la troisième et la quatrième vertèbre dorsale. L’apophyse ventrale des cinquième et sixième vertèbres reste indépendante dans chacune d’elles et ne se continue point avec ses voisines. La loge pulmonaire est constituée par les côtes et par la colonne vertébrale. On a vu que celle-ci forme dans sa partie ventrale une lame interpulmonaire qui sépare les deux poumons, mais elle contribue aussi à former la voûte de cette loge par l’intermédiaire d’une lame osseuse qui s’étend des vertèbres jusqu’au tubercule costal. Cette lame provient de l’ossification d’un ligament tendu entre les apophyses transverses des côtes. Elle forme, avec la lame résultant de la fusion des apophyses épineuses dorsales, une gouttière dans laquelle sont placés les muscles dorsaux. POUMON DES OISEAUX 227 Les côtes se divisent en fausses côtes qui ne se relient point au sternum et comprennent seulement une partie dorsale et en côtes vraies. Les fausses côtes, placées cranialement, sont au nombre de deux de chaque côté, chez le Poulet: la première très peu développée a la forme d’un simple stylet osseux , la seconde, plus longue, se rapproche par sa forme de la partie dorsale ou vertébrale des côtes suivantes, toutefois elle est encore peu développée, ses deux têtes sont peu séparées l’une de l’autre et son apophyse uncinée ne vient point s’appuyer sur la côte suivante comme elle le fait pour les troisième, quatrième et cinquième côtes. Les côtes vraies ont une forme absolument caractéristique pour les Oiseaux : elles comprennent deux parties, l’une dorsale ou vertébrale qui s’attache à la colonne, l’autre ventrale ou sternale qui s’attache au sternum. Ces deux parties de la côte sont toujours osseuses et s’articulent entre elles en faisant un angle obtus ouvert en avant. La portion dorsale de la côte, seule en rapport avec le poumon et même pas sur toute sa longueur, a la forme d’un quart de cercle et présente une extrémité proximale bifurquée en deux têtes. La tête dorsale ou tubercule s’articule avec l’apophyse transverse : elle se continue en dehors avec le corps de la côte qui est aplati dorso-ventralement. La tête ventrale, séparée de la dorsale par une vaste échancrure comblée pendant la vie par une lame fibreuse mince, s’articule avec le corps de la vertèbre qui présente, pour la recevoir, une légère saillie en forme de tubercule excavé à son sommet. Cette tête ventrale, contrairement à la précédente, est aplatie d'avant en arrière et se rattache au corps de la côte en formant une lame mince, perpendiculaire à la lame constituée par le corps, de telle sorte que sur une section perpendiculaire de la côte faite dans le voisinage de ses deux têtes, on observe la disposition connue sous le nom de fer à T. Pendant la vie le poumon entoure complètement la tête ventrale de la côte qui est comme enfouie dans son épaisseur, 228 A. JUILLET parce que les deux parties du poumon déprimées par la tête ventrale se rapprochent l’une de l’autre au-dessus de celle-ci, et viennent se toucher en se plaçant dans l’échancrure existant entre les deux têtes. La lame saillante qui s’étend de la tête ventrale à la tête dorsale et sur le coté externe de la côte, s’imprime à la surface du poumon en formant un sillon qui s’atténue progressivement en allant de dedans en dehors. Il y a chez le Poulet cinq côtes vraies (de la troisième à la septième incluse), dont les trois premières seules ont une apophyse uncinée ; les deux dernières dépourvues de cette apophyse sont en rapport, au voisinage de la colonne, avec lPilion qui atteint jusqu’au bord caudal de la sixième, tandis qu’il recouvre totalement la septième. Le poumon s’étend chez le Poulet adulte depuis la deuxième côte dont il atteint le bord caudal jusqu’en arrière de la sixième. Quatre côtes s’impriment fortement sur lui: les troisième, quatrième, cinquième et sixième (voy. fig. IT et fig. 14b). Dans d’autres cas, et en particulier chez les Oiseaux à corps plus allongé, le Canard, le nombre des côtes en rapport avec le poumon est plus considérable et s'étend jusqu’à six (fig. 31); il n’est que de cinq chez le Pigeon (fig. 20 b). Mais ces différences importent peu dans la structure générale qui reste la même dans ses traits essentiels. Forme des poumons La forme du poumon des Oiseaux que l’on peut étudier surtout sur les moulages métalliques (fig. 11 4, b, cet pl. XVIII à XX), présente ce caractère général que le poumon est toujours plus épais du coté médial en rapport avec la colonne, tandis qu'il s’amincit graduellement en s’avançant vers le dehors. Aussi, sur toutes les coupes transversales que lon peut faire d’avant en arrière, le poumon présente-t-il toujours l’aspect d’une faux, dont la pointe est tournée en dehors, le POUMON DES OISEAUX 299 talon en dedans, le dos du côté dorsal et le tranchant du côté ventral. En d’autres termes, le poumon présente trois faces : une face dorsale convexe, une face médiale à peu près plane, une face ventrale concave. Il faut remarquer toutefois que dans sa partie craniale, très courte, le poumon n'offre point cet aspect su les coupes : sa face ventrale n’est point ici concave, mais légèrement convexe. Cependant la brièveté de cette por- a FiG. II. — b c Poumon de Poulet Gallus domesticus (L.) ; moulage métallique (Darcet), Gr. nat. — dor«., face ventrale (poumon droit) ; b., face médiale (poumon gauche); e., face sale (poumon droit); an, circuits médiaux; an. #. anastomose transverse entre deux pente parabronches, B. bronche extra-pulmonaire; P.cr. pente craniale; P.ed., caudale ; $, e., sillons costaux et trous costaux. tion craniale, permet de conserver la description ci-dessus qui répond à la plus grande partie du poumon. La FACE DORSALE (c, fig. 11) est marquée par les sillons costaux. La direction de ces sillons est, comme celle des côtes, oblique de dedans en dehors et d'avant en arrière. Leur nombre varie naturellement avec celui des côtes rencontrées par les poumons. La FACE MÉDIALE (b, fig. 1) est verticale, en rapport avec la colonne vertébrale et la lame osseuse interpulmonaire dont nous avons parlé. Chez les types à poumon court, comme le Poulet par exemple, cette face est à peu près quadrilatérale à angles mousses, c’est-à-dire que son épaisseur est sensible- 230 A. JUILLET ment la même à ses extrémités qu’en son milieu. Dans les poumons plus allongés, comme celui du Canard, l’épaisseur va s’atténuant graduellement, depuis le milieu jusqu'aux extrémités craniale et caudale de cette face. Elle est occupée par des bronches verticales, de direction dorso-ventrale, toutes parallèles entre elles, et présente des trous arrondis, répondant aux têtes ventrales des côtes et circonscrits par les bronches verticales dont il a été parlé, ces bronches s’écartent au niveau des côtes pour les former, puis se rapprochent en dessus et en dessous d’eux, de façon à enfouir la tête costale dans le tissu pulmonaire. La FACE VENTRALE du poumon (a fig. 11), bien qu’elle soit d’une manière générale concave par rapport à la face dorsale, est en réalité assez compliquée et forme une pyramide triangulaire dont le sommet répond à l’entrée de la bronche dans le poumon : un des pans est formé par la face cranio-latérale du poumon un autre par une face médio-ventrale s’étendant de la bronche extra-pulmonaire à la face médiale ci-dessus décrite, le dernier enfin par la face caudale. Mais comme la face médiale de cette pyramide est très courte et que, au point de vue des rapports, tout ce qui est en avant de la bronche se comporte bien différemment de tout ce qui est situé caudalement à elle, on peut, pour plus de simplicité, diviser cette face en deux parties seulement : l’une craniale ou prébronchique, l’autre caudale ou postbronchique. Comme d’une manière générale aussi, le point de pénétration de la bronche dans le poumon répond à la partie la plus saillante de cette face ventrale et qu’à partir de lui toute la partie craniale du poumon est inclinée d’arrière en avant, et du ventre au dos tandis que la portion postbronchique est inclinée en sens inverse, on peut décrire simplement dans la face ventrale deux pentes, l’une craniale située en avant de la bronche, l’autre caudale placée derrière celle-ci. Ces deux parties, très visibles sur la fig. 14 a, sont d'autant plus légitimement distinguées que leurs rapports sont, comme on le verra plus loin, tout à fait POUMON DES OISEAUX 231 différents. Pour le moment on dira simplement que la pente craniale du poumon a une étendue assez variable dans les différents types. Elle est courte et presque verticale dans les poumons courts, comme celui du Geai (fig. 23 «). Elle est au con- traire allongée et faiblement inclinée, comme dans le poumon très étendu du Canard (fig. 30 et 32). Elle peut du reste offrir une disposition un peu différente chez l’oiseau qui vient d’éclore et chez l’adulte. Aïnsi, chez le petit Poulet à la naissance (fig. 12 a), elle est courte et verticale comme chez le Geai. Chez l’adulte, elle est faiblement inclinée et assez longue, ce qui est en accord avec le mode d’accroissement du poumon, qui paraît après la naissance s'étendre surtout du côté cranial, suivant gement de forme de l’ensemble du tronc, le chan- qui, d’abord très ramassé, s’allonge ensuite comme on sait (fig. 11 & - fig. 14 à). J’ai déjà signalé cette forme de la face ventrale du poumon des Oiseaux, (A. JUILLET, 1911. b). Diaphragme ornithique et plèvre Il importe de bien préciser les rapports des poumons avec les parties qui les entourent, afin de fixer dès maintenant certains points très intéressants pour la physiologie de ces organes. Les poumons sont logés exclusivement, comme on l’a vu, dans la portion dorsale de la cavité thoracique et ne s’avancent que très peu sur les côtés de celle-ci. On a vu aussi que les côtes s’enfonçaient profondément dans le tissu pulmonaire puisque leurs têtes ventrales sont entièrement enfouies dans ce dernier. Cette disposition, la présence des sillons costaux profonds et bien marqués, permettent déjà de penser que les poumons ne jouent point dans la cage thoracique comme ils le font chez les Mammifères et conduit à envisager la constitution des plèvres ornithiques. Cette constitution a été comprise de manières bien différentes par les divers auteurs qui ont traité ce sujet et dont les conceptions se ressentent forcément de l’état de la science au moment où ils ontécrit. Nous ne referons pas l’historique de ces différentes 232 A. JUILLET opinions qui à été déjà fait par BERTELLI (1905) et qui est sans intérêt, étant donné l’état très précaire des connaissances embryologiques à l’époque où toutes ces opinions contradictoires ont été émises. Aujourd’hui, grâce aux connaissances acquises sur le développement de la région thoracique, il est aisé de faire disparaître toutes ces contradictions et d’avoir une idée claire de la manière dont les choses, partant d’un état initial très voisin de celui que l’on observe chez les autres Amniotes où les poumons sont libres dans des cavités pleurales, sont arrivées à l’état qu’elles montrent chez les Oiseaux où les poumons sont plus ou moins soudés à la cage thoracique. Nous suivrons pour faire cet exposé, le résumé qu’en a donné M. VIALLETON (1910, p. 122) et qui permet de comprendre la disposition des principaux sacs aériens, telle qu’elle est aujourd'hui admise. « Chez l'embryon de huit jours, les poumons se sont déjà développés et chacun d’eux, bourgeonnant surtout du côté dorsal du méso latéral qui le renferme, présente une surface dorsale convexe et une surface plane tournée du côté ventral. Cette dernière est occupée par une épaisse lame mésodermique dérivée du méso latéral et qui vient se souder aux parois latérales du corps, soit directement, soit par l’intermédiaire des plis des reins primitifs ou du septum transverse suivant les points. Les poumons sont donc placés maintenant de chaque côté de la ligne médiane dans une cavité limitée en dedans par le mésentère dorsal, latéralement par les parois du corps, ventralement par la lame mésodermique épaisse dont on vient de parler et qui, continue en avant avec la paroi dorsale du péricarde, est libre en arrière où elle s’arrête tout d’abord. Cette lame laisse ainsi pendant un certain temps un orifice étroit qui fait communiquer la cavité renfermant le poumon avec celle qui contient les viscères abdominaux. On peut la comparer au septum décrit plus haut chez les Tortues. Elle se continue plus tard en arrière, de manière à fermer complètement la cavité réservée au poumon. POUMON DES OISEAUX 233 Elle à reçu de BErTeLrt le nom de diaphragme primaire. « Bientôt la disposition des parties change profondément par le développement des sacs aériens. Ces derniers sont des diverticules pulmonaires qui, comme l’a montré BERTELLTr, se développent à partir des bronches dans l'épaisseur du diaphragme primaire entraînant au-devant d’eux la face ventrale de ce dernier recouverte par le péritoine, tandis que sa face dorsale, le poumon, en contact avec continue à former un septum horizontal ayant les mêmes dispositions que le diaphragme primaire et qui constitue le diaphragme définitif. Ce dernier s’étend de la ligne médiane à la paroi latérale du coxps de chaque côté, séparant complètement les poumons, logés en dessus de lui, du reste de la cavité du tronc. Ce septum présente sur ses bords réunis à la paroi thoracique de petits faisceaux musculaires capables de l’abaisser légèrement ; il a reçu de SAPPEY le nom de diaphragme pulmonaire, BeRTELLI le nomme diaphragme ornithique. « Les sacs aériens s’étendent, d’une part, entre le diaphragme définitif contre lequel ils s’appliquent étroitement par leur face dorsale qui se confond avec lui et que leur épithélium tapisse, d'autre part ils s’accolent aux parois du corps ou à celles de différents viscères, comme on va le voir. « Les sacs aériens sont au nombre de cinq de chaque côté. On peut les diviser (BERTELLI) en trois groupes ; un groupe supérieur, comprenant deux sacs placés dans la partie craniale de la cavité du tronc (sacs supérieurs) ; un groupe moyen, composé également de deux sacs, et un groupe inférieur comprenant un seul sac. « Les sacs du groupe supérieur sont placés l’un dorsalement, l’autre ventralement. Le sac dorsal, petit, se glisse en avant et, dépassant la clavicule, vient se placer à la base du cou. Les deux sacs droit et gauche de cette paire constituent les sacs cervicaux de SApPpey. Les deux sacs supérieurs ventraux prennent un grand développement et occupent le côté ventral de la partie antérieure ou craniale du thorax. Ils se moulent 234 A. JUILLET sur les viscères contenus dans cette cavité, les entourent et les écartent des parties dont ils étaient primitivement voisins. Ils se réunissent sur la ligne médiane en s’accolant l’un à l’autre, et la lame formée à ce niveau par leurs parois fusionnées se résorbe, de telle sorte que leurs cavités se confondent en une seule, le réservoir interclariculaire de SAPPEY (considéré par lui comme un sac aérien impair). La trachée, les gros vaisseaux traversent librement cette cavité dans laquelle ils sont comme suspendus pour la raison indiquée plus haut. Le réservoir interclaviculaire émet un diverticule important, qui franchit la paroi latérale du thorax et vient s'épanouir entre elle et le muscle grand pectoral. On verra plus loin son rôle dans la respiration pendant le vol. « Les sacs moyens de BERTELLI, appelés aussi sacs diaphragmatiques (SAPPEY), sont au nombre de deux de chaque côté. Le sac moyen supérieur (diaphragmatique antérieur) forme une petite loge quadrangulaire sur les coupes frontales, et qui est placée entre la paroi du corps et le péricarde. Le sac moyen inférieur (diaphragmatique postérieur) plus allongé, le suit en arrière et s'étend assez loin au delà du bord du sternum. Les parois médiales de ces deux sacs sont situées exactement dans le prolongement l’une de l’autre et forment de chaque côté une lame verticale qui s’étend obliquement de la partie moyenne du péricarde en avant jusque vers l’extrémité postérieure du corps. Les viscères abdominaux sont donc refoulés sur la ligne médiane entre ces deux lames qui les séparent en même temps du diaphragme définitif et qui complètent avec ce dernier la paroi de la cavité péritonéale du côté cranial. A cause de cela on considère ces deux lames comme faisant partie de l’appareil cloisonnant du cœlome et on les a comparées au diaphragme. Ce dernier était alors formé chez les Oiseaux de deux parties, l’une pleurale répondant au diaphragme pulmonaire, l’autre abdominable représentée par la paroi médiale des deux sacs moyens et qui a reçu le nom de diaphragme thoraco-abdominal. Cette dénomination explique POUMON DES OISEAUX 235 l’épithète de «diaphragmatiques » donnée aux sacs moyens qui contribuent par leur paroi médiale à former cette cloison. «Le sac inférieur forme de chaque côté une poche très allongée qui dépasse encore en arrière les sacs moyens postérieurs, et atteint jusqu'aux vertèbres caudales. Il se place à la face ventrale des reins définitifs qu’il sépare des viscères intestinaux sous-jacents. «Les parois de ces différents sacs forment done des cloisons qui subdivisent d’une manière compliquée la cavité du tronc. Celles des sacs interclaviculaires et moyens antérieurs accolées limitent en arrière la cavité thoracique craniale dans laquelle sont placés la trachée, les gros vaisseaux et la base du péricarde. Les sacs moyens limitent de leur côté deux grandes cavités latérales droite et gauche dont chacune est cloisonnée par une lame formée par l’accolement de la paroi caudale du sac moyen antérieur avec la paroi craniale du sac moyen postérieur et une loge médiane dans laquelle sont renfermés les viscères abdominaux. Par conséquent, les parties antérieure et dorsale de la cavité du tronc des Oiseaux sont entièrement remplies d'air provenant soit des sacs supérieurs, soit des poumons et des cavités aériennes également ménagées sur les côtés (sacs moyens) et sur la paroi dorsale (sacs inférieurs) de la cavité viscérale de telle façon que le poids des viscères abdominaux est entièrement réparti sur la ligne médiane et du côté ventral. » Au début, la face médiale et la face dorsale du poumon embryonnaire sont absolument libres et indépendantes de la paroi thoracique qui les environne. Il existe une véritable cavité pleurale, mais celle-ci est située tout entière du côté dorsal du diaphragme primaire. Au fur et à mesure que l’oiseau se développe, cette cavité pleurale s’atrophie peu à peu ; des points de contact nombreux apparaissent entre la face dorsale des poumons et la paroi thoracique ; des adhérences s’établissent et l’endothélium pleural disparaît sur la plus grande partie de son étendue 236 A. JUILLET pour ne persister qu’en de rares points où la fusion des deux parois viscérale et thoracique n’a pas été complète. BERTELLI (1905 p. 827) donne pour les plèvres la description suivante : « Les poumons sont adhérents et unis plus ou moins étroitement aux parois de ces cavités (cavités pleurales). La plèvre pariétale comprend deux portions, costale et médiastinique, et il n’y a pas de plèvre diaphragmatique à cause de la fusion du poumon et du diaphragme. Les surfaces dorsales et médiastinales des poumons sont adossées et unies aux parois de la cavité thoracique. L’adossement se rencontre sur toute la surface, l’union s'effectue ou bien par des lamelles et des filaments conjonctifs qui laissent entre eux des surfaces libres des feuillets pleuraux ou par une fusion plus étendue de ces feuillets. Chez les Gallinacées la surface dorsale est unie au thorax par des lamelles ou filaments peu nombreux qui laissent de grandes surfaces libres de la cavité pleurale. Au voisinage du bord latéral du poumon les deux plèvres se fusionnent. En dehors des points ainsi fondus tout le reste de la plèvre est libre, revêtu d’épithélium. Dans les oiseaux où l’adhérence des plèvres se fait par fusion celle-ci, très étendue, est variable. Chez Numenius arcuata la surface des poumons est lâchement unie à l’extrémité supérieure des côtes où elle est lisse et largement revêtue d’épithélium. Il y a aussi des aires d’épithélium chez la Pie. Chez le Pigeon, la surface dorsale est fusionnée : il existe des aires épithéliales abondantes dans les sillons costaux, très rares ailleurs. Dans d’autres Oiseaux, la fusion est étendue et on ne peut mettre en évidence que de rares aires épithéliales (Anas boschas, Passer Italiæ, Hirundo rustica). La couche connective qui unit les poumons au thorax est bien développée dans Buteo vulgaris et encore mieux dans le Cygne. Chez tous les individus la partie sillonnée par les côtes est unie moins intimement. POUMON DES OISEAUX 237 Sur le médiastin, c’est la même chose. » La partie de la cavité pleurale appelée par BERTELLI « médiastine » répond à notre face médiale. Il importe de faire remarquer les différences qu’elle présente avec la partie médiastinale du poumon des Mammifères, et qui sont dues à la constitution spéciale du squelette thoracique. En effet, tandis que chez les Mammifères les poumons dépassent largement du côté ventral la colonne vertébrale au devant de laquelle se placent l’aorte et l’œsophage avec lesquels ils entrent plus ou moins en rapport, chez les Oiseaux, à cause de la crête osseuse ventrale placée sur le corps des vertèbres thoraciques, les organes qui étaient en rapport chez les Mammifères avec ces corps de vertèbres sont fortement repoussés du côté ventral. Comme la face ventrale des poumons ne dépasse point le bord de cette crête osseuse, il en résulte que l’aorte et l’œsophage au lieu d’être logés entre les deux poumons sont placés tout à fait ventralement par rapport à ceux-ci, et n’ont rien à faire avec leur face médiale qui est absolument contiguë avec celle du poumon opposé dont elle n’est séparée que par les crêtes osseuses. Ainsi sont déterminés les rapports des faces dorsales et médiales des poumons. Ceux de leur face ventrale sont beaucoup plus compliqués à cause du nombre des viscères avec lesquels elles entrent en contact. Rapports de la face ventrale des poumons Pour étudier convenablement les rapports de la face ventrale des poumons, il faut employer la méthode indiquée p. 216, c’est-à-dire, se servir d’un animal préalablement injecté à l'alcool et durcei par un séjour d’au moins quarante huit heures dans ce réactif. Les muscles des ailes et le bréchet ayant été enlevés avec soin, on ouvre le thorax en détachant avec beaucoup de précautions le plastron sternal, sciant avec une scie d’horloger les parties dures, de façon à suivre très exactement 238 A. JUILLET la face profonde du sternum et à ne point déchirer les cloisons séparatives des sacs qui viennent s’y attacher. Il est bon d'enlever d’abord seulement la partie caudale du sternum, en laissant intacte sa partie craniale en rapport avec les côtes et les coracoïdes ; puis on désarticule ces derniers et on les enlève en même temps que l’omoplate. Les viscères sont alors suffisamment dégagés pour que l’on ne risque point de déchirer leurs enveloppes ou leurs ligaments lorsqu'on achèvera l’ouverture du thorax, ce qui se fait en enlevant à la scie d’horloger le reste du sternum (1). On voit alors le péricarde qui est à peine ouvert sur sa face ventrale si l’opération a été bien conduite, puis, en avant du péricarde on découvre une sorte de cavité qui s’étend de chaque côté de la base du cœur, jusque sur les côtés du cou et qui répond au sac interclaviculaire. De chaque côté du péricarde on distingue deux lames qui s’insèrent sur lui à peu près au niveau de la base des ventricules et se dirigent de là, d’avant en arrière et de dedans en dehors. Ce sont les diaphragmes thoraco-abdominaux qui limitent en dedans les sacs diaphragmatiques antérieurs et postérieurs qui ne se distinguent tout d’abord pas l’un de l’autre parce qu’ils ne sont point ouverts. Entre le diaphragme thoraco-abdominal de chaque côté et le péricarde, se loge le lobe correspondant du foie qui remonte ainsi très loin du côté du cœur dont la pointe est entourée par les lobes hépatiques. Cette disposition mérite d’être signalée comme un trait reptilien de la topographie viscérale des Oiseaux En effet, elle ne se rencontre jamais chez les Mammifères où le cœur est toujours nettement séparé du foie par le diaphragme et n’est point entouré de tissu hépatique. Pour poursuivre l’étude des rapports, il faut maintenant enlever la plus grande partie du cœur et du foie puis ouvrir (1) Pour suivre cette description, il est bon de s’aider de la figure de STRASSER, reproduite par WIEDERSHEIM (fig. 297, 1906) et par VIALLETON (fig. 76, 1911) ou encore de la figure 3, pl. IV de SAPPEY (1847). N'ayant pu mieux faire que ces auteurs, dont les dessins ne sont d’ailleurs pas parfaits à cause des difficultés résultant des reliefs et de la transparence de certaines parties, comme a paroi dorsale du sac interclaviculaire, j’ai renoncé à donner une figure personnelle, POUMON DES OISEAUX 239 les sacs diaphragmatiques. Mais comme il importe avant tout de ne point déchirer les fines membranes qui cloisonnent la cavité viscérale, il faut enlever les organes avec beaucoup de précautions et le meilleur moyen d’opérer consiste à morceler les viscères en petits fragments que l’on enlève à la pince. On écarte ainsi les ventricules tout entiers, en gardant les gros troncs aortiques et pulmonaires dans leur trajet intrapéri- cardique et en conservant les oreillettes avec leurs orifices veineux. Le sang coagulé qui les remplit est enlevé par fragments à la pince et de cette façon les embouchures des gros troncs vasculaires restent parfaitement en place, tandis que le péricarde transparent laisse voir les parties placées sur son côté dorsal. Les lobes droit et gauche du foie sont enlevés de même par morcellement. Enfin les sacs diaphragmatiques sont ouverts. On voit dès lors que, en dehors du péricarde, les parties en rapport avec la face ventrale des poumons sont avant tout les sacs aériens avec les viscères qui les traversent comme c’est le cas pour le sac interclaviculaire. SAC INTERCLAVICULAIRE. — Le sac interclaviculaire, formé comme on sait par la réunion sur la ligne médiane de deux sacs primitivement distincts, occupe toute la partie ventrale de la cavité thoracique comprise entre la base du péricarde d’une part, et la clavicule de l’autre. Il se prolonge un peu latéralement du côté caudal, de chaque côté de la base du péricarde ou du cœur qu’il entoure jusque vers sa partie moyenne; il envoie aussi en dehors du thorax un prolongement très important, le sac sous-pectoral, mais nous n’avons pas à nous occuper de ce dernier. Il est limité en arrière dans sa partie médiale par le péricarde, dans ses parties latérales par une cloison formée par l’accolement de sa paroi avec celle du sac diaphragmatique antérieur. Sa voûte ou son toit, que l’on voit de face sur l’animal couché sur le dos, est à peu près plane, mais est parcourue par divers organes plus ou moins saillants sur elle et qui lui sont rattachés par des mésos complets ou fenêtrés. ARCH. DE ZOOL. EXP. ET GÉN. — 5€ SÉRIE, — T. IX. — (III). 16 240 A. JUILLET Sur la ligne médiane, on trouve d’abord la trachée fortement saillante et accolée à l’œsophage et aux muscles du cou que recouvre la paroi du sac interclaviculaire. Puis vient la bifurcation de la trachée en ses deux bronches extrapulmonaires qui, elles, passent librement sur la paroi dorsale du sac sans contracter adhérence avec lui. Avant la bifurcation de la trachée, on voit s’insérer sur elle deux muscles obliques de dedans en dehors et d'avant en arrière, qui traversent librement la cavité du sac interclaviculaire pour venir s’insérer sur la paroi latérale de la cage thoracique. Ce sont les muscles du syrinx. Ils constituent les organes les plus ventraux contenus dans la cavité du sac et se placent ventralement au tronc de l’artère axillaire. En dehors de la trachée et de ses muscles, la cavité du sac est encore traversée par les deux troncs artériels brachiocéphaliques qui font saillie en dedans d’elle jusque vers leur bifurcation en artère carotide et artère axillaire, les carotides se confondent peu à peu avec la paroi dorsale du sac. Plus dorsalement se trouvent les troncs droit et gauche des veines caves supérieures, qui font moins saillie dans la cavité du sac et sont plus intimement soudés à sa paroi dorsale. En avant, sur le toit, se trouve la glande thyroïde recouverte par la paroi dorsale du sac mais facile à reconnaître cependant à la saillie ovalaire qu’elle forme entre la veine jugulaire et la carotide. Toute la partie de la paroi dorsale du sac interclaviculaire, située en avant du tronc artériel brachio-céphalique n’est point en rapport avec le poumon ; elle est tendue ventralement à la cavité des sacs cervicaux qui occupent la portion dorsale et craniale de la cage thoracique. Le sac interclaviculaire n’est en rapport avec le poumon que sur une partie très limitée de ce dernier, à savoir au niveau du cul-de-sac latéral qu’il forme de chaque côté de la base du péricarde. Là, sur une très courte étendue comprise entre la paroi postérieure du sac interclaviculaire et la veine cave supérieure continuée par la veine axillaire, le toit du sac interclaviculaire s'applique sur la partie de la pente craniale qui confine immédiatement à l’arête POUMON DES OISEAUX 241 la séparant de la pente caudale. Mais ce contact est limité à une faible portion du poumon, la plus grande partie de la pente craniale étant en rapport immédiat avec le sac cervical du même côté, ce qui nous conduit à étudier la disposition des SACS CETVICAUX. SAC CERVICAL. — Il est facile d’apercevoir à travers la mince paroi dorsale du sac interclaviculaire la cavité des sacs cervicaux situés de chaque côté de la base du cou. Pour étudier ces sacs, on introduit d’abord dans leur cavité une sonde cannelée, que l’on y fait pénétrer par l’orifice créé dans ces sacs par la section transversale du cou, faite un peu en avant du thorax. On fait saillir la paroi du sac cervical avec l'extrémité mousse de la sonde et l’on se rend compte ainsi de l’étendue de chaque sac cervical, puis on coupe sur la sonde cannelée la membrane qui sépare les deux sacs interclaviculaire et cervical l’un de autre, et de chaque côté apparaît alors la cavité du sac cervical. Les rapports des sacs cervicaux sont un peu différents à droite et à gauche : à droite, le sac recouvre la plus grande partie de la pente craniale du poumon, puis s’étend entre la base du cou et l’extrémité de la cage thoracique par laquelle il passe dans le cou. Il est limité en dehors par la partie craniale de la cage thoracique, en dedans par l’œsophage rattaché aux muscles prévertébraux par un court méso. En arrière il présente un petit cul-de-sac latéral court qui s’applique contre la convexité de la crosse aortique et revêt la face dorsale de la veine cave supérieure droite ; en dedans il se prolonge beaucoup en arrière sous la forme d’un conduit cylindrique large qui s’enfonce entre la face médio-ventrale du poumon droit et l’œsophage (voir fig. III). A gauche, la portion craniale du sac est en rapport avec le bord gauche de l’œsophage rejeté plus fortement à droite, le sac paraît moins volumineux et présente comme le sac correspondant deux culs-de-sac postérieurs, l’un latéral plus petit, s'étendant dorsalement au tronc de la veine cave supérieure de conduit, glissant sur le côté de gauche, l’autre en forme l’œsophage et terminé en 242 A. JUILLET cul-de-sac comme son homologue du côté opposé. Ce conduit est séparé du conduit droit par le méso œsophagien dont il à été question. Mais par suite de la situation plus à droite de l’œsophage, le méso s’insère sur le côté gauche de cette portion du tube digestif, de ‘sorte que la paroi ventrale de ce cul-de-sac est moins nettement formée par l’œsophage qu’elle ne l’est du côté droit. Les deux sacs cervicaux peuvent communiquer entre eux sur la ligne médiane, dorsalement à l’œsophage, comme la déjà dit CAMPANA (1875). Cette situation du sac cervical sur le côté dorsal du sac interclaviculaire est très importante et doit être bien retenue pour comprendre exactement les rapports de la plus grande partie de la pente craniale du poumon. Cette disposition tout à fait dorsale du sac cervical est bien exprimée par le nom même imposé à ce sac par BERTELLI qui l’a appelé « sac supérieur dorsal » mais qui néanmoins l’a méconnu dans sa figure 1 Tav. LXXIX qui représente le diaphragme ornithique. NaraLts GUILLOT (1846) seul a bien représenté ce point important de la disposition des sacs, lorsque dans sa figure 2, PI. IV, de beaucoup la plus exacte que nous possédions du toit du sac interclaviculaire, il montre, en incisant ce dernier du côté gauche, la cavité du sac cervical qui lui est sous jacent. La pente craniale du poumon est assez courte, elle ne s’étend pas au delà du tronc de l’artère axillaire qui forme sa limite antérieure, sa surface n’est point concave et lisse comme celle de la pente caudale, mais elle présente une saillie médiane formée par la bronche qui donne naissance au sac cervical antérieur. De plus, sur le poumon droit, elle présente à son bord médial l'empreinte très marquée de la crosse aortique. SAC DIAPHRAGMATIQUE ANTÉRIEUR. — Sur les coupes frontales qui ouvrent largement la cavité du sac diaphragmatique antérieur, ce dernier a la forme d’un rhomboëdre présentant un bord cranial et un bord caudal obliques de dedans en dehors POUMON DES OISEAUX 243 et d'avant en arrière, puis deux bords, latéral et médial, paral- lèles entre eux. Ce’sont la paroi latérale du thorax d’une part, le diaphragme thoraco-abdominal d'autre part. Le sac dia_phragmatique antérieur recouvre à peu près toute la pente caudale du poumon, le sac diaphragmatique postérieur n’étant en rapport avec elle que par une partie très étroite, sur le bord latéral et caudal de cette face, comme on peut le voir sur la fig. III qui représente schématiquement les rapports des sacs avec la face ventrale. Le toit de la cavité du sac diaphragmatique antérieur a donc la forme d’une voûte à concavité tournée ventralement et qui répond à la pente caudale du poumon. Cette pente est en rapport avec la face dorsale des lobes hépatiques immédiatement sous-jacents aux sacs et qui, lorsque ceux-ci ne sont pas distendus d’une manière forcée, doit se mouler en quelque sorte sur le poumon dont elle n’est séparée que par une mince lame d’air. OWEN (1835 et 1866) avait signalé ce rapport des sacs diaphragmatiques en l’exprimant par le terme de «sacs hépatiques » à l’aide duquel il les désignait. (La paroi craniale des sacs diaphragmatiques antérieurs mérite d'attirer l’attention premièrement parce qu’elle est en rapport très intime avec la crête qui sépare les deux pentes pulmonaires l’une de l’autre, et a par conséquent une relation topographique très importante avec la configuration générale du poumon, en second lieu, parce que dans sa partie dorsale et médiane, elle donne passage au conduit aérien du sac interclaviculaire. On peut lui décrire deux parties. L’une latérale qui s’étend de la paroi thoracique au péricarde, et qui répond au bord cranial oblique du rhomboëdre, formé par la coupe frontale du sac diaphragmatique antérieur, l’autre dorso-médiale, comprise entre les vaisseaux pulmonaires et le péricarde, et qui donne précisément passage au conduit aérien sus-nommé. La partie latérale formée par l’adossement de la paroi caudale du sac interelaviculaire et de la paroi craniale du sac diaphragmatique antérieur ne nécessite pas de description spéciale. 244 A. JUILLET C’est une lame oblique simple. Mais il n’en est plus de même de la paroi dorso-médiale qui est extrêmement compliquée. Elle est formée en effet par le paquet vasculaire qui s’étend du Cramal Lateral Ra Rda. O.ab. Fi. ui. — Caudal Schéma montrant les rapports des sacs aériens et de leurs orifices avec la face ventrale du poumon chez le Poulet. Gr. = 1 x 1,5. — B. bronche extra-pulmonaire ; C.d.p., canal direct du sac diaphragmatique postérieur ; C'4., canal du sac interclaviculaire; E,, E,, E,, E,, première, deuxième, troisième et quatrième entobronches ; Mes. mésobronche ; O.ab., orifice direct du sac abdominal; O.c., orifice direct du sac cervical ; O.d.a., orifice direct du sac diaphragmatique antérieur ; O.d.p., orifice direct du sac diaphragmatique postérieur ; R.ab., orifices récurrents du sac abdominal; ÆR.d.a., orifice récurrent du sac diaphragmatique antérieur ; ÆR.d.p., orifices récurrents du sac diaphragmatique postérieur ; R.i., orifice récurrent du sac interclaviculaire. cœur au poumon, et qui comprend : l’artère pulmonaire cra- nialement, la veine pulmonaire du côté caudal et entre les deux la bronche extrapulmonaire qui pénètre avec les vaisseaux dans le poumon. De plus elle est incomplète, puisqu'elle présente comme on l’a déjà dit un orifice, le conduit aérien du sac POUMON DES OISEAUX 245 interclaviculaire, qui mène dans ce dernier et fait communiquer les deux sacs interclaviculaire et diaphragmatique antérieur (voy. fig. [IT). Pour voir cette communication, on coupe les gros troncs artériels transversalement au niveau de la bifurcation de la trachée et on les relève de façon à mettre en évidence ce qu'ils cachaient. On aperçoit alors la face dorsale du péricarde séparée des bronches extrapulmonaires et des organes sous-jacents à celle-ci par un espace appartenant au sac interclaviculaire. Cet espace continu et impair dans sa portion craniale qui fait immédiatement suite à la bifurcation de la trachée, est au contraire cloisonné dans sa partie caudale par une lame médiane qui s’attache d’une part au péricarde, d’autre part au tube digestif. Du côté caudal, cet espace se prolonge par une sorte de canal ou de tunnel, qui passe dorsalement à la veine pulmonaire et qui vient s'ouvrir dans le sac diaphragmatique antérieur au niveau de son orifice dont il semble constituer en quelque sorte la continuation. C’est le conduit aérien du sac interclaviculaire ou canal interclaviculaire. I a été décrit pour la première fois par CAMPANA (1875). On le voit bien sur la fig. 17 où il se présente sous la forme d’une tige verticale placée en dedans de la bronche extra-pulmonaire. Il est représenté également d’une manière schématique dans les fig. XV, XVI et XVII. Pour terminer la description de la paroi caudale du sac interelaviculaire, il ne reste plus qu'à mentionner sa partie médiale située en dedans du canal interclaviculaire et qui est constituée en majeure partie par la portion craniale de la base du péricarde. Il est à remarquer que la bronche extrapulmonaire passe librement en avant de cette paroi, ce qui, étant donnée l’indépendance de la bronche du côté dorsal, indique que cette bronche a du être enserrée entre deux bourgeons du sac interclaviculaire placés l’un dorsalement, l’autre ventralement à elle, et qui ont été certainement indépendants durant un certain temps, avant l'accroissement des cavités qu’ils renfer- 246 A. JUILLET maient et leur confluence autour de la bronche qu’elles enveloppaient. L’embryologie confirmera d’ailleurs cette interprétation. SAC DIAPHRAGMATIQUE POSTÉRIEUR. — Le sac diaphrag- matique postérieur a, sur les coupes frontales une forme triangulaire avec une base tournée cranialement, et un sommet caudal. La base est formée par l’accollement de la paroi caudale du sac diaphragmatique antérieur avec la paroi craniale du sac diaphragmatique postérieur. Elle n’est autre que le bord postérieur du rhomboëdre précédemment décrit. Le bord médial est formé par le diaphragme thoraco-abdominal, le bord latéral par la paroi thoracique. La voûte de ce sac n’est en rapport, comme il a été dit, qu'avec une étendue très restreinte de la face ventrale du poumon et en particulier avec un petit espace triangulaire dont la base est tournée du côté de la paroi thoracique et dont le sommet est sur le bord caudal du poumon (voy. fig. IT). SAC ABDOMINAL. — Le sac abdominal, situé dorsalement par rapport à ceux qui viennent d’être étudiés jusqu'ici, n’a aucune relation avec la face ventrale du poumon et se trouve tout entier sur le bord caudal de cet organe. Il est séparé du sac diaphragmatique postérieur par une crête fibreuse saïllante qui s'attache au bord caudal de la face ventrale du poumon : puis il s'étend de là en arrière et perd presque immédiatement contact avec le tissu pulmonaire ; il n’a par conséquent pas d'intérêt au point de vue où nous nous sommes placés. RÉSUMÉ Après la description qui vient d’être donnée des rapports des différents sacs avec le poumon, il est facile de reprendre l’étude de la face ventrale de cet organe envisagée dans son ensemble. Cette étude est d'autant plus nécessaire que les auteurs, même POUMON DES OISEAUX 247 les plus récents, ont très mal représenté les choses dans leurs descriptions et dans leurs figures. Ceux-là seuls se sont occupés de cette question qui ont traité de l’anatomie du diaphragme ornithique. Sans reprendre une à une leurs observations, il suffit de montrer l’erreur commise par deux des auteurs qui ont le plus exactement étudié la question pour faire voir combien notre travail conduit à une conception différente de celle admise jusqu'ici. B&rTELLI (1905) aussi bien que SAPPEY (1847) représentent tous deux la face ventrale du poumon comme occupée par une lame horizontale formant une sorte de voûte à concavité ventrale qui divise la cavité thoracique, en une partie dorsale renfermant le poumon et une partie ventrale occupée par les sacs aériens et les viscères. A cette lame ils ont donné le nom de diaphragme : « diaphragme ornithique » (BERTELLI), « diaphragme pulmonaire » (SAPPEY). Si Pon compare la figure 1 Tav. LXXIX de BERTELII qui se rapporte au Poulet et les figures 3, PI. 1 et fig.1, PI. 2, de SAPPEY qui concernent toutes deux le Canard, on voit que pour ces deux auteurs, le diaphragme horizontal tendu sur la face ventrale des poumons va depuis la première côte en avant jusqu’au bord caudal du poumon en formant une lame absolument continue. Or, il est clair que cette lame n’est point du tout une unité anatomique, qu’elle ne forme point un organe distinct, mais qu’elle résulte de la réunion de plusieurs lames différentes décrites séparément ci-dessus. Le diaphragme représenté par SAPPEY et par BERTELLI est un artefact anatomique résultant d’une dissection trop complète des sacs, et évidemment aussi de la tendance à retrouver dans l'appareil pulmonaire des Oiseaux, une membrane capable de jouer le rôle du diaphragme musculaire des Mammifères. La complication des parties en présence, la difficulté de représenter par le dessin les cavités des sacs et les accidents très nombreux de surfaces avoisinantes expliquêént aisément la facilité de créer des lames en partie artificielles comme l’est le diaphragme total de ces auteurs; et si nous comparons la figure 3, PI. IV, de 248 A. JUILLET SAPPEY qui représente les viscères en place et les sacs ouverts, avec sa figure 3, PI. I, qui représente le diaphragme, nous voyons clairement que toute la surface qui entoure les bronches, pour ne parler que de celle-là, a été fortement schématisée dans cette dernière figure, les saillies des troncs carotidiens et de la thyroïde ne sont plus représentées et les troncs vasculaires qui pénètrent dans le poumon sont évidemment eux-mêmes schématisés, enfin l’œsophage n’est point figuré. Il ne l’est pas davantage dans le dessin de BERTELI. ILest clair que dans les figures de ces auteurs, la lame formée par l’accolement des parois caudales du sac interclaviculaire et craniale du sac diaphragmatique antérieur à été détruite sans laisser de traces, ce qui pourrait tenir du reste à ce que la préparation à été faite sur le frais où la rétractibilité des membranes fait aisément disparaître des lambeaux coupés à quelque distance de leur insertion. Il est clair aussi que toute la partie craniale du diaphragme décrit par SAPPEY et BERTELLI, c’est-àdire toute la partie située en avant du hile du poumon, n’est point comme la partie caudale du même diaphragme, en rapport direct avec la surface pulmonaire ; elle en est séparée par toute la cavité du sac cervical de chaque côté, et cette prétendue partie craniale n’est autre chose que la paroi dorsale du sac interelaviculaire, unie à la paroï ventrale du sac cervical et formant la voûte du sac interclaviculaire. BERTELLI, comme SAPPEY, a absolument méconnu la direction de la pente craniale du poumon, et ne s’est point rendu compte de l'intervalle qui la sépare de cette voûte. Or, la pente craniale du poumon qui est souvent si courte que l’on ne peut lui attribuer une longueur dans le sens longitudinal, n'offre point comme la pente caudale de revêtement comparable à un diaphragme, elle est recouverte simplement par la paroi dorsale du sac cervical, dans laquelle rien ne peut être représenté comme un diaphragme véritable. En effet la forme légèrement convexe ventralement de la face ventrale du poumon, dans sa pente craniale, s'oppose complètement à ce que l’on puisse comparer POUMON DES OISEAUX 249 le feuillet dorsal du sac cervical qui la revêt, au diaphragme qui recouvre la pente caudale. Pente craniale et pente caudale de la face ventrale du poumon ont donc toutes deux des rapports spéciaux et très différents pour lune et pour l’autre. La pente caudale seule est en rapport avec le diaphragme ornithique qui, chez le Poulet, est formé exclusivement par la voûte du sac diaphragmatique antérieur, et d’ailleurs le rôle même de ce diaphragme est des plus douteux. Les principaux résultats exposés dans ce résumé ont déjà été publiés dans ma note du 29 juillet 1911 (A. JUILLET, 1911 d). PRINCIPALES VOIES CHAPITRE II AÉRIENNES INTRAPULMONAIRES Étude macroscopique Lorsqu'on isole un poumon sur un animal injecté à l’alcool selon le procédé indiqué p. 210, il forme un organe spongieux de consistance faible, revêtu sur toute sa surface d’une mince couche conjonctive lisse et continue, sauf au niveau des orifices des sacs aériens dont on parlera plus tard. À travers cette mince paroi conjonctive, on aperçoit sur toute la surface du poumon et particulièrement sur la face ventrale, un dessin formé par les conduits aériens vides et par le parenchyme pulmonaire de couleur jaunâtre qui leur est interposé. Sur la pente craniale, les voies aériennes forment des conduits disposés en éventail. Sur la pente caudale au contraire, ces conduits très flexueux s’ouvrent les uns dans les autres en formant un véritable laby- rinthe de voies aériennes séparées par des bandes également flexueuses et entrelacées de parenchyme pulmonaire. Cet aspect si particulier, si différent de celui des autres poumons qui mon- 250 A. JUILLET trent toujours une tendance à la formation de lobules polygonaux, si nets chez les Mammifères, est encore augmentée par le fait que plusieurs gros troncs bronchiques se distinguent aisément sur la face ventrale et montrent des caractères tout à fait particuliers. En effet, immédiatement sous-jacents à la membrane connective, ils n’offrent du côté ventral qu’une paroi très mince, absolument dépourvue de canalicules aérifères (parenchyme pulmonaire). A travers cette paroi, on aperçoit facilement de nombreux orifices, très visibles à l’œil nu, qui criblent leur face dorsale parenchyme pulmonaire qui la revêt. et conduisent dans le Ainsi un premier trait caractéristique du poumon des Oiseaux consiste dans la position très superficielle des grosses bronches, et dans la présence de bronches ou de conduits aériens tout à fait périphériques, de calibre assez développé cependant pour être suivis à l’œil nu et pouvoir être rattachés à leurs troncs originels sans autre préparation. La minceur de la paroi connective qui recouvre à la surface du poumon ces voies aériennes de différents calibres, et la facilité avec laquelle on les déchire, permettent de saisir tout d’abord un caractère très particulier du poumon des Oiseaux, à savoir que ce poumon est perforé par une série de conduits grossiers qui viennent jusqu’auprès de sa surface et que par le fait, son parenchyme est beaucoup plus perméable que celui des poumons des Mammifères. Mais les préparations simplement fixées à l’alcool, selon le procédé indiqué, sont insuffisantes pour permettre d’étudier l’anatomie de ces voies aériennes. Elles ne peuvent être employées que pour l’étude des gros troncs qui font immédiatement suite à la bronche extra-pulmonaire lorsqu'elle est entrée dans le poumon. Si on ouvre le poumon sur une sonde cannelée enfoncée dans la bronche intra-pulmonaire, on distingue facilement les gros troncs qui naissent sur cette dernière, et l’on voit aussi que ces troncs se dirigent immédiatement vers la périphérie POUMON DES OISEAUX 251 2 où ils s’étalent, tandis que dans le centre du poumon il en existe un seul qui continue à peu près la direction de la bronche extra-pulmonaire et forme la bronche souche ou «mésobronche » des auteurs. La bronche extra-pulmonaire est dirigée de dedans en dehors. Dès qu’elle est entrée dans le poumon elle est remplacée par le vestibule. VEsTIBULE. — Le vestibule est un conduit court, dirigé ventro-dorsalement et cranio-caudalement, qui ne continue pas tout à fait la direction de la bronche extra-pulmonaire, mais se rapproche un peu plus de la ligne médiane, formant avec la bronche un angle très obtus pour se rapprocher de la partie médiale du poumon, dont il s’écarterait s’il continuait à cheminer dans le même sens. Pour certains auteurs il constitue un léger renflement ovalaire : chez le Poulet il ne se distingue point ainsi par un changement de calibre, mais seulement par la constitution histologique de ses parois, comme on le verra. Il répond à la partie du tronc aérien intrapulmonaire où naïssent les entobronches (fig. 1). Le vestibule se termine au bord cranial de la première ectobronche, et se distingue très bien des autres voies aériennes parce que dans toute son étendue, mesurant chez le Poulet sept millimètres environ, sa surface interne est parfaitement lisse et continue, et ne présente pas d’autres trous que ceux des entobronches. Il est muni du côté dorsal d’un certain nombre d’anneaux cartilagineux comparables à ceux de la bronche extra-pulmonaire. Sur son bord médial, le vestibule porte d'habitude quatre gros orifices bronchiques disposés d'avant en arrière et séparés l’un de l’autre seulement par un éperon vertical qui ne fait point saillie dans la lumière du vestibule : ce sont les orifices des entobronches. MésoBroNCHE. — Le vestibule est continué par la mésobronche, laquelle située dans la profondeur du poumon, beaucoup plus près du côté dorsal que du côté ventral, se porte vers le bord caudal du poumon en décrivant une ligne 252 A. JUILLET légèrement courbe et vient se terminer sur ce bord au niveau de l’orifice du sac abdominal (ostium posterius.) La mésobronche se distingue du vestibule non seulement par son calibre régulièrement décroissant, mais par sa paroi interne, non plus lisse et continue comme celle du vestibule, mais criblée d’orifices plus ou moins larges formés par les parabronches. D’autres orifices plus importants se trouvent encore sur le bord médio-dorsal de la mésobronche : ce sont les orifices des ectobronches, au nombre de six à huit chez le Poulet (fig. 3). Ces orifices se succèdent dans l’ordre suivant : le premier, à partir du côté cranial est large, arrondi et placé bien exactement sur la face dorsale de la mésobronche: deux autres conduits à peu près du même diamètre le suivent, séparés l’un de Pautre par une bande étroite qui ne fait pas saillie dans la lumière de la mésobronche. Ces orifices ne sont pas placés exactement sur la même ligne droite que le précédent, mais se détachent en dedans de cette ligne, se rapprochant ainsi du bord médial de la mésobronche, et cette disposition se poursuit jusqu’au cinquième orifice qui est placé tout à fait sur le bord médial. La quatrième ectobronche est séparée de la troisième par un intervalle plus large que celui qui est compris entre les premières ectobronches. L’intervalle situé entre les quatrième et cinquième est encore plus développé. Après la cinquième ectobronche on distingue encore deux ou trois orifices de grosseur intermédiaire entre ceux des ectobronches précédentes et les orifices des parabronches qui criblent le reste de la mésobronche. Ces orifices appartiennent à la sixième ectobronche et à quelques bronches dorsales qui se distinguent mal des parabronches avoisinantes. Elles deviendront peut-être, chez les espèces à poumon plus allongé, des troncs bien distincts à ajouter aux six premières ectobronches. Les faces ventrales et latérales pe la mésobronche qui sont en dehors de l’insertion des grosses bronches dont il vient d’être question, se montrent criblées d’une série de trous de diamètre POUMON DES OISEAUX 253 inférieur à celui des ectobronches, ce sont les orifices de parabronches dont on parlera plus loin. Sur le bord externe de la mésobronche on distingue, parmi ces orifices, quelques-uns plus volumineux et de dimensions intermédiaires entre celles des orifices ectobronchiques et des orifices parabronchiques ordinaires. [ls appartiennent à un petit nombre de bronches dirigées vers le bord latéral du poumon et qui forment pour CAMPANA (1875) un groupe bronchique particulier auquel il a donné le nom de « bronches secondaires externes ». A cause de leurs faibles dimensions, ces bronches sont difficiles à dis- tinguer chez l’adulte, au milieu des bronches qui les environnent ou qui en partent, mais elles ont évidemment une impor- tance particulière parce qu’elles apparaissent de bonne heure chez l’embryon, sur la face externe de la mésobronche : on en reparlera plus loin. L’une d’entre elles forme le canal direct du sac diaphragmatique postérieur. Ce canal est caractérisé par ce fait que son calibre croît d'avant en arrière, si bien que l’on pourrait considérer sa portion distale comme une partie du sac diaphragmatique postérieur enfermée dans le poumon et cela pour deux raisons, premièrement à cause de sa largeur qui contraste vivement avec celle des grosses bronches qu’elle dépasse beaucoup lorsqu’elle arrive vers le diaphragme pulmonaire, et secondement parce que ses dimensions sont en quelque sorte inverses de celles des grosses bronches, puisqu'elles vont en croissant à mesure que ce canal s'éloigne de son point d’origine. Nous reviendrons plus loin sur ce canal. L'injection à l’alcool permet de suivre l’origine des troncs bronchiques, mais ceux-ci ont une paroi trop faible, lorsqu'ils touchent la périphérie et une individualité trop peu accusée, par suite de l’absence d’anneaux cartilagineux, pour qu'il soit possible de les suivre dans leur trajet ultérieur. Il faut absolument recourir à un artifice de technique pour en obtenir des préparations exactes et permanentes. Le meilleur moyen consiste certainement dans les injections métalliques dont il a été 254 À. JUILLET question. Grâce à celles-ci, on peut étudier en détail toutes les voies aériennes intra-pulmonaires et en donner une description exacte. DESCRIPTION DES GROS TRONCS BRONCHIQUES Les canaux aériens qui font suite au vestibule ne présentent point une structure différente selon leur parcours et sont essentiellement constitués de la même façon tout près de leur origine sur le vestibule ou sur la mésobronche et dans le reste de leur trajet. Au point de vue histologique, tous ces canaux mériteraient done le même nom de « bronches ». Cependant comme ils n’ont pas tous la même grosseur et que certains d’entre eux servent de troncs à un plus grand nombre de canaux aériens que les autres il est bon de les distinguer par quelques noms particuliers comme l’ont fait différents auteurs. SappEy (1847) avait divisé les bronches du poumon des Oiseaux en deux groupes : 19 Celles qui s'étendent sur la face ventrale ou diaphragmatique, « bronches diaphragmatiques » ; 29 celles qui s'étendent sur la face dorsale, sous les côtes, « bronches costales ». HuxLey (1882) proposa le nom d°’ « entobronches » pour les bronches diaphragmatiques et d’ « ectobronches » pour les costales. Plus commodes que les précédents à cause de leur unité, ces termes ne sont pas sans défauts, parce que les préfixes ento et ecto n’ont pas une signification suffisamment précise et pourraient être confondus avec médial et latéral, ce qui serait une erreur au point de vue de la position relative des troncs qu’ils désignent. Nous les conserverons cependant pour leur brièveté et à cause de l’usage, en faisant bien remarquer cependant que le mot ectobronches désigne les bronches les plus extérieures par rapport à l’ensemble de l'animal, celles qui sont les plus voisines des téguments, tandis que le mot entobronches désigne les conduits aériens qui sont dirigés vers la profondeur de l'animal. POUMON DES OISEAUX 255 Cette explication était d'autant plus nécessaire qu’elle répond encore à une autre objection que l’on pourrait faire à ces termes, à savoir que le mot entobronches est susceptible de faire penser à des bronches internes par rapport au poumon et qui s’opposeraient ainsi aux bronches externes ; or le poumon des Oiseaux se distingue précisément de celui de tous les autres animaux en ce que ses gros troncs bronchiques deviennent très vite superficiels et ne se ramifient qu’arrivés à la surface même du poumon où on les aperçoit par transparence. A cause de cette disposition, nous décrirons tout d’abord la manière dont les principaux troncs bronchiques s’étalent et se ramifient sur toutes les faces de l’organe, pour parler ensuite des rameaux qu'ils envoient dans sa profondeur. Ectobronches et entobronches sont donc les principaux troncs originels des bronches qui courent à la surface des poumons, aussi bien que de celles qui s’enfoncent dans leur épaisseur. Mais si elles engendrent la plupart des bronches superficielles, elles ne les donnent cependant pas toutes et un certain nombre de celles-ci, par une exception unique, loin de tirer leur source de bronches plus centrales qu’elles, viennent au contraire de portions de voies aériennes tout à fait périphériques, extrapulmonaires, et en réalité des sacs aériens. Ces dernières bronches que je crois être le premier à décrire dans l’appareil pulmonaire des Oiseaux en leur donnant leur véritable signification, méritent donc le nom de bronches récurrentes et se caractérisent par le fait qu’elles sont plus larges à leur extrémité distale qu’à leur extrémité proximale. Mais elles se ramifient abondamment comme les autres bronches superficielles du type commun et s’anastomosent avec elles de manière à former un réseau de voies aériennes absolument continu. Ces bronches récurrentes occupent une surface assez grande dans la moitié postéro-latérale du poumon des Oiseaux. Nous les étudierons plus tard en indiquant le sac aérien auquel chaque groupe d’entre elles appartient. ARCH. DE Z00L, EXP. ET GÉN. — 5° SÉRIE, — T, IX. -— (I). 17 256 A. JUILLET CAMPANA (1875), dans sa description si fouillée du poumon du Poulet, décrivit trois sortes principales de bronches : « la bronche primaire » ou « bronche souche » qui traverse le poumon d'avant en arrière et qui correspond à la mésobronche, des «bronches bronches secondaires », enfin des « bronches tertiaires ». Les secondaires forment pour CAMPANA quatre groupes qui sont : 19 Le système des grandes bronches divergentes ; 20 le système des bronches internes ; 39 le système des bronches externes ; 4° le système des bronches postérieures ou dorsales. Le « système des grandes bronches divergentes » répond aux bronches diaphragmatiques de SAPpPEyY (1847). Le « système des bronches secondaires internes » répond au système des ‘bronches costales du même auteur. Le « système des bronches secondaires externes » répond à ce petit groupe de bronches formées par les troncs nés sur le bord externe de la mésobronche et dont nous avons parlé plus haut. On les voit bien dans la fig. 2 où elles sont représentées par trois troncs naissant sur la mésobronche du côté opposé aux ectobronches. Enfin le «système des bronches secondaires postérieures » ou « dorsales » est constitué par une série de bronches faisant transition entre les secondaires et les tertiaires et qui naissent soit sur la mésobronche, soit sur les secondaires externes et les ectobronches qu’elles relient entre elles, en passant sur la surface dorsale du poumon, comme on le voit dans la fig. 2. Ce dernier système de l’aveu même de CAMPANA (1875, p. 42) ne peut pas se rattacher à de véritables bronches secondaires, parce qu’il renferme beaucoup de conduits nés sur les bronches secondaires ellesmêmes, et appartenant par conséquent au système tertiaire. On peut done sans inconvénient n’en point tenir compte, et, à cause de la profondeur du système des bronches secondaires, à cause de leur faible diamètre et des transitions insensibles qu’elles montrent avec les parabronches, on peut aussi laisser de côté ce groupe bronchique, pour n’admettre à une descrip- POUMON DES OISEAUX 257 tion particulière que celles répondant aux bronches diaphragmatiques et costales de SAPPEY. ENTOBRONCHES. — Les entobronches sont toutes situées sur la face ventrale des poumons (voy. fig. 1). La première entobronche (fig. 1, Æ1) est formée par un gros tronc court dirigé cranialement qui s’épanouit bientôt en trois rameaux principaux : un latéral qui vient sur le bord latéral du poumon et présente une direction plus ou moins transversale : il s’étale en un éventail qui couvre le tiers latéral de la pente craniale du poumon et fournit des rameaux qui s’anastomosent avec les conduits les plus antérieurs de la pente caudale. Une autre branche également transversale se dirige sur le bord médial du poumon et fournit les parabronches de la face médiale qui entourent la troisième côte (rameau médial). Le troisième rameau ou cranial engendre toutes les bronches du sommet du poumon en se divisant en éventail d’une manière plus ou moins pennée. Le tronc de ce rameau principal porte l’orifice du sac cervical (0.c. fig. 1). Dans ce cas, on voit que la première entobronche fournit tous les rameaux bronchiques de la face ventrale du sommet du poumon ou en d’autres termes toutes les bronches de la pente craniale. Parfois, la partie de cette pente craniale qui est formée par le rameau transverse médial de la première entobronche vient au contraire de la deuxième entobronche fig. II &.) (voir La deuxième entobronche (fig. 1 Æ2) varie un peu dans sa distribution suivant le mode de ramification de l’entobronche précédente et parce qu’elle peut ou non se charger du territoire pulmonaire appartenant dans certains cas au rameau trans- verse médial de l’entobronche précédente. Dans le cas où elle est la plus réduite, la deuxième entobronche fournit seulement quatre parabronches consécutives qui se dirigent sur la face médiale du poumon où nous les retrouverons plus tard. Si sa distribution est plus étendue, elle peut remplacer trois ou quatre parabronches du rameau transverse médial de la première 258 entobronche. A. JUILLET La deuxième entobronche ne se borne pas du reste à se distribuer dans le territoire médial, mais elle envoie du côté latéral un rameau qui contourne dorsalement le vestibule et vient s’épuiser dans le reticulum bronchique de la partie latérale du poumon au voisinage immédiat de l’orifice récurrent du sac interelaviculaire. Cette branche n’a pas été représentée dans la figure 1 pour éviter une complication qui aurait beaucoup embrouillé le dessin. Comme elle passe sur le côté dorsal du vestibule ou de la bronche prolongeant la trachée, on peut l’appeler rameau dorsal de la deuxième entobronche. Ce rameau se développe de bonne heure chez l'embryon et il est bien visible sur la figure 4 Æ2, r. d. La troisième entobronche (fig. 1 Æ3) s'étend depuis le voisinage du point d’entrée de la bronche extra-pulmonaire jusque vers l'extrémité caudale du poumon. Elle se dirige obliquement d’avant en arrière et de dehors en dedans :elle émet seulement six à sept parabronches rangées toutes sur son bord médial et présente par suite une disposition semi-pennée. Sur son bord latéral, elle émet une bronche oblique qui Punit à la quatrième entobronche. Près de son origine elle porte un orifice pour le tronc commun des deux sacs interclaviculaire et diaphragmatique antérieur ©. c.i.d. La quatrième entobronche (fig. 1. Æ4) à une distribution assez étendue. Elle comprend un tronc accolé à celui de la troisième entobronche sans l’interposition d’aucun tronc bronchique, et couvre de ses ramifications la plus grande par- tie de la pente caudale du poumon. Pour cela, elle émet au voisinage même de son origine un tronc transverse (fig. 1. E4r.t.) se dirigeant latéralement et croisant ventralement la mésobronche et qui vient se terminer sur le bord latéral du poumon, un peu en arrière de la moitié de cet organe. Ces deux branches de la quatrième entobronche, tronc principal et branche transverse, circonscrivent un peu plus d’un angle droit dont toute la surface est occupée par un réseau de bronches très fréquem- ment anastomosées entre elles et avec les bronches récurrentes POUMON DES OISEAUX 259 venues du sac diaphragmatique postérieur et du sac abdominal. Entre le rameau transverse de la quatrième entobronche, et celui de la première se trouve également une surface occupée par un réseau bronchique venu de plusieurs sources, à savoir des rameaux transverses latéraux de la première et de la quatrième entobronche et des rameaux récurrents qui se trouvent dans cette région venant du sac diaphragmatique antérieur et du sac interclaviculaire. G. FIsCHER (1905) à donné des bronches diaphragmatiques une description assez confuse et à laquelle il y a plusieurs reproches à faire. Dans la première entobronche, il distingue le rameau cranial sur lequel s’ouvre le sac cervical, sous le nom de « Bronchus cervicalis ». Ce nom pourrait être conservé. Il ne signale pas le rameau médial, mais il décrit le rameau latéral sous le nom de « Bronchus clavicularis », qu'il fait se prolonger jusque vers le bord caudal de la bronche extra-pulmonaire. Le sac interclaviculaire s’ouvre d’après lui sur ce rameau bronchique : l’orifice qu’il signale ici est très certainement. l’orifice récurrent de ce sac dont il à mal saisi les rapports exacts. Ce rameau bronchique est mis d’autre part en relation à son extrémité avec le sac diaphragmatique antérieur par une étroite ouverture qu'il désigne dans la figure 2, Taf. I, sous le nom d’ «Ostium intermedium anterius », c’est-à-dire comme, orifice direct de ce sac, qui naîtrait ainsi sur la première entobronche, quoique dans son texte (p. 11), il fasse naître ce sac sur « Bronchus diaphragmaticus anterior », (troisième entobronche). Le rameau médial de la deuxième entobronche est appelé par lui « Bronchus medialis » et il réserve le nom de « Bronchus clavicularis dorsalis » au rameau dorsal de cette entobronche. La troisième entobronche est divisée par G. FISCHER en deux parties : il l’appelle « Bronchus diaphragmaticus anterior » dans la zone où elle voisine avec le vestibule et « Bronchus caudalis » dans sa partie caudale. « Bronchus diaphragmaticus anterior » présente d’autre part dans la figure 5, Taf. I, un orifice 260 A. JUILLET qu’il ne désigne par aucun signe, mais qui d’après le texte est l’orifice direct du sac diaphragmatique anté- probablement rieur. Enfin, il donne le nom de « Bronchus lateralis » au tronc principal et au rameau transverse de la quatrième entobronche. Si l’on peut accepter, à la rigueur les dénominations que cet auteur attribue aux deuxième, troisième et quatrième entobronches, je ne puis admettre sa conception d’un sac interclaviculaire et d’un sac diaphragmatique antérieur, s’abouchant directement dans le rameau transverse latéral de la première entobronche. Je ne sais, il est vrai, à quel Oiseau l’auteur a voulu faire allusion : les figures précitées ne portent aucune indication à ce sujet, elles sont entièrement schématiques, et la description que l’auteur donne de ces bronches ventrales dans son texte est, comme nous l’avons signalé, en désaccord avec ses figures. ECTOBRONCHES. — Les ectobronches sont difficiles à séparer les unes des autres sur la face dorsale après les injections métalliques. Cela tient : 19 à ce qu'il n’y à pas une opposition aussi marquée qu’à la face ventrale entre les troncs d’origine et leurs premières ramifications dont le diamètre se rapproche plus du leur ; 20 à ce que les sillons déterminés par les côtes divisent parfois profondément certaines ectobronches, au point de faire croire à deux rameaux bien distincts, alors qu’il s’agit en réalité du même. Dans le cas où l’injection métallique est moins bien réussie, et où le réseau superficiel des petites bronches latérales qui recouvrent les origines des ectobronches n’a pas été conservé dans son entier, on peut voir que les ectobronches naissent toutes dans la profondeur du poumon et viennent s'épanouir sur la face dorsale en se dirigeant aussi bien du côté médial que vers le sommet du poumon, de telle sorte qu’elles fournissent seulement la partie médiale de cet organe et son sommet (fig. 2). La partie latérale est en dehors de leur domaine, sauf POUMON DES OISEAUX 261 par les connexions anastomotiques qu’elle peut prendre avec ce dernier. Des troncs anastomotiques courts (fig. 2 et fig. 11 an.) s’observent encore très aisément entre les gros rameaux des ectobronches sur la face dorsale et permettent une communication facile entre elles. La première ectobronche (fig. 2 Æcti) fournit des rameaux à toute la pointe du poumon. Ces rameaux dorsaux s’anastomosent avec des rameaux ventraux venus de la première entobronche, et complètent ainsi le cercle des voies aériennes de ce côté. Ces anastomoses forment une ligne parallèle au bord latéral du poumon et qui se voit très bien sur toutes les préparations (fig. 2 an). La deuxième ectobronche (fig. 2 Zct:) fournit toute la partie des bronches de la face dorso-médiale comprise entre la troisième et la quatrième côte ; un rameau même de cette ectobronche est situé caudalement à la quatrième côte. La troisième ectobronche (fig. 2 £cts) a un territoire plus réduit et ne donne au poumon que trois à quatre grosses parabronches, qui vont sur la face médiale et ne dépassent pas en arrière la cinquième côte. Le territoire compris entre la cinquième et la sixième côte est fourni par les quatrième et cinquième ectobronches (fig. 2, Ects et 5), peu ramifiées. La sixième ectobronche (fig. 2 Æcts) se dirige presque perpendiculairement à la sixième côte, qu’elle croise et fournit les quelques tubes bronchiques situés sur la face dorsale du poumon en arrière de cette sixième côte. On rencontre parfois une septième ectobronche dont les rameaux se portent principalement sur l’extrémité caudale du bord médial et entrent en rapport direct avec les bronches récurrentes du sac abdominal. Comme il a été dit, la présence des sillons costaux gêne beaucoup pour suivre le trajet des diverses ectobronches, et pour avoir une bonne idée de leur disposition nous prions de comparer les photographies des moulages métalliques (fig. 14 b) à la figure schématique précitée. 262 On A. JUILLET voit par cette description que les premiers conduits aériens qui naissent sur le vestibule ou sur la mésobronche, se dirigent sur les faces opposées du poumon et s’épanouissent sur ces faces en rameaux plus petits : les parabronches. Entobronches, ectobronches et parabronches ont exactement la même structure : elles n’ont pour ainsi dire pas de paroi propre isolable leur lumière n’étant limitée que par les éperons que forment les parois des conduits aériens de petit calibre, répandus dans le parenchyme pulmonaire et qui s’ouvrent perpendiculairement dans cette lumière. Nulle part on ne voit d’anneaux cartilagineux sur le trajet des entobronches et des ectobronches, et leur structure ne diffère pas à leur racine, où elles sont très larges, et à leur extrémité où elles se continuent sans transition brusque avec les parabronches qui en naissent. Celles-ci présentent exactement les mêmes caractères et sont comme les bronches principales de calibre bien différent, étant en général plus larges à la périphérie que dans la profondeur ou chez certains Oiseaux que chez d’autres. Ainsi les parabronches superficielles sont particulièrement puissantes chez le Poulet où leur calibre atteint Sur les faces pulmonaires jusqu’à 2 millimètres, tandis que dans la profon- deur du poumon il n’est plus que de 1 millimètre. Dans tous les autres Oiseaux que j’ai examinés, les parabronches superficielles sont beaucoup plus grêles que chez le Poulet et se distinguent par là plus facilement des ectobronches, comme on le voit chez le Canard et le Pigeon (fig. 20, 30, 31, 32). Sur les moules métalliques, le parenchyme pulmonaire, évidemment comprimé par le poids de la masse, est un peu réduit dans ses dimensions, et les parabronches se touchent presque toutes, laissant à peine entre elles des interstices étroits, de sorte que le poumon paraît être presque exclusivement constitué par un lacis serré de voies aériennes. L’examen des coupes de poumons injectés à l’alcool ou par les fixateurs ordinaires, permet de rectifier ce que ces préparations ont d’un peu défectueux : elles montrent bien d’ailleurs, que les moules POUMON DES OISEAUX 263 métalliques ne font qu’exagérer un peu la disposition fondamentale des poumons des Oiseaux, dans laquelle les conduits aériens tiennent une si grande place par rapport au parenchyme (voir fig. xt). Il semble aussi que la masse métallique tende à se dilater un peu par le refroidissement : elle se moule en effet exactement sur les moindres accidents de la surface des conduits aériens, et montre le plus souvent sur ces derniers, sous la forme d’une petite mosaïque saillante (voir en particulier fig. 23 b) les orifices des premiers conduits aériens du parenchyme qui mesurent seulement 0 mm. 09 à 0 mm. 12. Cette discussion met en relief les avantages et les défauts que présentent les moulages métalliques ; elle permet aussi de se rendre compte que, pour la description macroscopique des voies aériennes, ils ne présentent que des avantages et peuvent être employés non seulement sans aucun inconvénient, mais avec le plus grand profit. CIRCUITS BRONCHIQUES. — Les voies aériennes remplies par la masse métallique forment des tubes légèrement flexueux, et de diamètre progressivement décroissant jusqu’à une certaine limite cependant, à partir de laquelle ils se continuent sous la forme de cylindres parfaits pour s’anastomoser avec des tubes de même diamètre venus d’autres points de la surface pulmonaire et fermer ainsi le circuit des voies aériennes. Tous ces tubes sont fréquemment unis les uns avec les autres par des anastomoses transverses : ils ne se terminent jamais en cul-de-sac, mais se continuent régulièrement les uns dans les autres, ceux qui viennent de la face dorsale s’unissant avec ceux de la face ventrale, et il en est de même dans l’épaisseur du poumon. Cette disposition doit être étudiée avec soin pour ne laisser aucun doute sur son existence et pour préciser la manière dont s’effectuent les communications entre les différents canaux venus des principales sources précitées. Pour cela, examinons la surface pulmonaire où les parabronches sont plus volumineuses grande et où ces communications importance. Cette surface peut ont, par suite, une être grossièrement 264 A. JUILLET divisée en trois territoires : la face ventrale, la face dorsale et la face médiale. À la face ventrale (voir fig. 1 et 14 a), les parabronches sont toutes placées les unes à côté des autres de façon à recouvrir toute la surface d’une manière parfaitement continue. Les parabronches de la pente craniale en particulier, sont d’un calibre plus volumineux, en rapport avec leur origine sur les gros rameaux de la première entobronche. Elles sont très serrées les unes contre les autres, ne laissant presque plus de place au parenchyme pulmonaire très réduit dans cet endroit, et s’'étalent à la surface du sommet du poumon en ne montrant que de très rares anastomoses. sur le côté latéral, arrivées sur Les parabronches situées le bord cranial, se recourbent brusquement et passent sur la face dorsale du poumon: qu'elles suivent sur une certaine longueur puis, elles viennent s’unir avec les parabronches de la première ectobronche suivant une ligne d’anastomoses, merveilleusement dessinée sur certains moulages (fig. 14 b et 23 b.) et représentée sur la figure 2 an. Les parabronches du côté médial comme la plupart des parabronches issues des trois entobronches suivantes, se recourbent à angle droit lorsqu'elles arrivent sur le bord médial du poumon, puis passent sur la face médiale (fig. 3). On observe donc sur cette dernière, une série de parabronches issues des quatre entobronches successives et dirigées de bas en haut ou ventro-dorsalement. Ces parabronches en majeure partie parallèles entre elles, présentent cependant des inflexions assez marquées au niveau des côtes qu’elles enveloppent : elles sont unies entre elles par une série de petites anastomoses transverses, étroites, que l’on observe très facilement sur le moulage, fig. 11 b. Arrivées vers le bord dorsal du poumon, elles s’unissent avec les parabronches issues des ectobronches, suivant une deuxième ligne d’anastomoses, très facile à suivre sur la photographie précédente et sur la fig. 3 an. Toutes ces anastomoses sont très visibles par suite d’une EE 24 POUMON DES OISEAUX 265 disposition spéciale qui permet à défaut d’autres caractères et de tout changement de calibre en particulier, de reconnaître que l’on abandonne une espèce de rameau pour passer à une autre. Ce caractère est que l’anastomose s’effectue toujours par une double bifurcation appartenant à chacune des parabronches correspondantes. Les parabronches ventrales et les parabronches dorsales qui vont s’anastomoser ne sont pas dans le prolongement l’une de l’autre, mais alternent, se bifurquent à leur extrémité et ce sont ces bifurcations très courtes qui établissent ces anastomoses. La démonstration des anastomoses entre les bronches des deux faces pulmonaires est extrêmement importante parce qu’elle montre bien un des caractères fondamentaux du poumon des Oiseaux, qu’il importe absolument de connaître si on veut comprendre cet organe. Ce caractère consiste dans ce fait que nulle bronche ne se termine en cul-de-sac dans l’appareil pulmonaire et que toutes, au contraire, se continuent les unes dans les autres par des anastomoses constantes, qui les lient en un ensemble de conduits communicants, contrairement à ce qui se passe chez les autres animaux où l’arbre bronchique forme des ramifications toujours closes à leur extrémité et sans communications entre elles. Ces anastomoses bronchiques constituent les circuits aériens, déjà décrits par CAMPANA, mais qui semblent avoir été négligés par tous les auteurs qui lui ont succédé, car on ne peut considérer comme suffisante la description de l’anastomose des parabronches donnée par G. FiscHErR (1905). Les anastomoses bronchiques ne se voient pas seulement aux extrémités des conduits provenant des grosses bronches : elles s’observent également sur le parcours de ces conduits eux-mêmes. Nous avons précisément signalé comme un caractère des bronches de la pente craniale de ne s’anastomoser que rarement entre elles. C’est là une véritable exception dans la constitution de l’arbre aérien qui s’expliquera plus tard par un trait de cette 266 A. JUILLET : région : absence de bronches récurrentes qui permet au point de vue physiologique l’absence de ces anastomoses. Mais dans tout le reste de la surface du poumon les anastomoses sont extrêmement nombreuses et multipliées. On les voit s’établir d’après certains moulages, sur les premières parabronches à leur naissance des ectobronches. Ces anastomoses sont courtes, transversales ou obliques et elles se multiplient sur toute la surface du poumon comprise en dehors des gros troncs bronchiques, aussi bien sur la face dorsale que sur la face ventrale. Les deux moulages à, db, fig. 14, montrent avec assez d’évidence ces anastomoses pour qu’il soit inutile d’insister davantage pour le moment, car nous aurons à revenir sur ce point, lorsque nous traiterons des bronches récurrentes. Il suffit de dire que dans ces points, les bronches décrivent à la surface un véritable réseau inextricable. La distribution des bronches que nous venons de décrire à la surface pulmonaire se rencontre aussi dans l’intérieur du poumon, c’est-à-dire que des faces profondes ou, si l’on préfère, intra-pulmonaires des ectobronches et des entobronches partent des parabronches qui traversent le poumon, en établissant de toute part des communications entre les gros troncs des deux faces (fig. 19). La direction de ces parabronches intra-pulmonaires est facile à établir. Si l’on part du bord médial du poumon, on voit que les parabronches unissant les deux faces ont une direction à peu près verticale : cette direction est gardée par les premières parabronches les plus voisines de la face médiale. Mais au fur et à mesure que l’on s’avance vers le bord latéral, ces parabronches s’inclinent en décrivant une courbe allant de dedans en dehors et d’avant en arrière. Cette courbe à concavité ventrale répond à peu près à celle de la pente caudale du poumon, avec laquelle les parabronches intra-pulmonaires de la deuxième entobronche coïncident presque. En outre dans la moitié craniale du poumon la direction des parabronches se fait d'avant en arrière et du ventre au dos, de telle façon POUMON DES OISEAUX 267 qu’elles ont en général une direction parallèle aux rameaux de la première ectobronche. Toutes ces parabronches s’anastomosent dans l’intérieur du poumon par des bifureations analogues à celles de sa surface. Comme le disait déjà CAMPANA (1875 p. 45), elles « sont disposées en couches parallèles concentriques se recouvrant immédiatement l’une l’autre. » Ces parabronches internes forment des circuits profonds analogues aux circuits superficiels établis entre les premiers troncs bronchiques. C’est probablement cette ligne d’anastomoses profondes ou internes qu'a voulu représenter G. FISCHER dans ses figures 7 et 8, Taf. I (1905). Mais ces schémas sont loin de donner une idée suffisante de l’étendue des anastomoses parabronchiques et de faire bien comprendre les circuits intrapulmonaires signalés par CAMPpANA. Nos trois schémas (fig. 1, 2, 3.) montrent qu'il existe dans le poumon des Oiseaux deux sortes de circuits bien distincts qui se répètent de la même manière à la surface et dans la profondeur du poumon. Ce sont : 10 Les circuits médiaux de la face médiale, qui sont formés par la rencontre des parabronches venues des quatre entobronches et des ectobronches. Ces circuits se ferment au niveau de la ligne d’anastomoses signalée près du bord dorsal de la face médiale (fig. 3) ; 20 Les circuits latéraux qu’il faut subdiviser en : a, circuits latéraux ceraniaux, formés entre la première entobronche et la première ectobronche, et fermés suivant la ligne * anastomotique dorsale qui suit le bord cranial et latéral sur la face dorsale du poumon (fig. 2 an); b, circuits latéraux caudaux (fig. 1 et 2), ainsi nommés parce qu’ils occupent la portion latérale et caudale des faces ventrale et dorsale du poumon. Ces circuits s’établissent entre les parabronches issues de la quatrième entobronche (fig. 1) et des bronches secondaires externes (fig. 2), et celles qui naissent à la face dorsale de la plupart des ectobronches pour se porter, contrairement au trajet principal de lPectobronche, sur 268 A. JUILLET le bord latéral du poumon. Aïnsi se ferment dans cette région des circuits ento-ectobronchiques comme dans les autres territoires du poumon. Ces circuits se compliquent beaucoup, parce que les parabronches qui les forment sont disposées en un véritable réseau, au lieu de rester à peu près parallèles entre elles avec de petites anastomoses transverses. Ces circuits latéraux sont d'autre part alimentés par les bronches récurrentes des sacs diaphragmatiques antérieur et postérieur et par celles du sac abdominal, comme on le voit bien sur les fig. 1 et 2. Cette «partie du poumon est celle qui se moule le plus mal, surtout lorsque la quantité de masse injectée est trop faible pour remplir parfaitement les sacs. Au contraire, lorsque les injections métalliques ont été complètes ces rapports rendent très difficile l’isolement des moulages de certains Oiseaux (Canard, Mouette, etc.). Chez ces derniers, en effet, les bronches récur- rentes issues des sacs précités forment un paquet très volumineux qui entraîne souvent avec lui les bronches qui en partent lorsqu'on veut l’isoler des sacs aériens auxquels il se rattache. Ainsi s'explique le morcellement du bord latéral des moulages de poumons de Canard (fig. 30 et 31). Il résulte de cette réticulation des voies aériennes dans toute l’étendue de la face ventrale et d’une partie de la face dorsale du poumon, que l’air amené dans ce réseau par les bronches récurrentes qui contribuent à le former rencontre des conditions très favorables à sa diffusion dans la masse du paren- chyme pulmonaire. POUMON DES OISEAUX CHAPITRE RAPPORTS DES SACS AÉRIENS 269 III ET DES BRONCHES Il faut maintenant parler des rapports des sacs aériens avec les poumons. On admet généralement que ces sacs représentent l’extrémité de certaines bronches qui, arrivées à la surface ventrale du poumon se dilatent beaucoup, et s’interposant entre les poumons et les viscères les plus voisins, déterminent la production de vastes loges aériennes qui occupent une grande partie de la cage thoracique, une partie de la voûte de l’abdomen et des flancs, et contribuent ainsi à alléger le corps de l’oiseau et à améliorer les conditions de son équilibration (voy. p. 287). BERTELLI (1905) à bien montré comment ces sacs, naissant sur la face ventrale des poumons, se développent au milieu de la lame mésodermique qui ferme de bonne heure la loge pulmonaire du côté ventral, repoussent au devant d’eux une partie de cette lame mésodermique à l’aide de laquelle se forment les différentes cloisons connues dans le tronc et constituées par la paroi distale des sacs. Mais cette description, qui nous à apporté de grandes clartés sur la formation des différentes cloisons qui délimitent les compartiments du corps de l’adulte, n’épuise pas complètement la question des rapports des sacs aériens avec le tissu pulmonaire. On sait déjà depuis longtemps que l’air contenu dans les sacs aériens, et qui reste pur parce que les parois de ces sacs n’ont aucune fonction respiratoire, peut être chassé par ces sacs dans les poumons et remplir un rôle utile dans l’hématose. Mais on ne s’est pas beaucoup préoccupé des rapports que les orifices des sacs aériens creusés sur le diaphragme pulmonaire, offrent avec le tissu du poumon et surtout avec les conduits aériens d’un certain calibre qui se trouvent dans leur voisinage. Mes premières injections métalliques m'ont permis de remarquer certains faits assez inattendus et qui ont attiré mon atten- 270 A. JUILLET tion d’une manière tout à fait particulière. C’est la présence en certains points du poumon et surtout à son extrémité caudale et au voisinage de son bord latéral, de bronches d’une forme particulière, plus volumineuses à leur extrémité distale qu'à leur extrémité proximale ou centrale, et se ramifiant elles-mêmes à partir de cette extrémité distale dans le tissu pulmonaire, comme le feraient des bronches au voisinage de leur origine sur la trachée. Mais étant donné que le gros bout de ces bronches était aussi éloigné que possible de la trachée et qu’elles étaient en réalité dirigées en sens inverse des bronches voisines, on pouvait les désigner d'emblée comme des bronches récurrentes. BRONCHES RÉCURRENTES. — Ces bronches n’ont pas encore attiré l’attention des auteurs ou du moins n’ont pas été comprises par CAMPANA et par G. FIsCHER qui les ont cependant entrevues. En effet, ce dernier à pu apercevoir dans ses mou- lages à la celloïdine quelques-unes des bronches récurrentes les plus fortes et les plus caractéristiques, et notamment la bronche récurrente du sac abdominal (fig. 5, Taf. IV) dans le Canard, la bronche récurrente du sac abdominal et du sac dia- phragmatique postérieur chez le Pigeon (fig. 16, Taf. V) et sans doute les bronches récurrentes du sac diaphragmatique antérieur dans le Nisus communis (fig. 22, Taf. V), mais ce moulage est trop incomplet pour que l’on puisse être bien affirmatif à cet égard. En tout cas, ceux des figures 5 et 16 ne laissent aucun doute ; mais G. FISCHER n’a certainement pas compris leur signification, comme le montre la seule mention qu’il fasse de ces bronches et qui est une simple note dans l’explication de la figure 5 où il dit que, dans le réseau de conduits aériens qui s'étend à la surface dorsale du poumon sur son bord laté- ral et sur sa partie caudale « on observe un tronc bronchique plus gros qui, d’ailleurs, n’a été signalé nulle part, et qui se dirige vers le sac abdominal en s’anastomosant en chemin avec les nombreuses voies aériennes voisines. » Cette phrase de G. FISCHER montre clairement qu’il n’a pas compris du POUMON DES OISEAUX 271 tout la valeur de ces bronches, puisqu'il considère les bronches récurrentes du sac abdominal comme se rendant à ce dernier, tandis qu’elles en proviennent au contraire. CAMPANA a représenté et mentionné dans ses figures 11 et 13 du texte et dans ses photographies 11 et 13, PI. IV, les bronches récurrentes du sac diaphragmatique antérieur, du sac diaphragmatique postérieur, et du sac abdominal, sans reconnaître d’ailleurs leur nature, puisque, comme on le verra plus loin, il les à considérées comme résultant de la reconstitution en un seul tronc de plusieurs bronches tertiaires convergeant pour venir s'unir les unes avec les autres. Ces bronches récurrentes sont cependant des formations constantes dans les poumons des Oiseaux et que l’on retrouve dans la plupart des sacs aériens. Ce sont des bronches engendrées par les sacs aériens eux-mêmes et qui, suivant un trajet récurrent par rapport à ces derniers, rentrent dans le poumon qu'ils ont abandonné. Elles contribuent ainsi à former une partie du parenchyme pulmonaire et, pour le dire tout de suite, elles font passer dans la périphérie du poumon, c’est-à-dire dans le territoire le plus éloigné qu’il soit possible de l’origine trachéale des bronches, l’air contenu dans les sacs aériens qui, au lieu de repasser directement par les gros troncs qui l’ont amené dans les sacs et d'arriver presque directement à la trachée, est ainsi contraint de traverser le tissu respiratoire et d’y jouer un rôle utile pour l’hématose. J'ai suivi en détail la formation de ces bronches récurrentes par les sacs, au cours du développement chez le Poulet et cette formation sera décrite plus loin avec toutes les explications nécessaires. Pour le moment, j'indiquerai l’insertion des sacs sur l’arbre aérien, la forme des orifices qui les font communiquer avec le parenchyme pulmonaire et celle des bronches récurrentes qui s’y rattachent. J’ai déjà exposé les faits essen- tiels de cette description. (A. JUILLET, 1911, à). La topographie générale des sacs étant connue (voir p. 239), il est facile d’étudier les rapports de chacun d’eux avec le pouARCEH. DE ZOOL. EXP. ET GÉN. — 5° SÉRIE, — T. IX. — (I). 18 272 A. JUILLET mon. Pour la facilité de la lecture, j'ai reproduit au milieu de cette description le schéma IIT. SAC CERVICAL. — Le sac cervical naît sur la première entobronche et plus précisément sur le tronc cranial de cette entobronche par un petit conduit court (0. c. fig. 1). Cet orifice est arrondi, unique, c’est-à-dire non subdivisé par des éperons s’avançant à peu près jusqu’au plan de son insertion sur la surface pulmonaire et formés par les intervalles séparant des bronches situées plus profondément, comme cela s’observe en maints autres points. IL est situé à peu près au niveau du tiers postérieur de la pente craniale et, chez le Poulet, il correspond à la veine axillaire. Il est placé très profondément, bien au-dessous du toit du sac inter-claviculaire, qui le cache totalement lorsqu'on observe l’oiseau couché sur le dos. C’est pourquoi la figure 1 de BERTELLI (1905) représentant le diaphragme ornithique, et où cet orifice est placé exactement vers le sommet de ce diaphragme dans l’angle formé par les muscles prévertébraux et par la première côte, est absolument fautive comme nous l’avions déjà signalé pour le diaphragme lui-même. Celles de SapPEy (1847, fig. 3, PI. I et fig. 1, PI. IT) qui représentent le même orifice à la même place, sont également inexactes. En effet, bien que la pente craniale du poumon soit chez le Canard beaucoup plus allongée que chez le Poulet et que, par suite, l’orifice du sac cervical soit beaucoup plus cranial, il n’en est pas moins vrai que la position de cet orifice sur les figures incriminées est révélée fausse par les dessins de SAPPEY lui-même. Si l’on examine, par exemple, sa figure 1, PI. I, où les poumons sont représentés dans leurs rapports avec les bronches extra-pulmonaires et la trachée, on voit que l’orifice du sac cervical, correctement représenté, cette fois, sur le poumon lui-même à droite, est situé très cauda- lement par rapport à la bifurcation de la trachée, tandis qu'il est exactement au niveau de cette bifurcation sur la figure 3. SAC INTERCLAVICULAIRE. — Le sac interclaviculaire présente chez le Poulet, des rapports passablement différents de ceux POUMON DES OISEAUX 273 qui ont été décrits par les auteurs ; mais les ayant vérifiés à la fois sur l’adulte et au cours du développement embryonnaire, je puis affirmer leur exactitude et les décrire de la façon suivante : Le sac thoracique prend naissance sur le tronc d’origine du sac diaphragmatique supérieur qui naît à son tour de la troisième entobronche (fig. IV); le conduit qui va donner naissance au sac se dirige cranialement, contourne la bronche extrapulmonaire et se place ventralement à la surface pulmonaire, où il constitue un canal compris entre la face ventrale du poumon et le tronc de la veine pulmonaire, dans la partie du trajet qu’elle parcourt entre le poumon et l'oreillette. Ce canal C. à s'ouvre cranialement sur la face postérieure du sac interclaviculaire dont il forme le conduit direct, qui y amène l'air inspiré. Ce canal n’est autre chose que le canal interclaviculaire décrit plus haut (p. 245) : il est très bien conservé sur certains moulages métalliques où ses rapports avec le tronc d’origine du sac diaphragmatique antérieur sont indubitables et plus faciles à voir que sur les pièces fixées à l’alcool (voir en particulier fig. 17). Ce canal a été signalé par CaAMPANA (1875, p. 52), comme une communication entre le sac diaphragmatique antérieur et le sac interclaviculaire et par conséquent il ne paraît pas avoir été bien compris par cet auteur, ce qui explique qu’il ait été oublié ou méconnu par les auteurs qui ont suivi, et en fait la plupart d’entre eux, lorsqu'ils veulent indiquer les communications du sac interclaviculaire avec l’arbre bronchique, les cherchent non à ce point, mais vers l’orifice externe qui fait communiquer le poumon avec ce sac et qui constitue pour nous son orifice récurrent. ROCHÉ (1891) signale simplement ce canal comme une communication entre les deux sacs interclaviculaires et diaphragmatique antérieur (p. 10) et le reste de son travail montre combien il a peu compris la véritable disposition des orifices pulmonaires dans les sacs aériens puisqu'il les limite au nombre de cinq: « bien que ce nombre cinq ne soit pas 274 A. JUILLET absolument constant et puisse être dépassé, un réservoir ayant parfois deux ouvertures pulmonaires, nous exciperons de sa grande généralité pour la commodité de la description. » (p. 25). Or comme CAMPAXNA insiste à différentes reprises pour décrire sept orifices pulmonaires, l’exposition de ROCHÉ montre combien peu avait été compris le mémoire de CAMPAXA et l’obscurité qui régnait encore sur la question de ces rapports : le travail le plus récent, celui de G. FISscHER (1905), est encore bien imparfait sur ce point. En effet, le conduit du sac intercelaviculaire n’a été représenté par lui dans aucune des vingt-sept figures de son travail qui, d’ailleurs, se rapportent presque toutes à la face dorsale de divers poumons, trois seulement étant consacrées à la face ventrale, sur laquelle seule ce canal peut être aperçu. Comme il existe dans le territoire du poumon en rapport avec le sac interclaviculaire un autre orifice conduisant dans ce sac, les auteurs l’ont considéré comme représentant l’orifice bronchique du sac et se sont par suite désintéressés de la recherche de l’orifice direct qui leur a totalement échappé. L’orifice du sac interclaviculaire des classiques ou orifice récurrent de ce sac à mon avis est placé immédiatement en dehors de la veine cave antérieure et cranialement à la cloison qui sépare l’interclaviculaire du diaphragmatique antérieur. Il est à peu près arrondi et il paraît divisé en deux moitiés inégales par un éperon saillant, issu du parenchyme pulmonaire, et qui semble arriver jusqu’au niveau même de la face ventrale du poumon. Les deux moitiés de l’orifice ainsi subdivisé présentent elles-mêmes dans la profondeur une série de trous arrondis, de diamètre plus restreint, et qui sont des orifices de bronches. Par conséquent, l’orifice du sac interclaviculaire paraît beaucoup moins conduire dans un tube unique qui le raccorderait à l’arbre trachéal que dans un carrefour servant d’aboutissant à un certain nombre de bronches. La présence de nombreux orifices bronchiques dans le territoire d’un orifice de sac a été déjà signalée, à propos de l’ou- POUMON DES OISEAUX 275 verture de divers sacs, par quelques auteurs et en particulier ; par SAPPEY (1847) qui a figuré les orifices bronchiques, situés . - dans la profondeur de l’orifice des sacs interclaviculaire, diaphragmatique postérieur et abdominal du Canard. Nararis Cranial Po L \| e ls ;7 4 A iQ / NN LA LA (1h ANT NES Ë : À À ; \ AK a : O.c Le Latéral 1,Médial Ro E2 de. O.d.a. R (be 3 Be Mes Caudal FIG. IV. — Schéma montrant les rapports des sacs aériens et de leurs orifices avec la face ventrale du poumon chez le Poulet. Gr. — 1 x 1,5. — B, bronche extra-pulmonaire ; C.4.p., canal du sac diaphragmatique postérieur; C2, canal du sac interclaviculaire ; E,, E,, E,, E,, première, deuxième, troisième et quatrième entobronches ; Mes mésobronche ; O.ab., orifice direct du sac abdominal ; O.c., orifice direct du sac cervical ; O.d.a., orifice direct du sac diaphragmatique antérieur ;O.d.p., orifice direct du sac diaphragmatique postérieur ; R.ab., orifices récurrents du sac abdominal, R.d.a.., orifice récurrent du sac diaphragmatique antérieur ; R.d.p., orifices récurrents du sac diaphragmatique postérieur ; R.i., orifice récurrent du sac interclaviculaire: GUILLOT, dans ses belles figures des sacs du Coq, a représenté aussi plusieurs orifices bronchiques dans l’étendue de louver- ture latérale du sac diaphragmatique antérieur (1846, fig. 2, PI. IV). Les autres auteurs, Voar C.et YUNG (1894), BERTELLI 216 A. JUILLET (1905), G. FISCHER (1905), n’ont pas représenté ces multiples orifices bronchiques, en rapport avec l’ouverture du sac. CAMPANA (1875), au contraire, à parfaitement distingué (p. 211 et suivantes) ces orifices bronchiques, et a remarqué qu'ils ne se rencontraient point indistinctement dans toutes les ouvertures des sacs aériens, mais seulement dans quelquesunes d’entre elles. Il a distingué, par suite, les orifices des sacs aériens ou « infundibula » comme il les appelait, en « monobronchiques » et « polybronchiques ». Les monobronchiques présentent un orifice unique conduisant directement dans une grosse bronche : ils sont situés près du bord interne (médial) du poumon : il y en a deux pour chaque poumon : le premier n’est autre que l’orifice du sac cervical : le deuxième est celui du diaphragmatique antérieur. Les orifices polybronchiques se distinguent parce qu’au lieu d’être uniques, ils sont subdivisés en une série de pertuis secondaires qui conduisent dans les bronches ; CAMPANA divise ces orifices polybronchiques en deux catégories : les «polybronchiques simples » et les « polybronchiques mixtes ». Nous verrons, à propos des orifices auxquels ces dénominations se rapportent dans la description de CAMPANA, qu’elle était leur constitution pour cet auteur et s’il y a lieu de conserver ces expressions. Mais pour le moment, il suffit de faire remarquer combien peu, on a tenu compte jusqu'ici du mémoire si important de CAMPANA bien qu’il soit toujours cité dans les bibliographies, puisque des expressions si commodes et qui font bien ressortir une des particularités les plus frappantes des divers orifices des sacs n’ont même pas été reproduites par les auteurs qui se sont succédés depuis 1875. L’orifice récurrent (A. 1. fig. 1 et fig. IV, et fig. 14 a) du sac interclaviculaire, dont le développement embryologique sera décrit plus loin, donne naissance à une série de bronches récurrentes qui répondent justement aux orifices signalés dans sa partie profonde, mais qui attirent peu l’attention et sont assez ES Le POUMON DES OISEAUX , 277 difficiles à bien distinguer, à cause de circonstances sur lesquelles nous nous étendrons quelque peu. Ces bronches, en effet, au lieu de courir sur une certaine longueur à la surface ventrale du poumon, s’enfoncent perpendiculairement dans son épaisseur et s’anastomosent très rapidement avec les parabronches voisines. Aussi leur longueur est-elle insignifiante, par suite leur calibre est beaucoup moins considérable que dans d’autres bronches analogues. De plus, sur les moulages métalliques, elles sont souvent enlevées avec le sac interclaviculaire et on ne trouve plus à leur place qu’une lacune dans le moulage. J’en ai obtenu cependant de bonnes préparations dans des injections métalliques, faites chez des Poulets de quinze jours, et qui confirment tout à fait ce que m’avaient montré l’embryologie et la reconstruction partielle d’un poumou d’embryon au dixième jour. SAC DIAPHRAGMATIQUE ANTÉRIEUR, — Ce sac naît sur la troisième entobronche sous la forme d’un conduit cylindrique, qui s’ouvre perpendiculairement à la face ventrale du poumon, sur le point même où la troisième entobronche arrive sur cette face et s’y étale d'avant en arrière (O0. d. a, fig. 1v). L’orifice de ce conduit est donc placé sur une ligne transversale, passant par le bord caudal de la bronche extra-pulmonaire, au point où celle-ci pénètre dans le poumon et médialement à cette bronche. Cet orifice est toujours monobronchique simple. Il existe à la face ventrale du poumon, dans le territoire du sac diaphragmatique antérieur, un autre orifice qui fait communiquer ce sac avec le tissu pulmonaire. Par la nature des conduits qui y aboutissent, cet orifice conduit certainement du sac dans le poumon, c’est donc un orifice de bronche récurrente. En effet, il aboutit à quelques troncs très courts, qui se bifurquent bientôt en Y et donnent des rameaux divergeants appliqués contre la face ventrale du poumon À. d. a. (fig. 1V). Ces bronches ne tardent pas à s’anastomoser avec les autres conduits bronchiques de cette face. Elles ont tout à fait les caractères de bronches récurrentes, car elles se ramifient 218 A. JUILLET proximalement comme il à été dit ci-dessus et les rameaux qui en partent sont plus gros à leur origine et vont en diminuant à mesure qu'ils s’enfoncent dans le tissu pulmonaire ou s’étalent à sa surface (dr. d. a, fig. 1). L’orifice des bronches récurrentes ou, si l’on préfère, le deuxième orifice du sac diaphragmatique antérieur n’a été décrit jusqu'ici que par CAMPANA, qui le considère comme un orifice polybronchique simple, c’est-à-dire comme un orifice recevant des bronches tertiaires (parabronches) : les auteurs qui l’ont précédé ou qui l’ont suivi n’en ont pas fait mention. On ne peut en effet regarder comme une description, ces mots de SAPPEY (1847), à propos du réservoir diaphragmatique antérieur (p. 34) que « souvent il existe une seconde ouverture de communication en dehors de l'embouchure du tronc aérifère» (chez le Canard). Cette indication n’a aucune précision : l’auteur n’a représenté nulle part cette seconde ouverture, de telle sorte que l’on ne peut savoir s’il a bien en vue un orifice répondant à celui dont nous parlons ici. Enfin les mots qu’il ajoute, « ce réservoir est le seul qui reçoit l’air du poumon par un double orifice », indiquent bien qu’il n’a point du tout saisi l'importance de la double communication des sacs avec le poumon et le rôle que nous lui attribuons dans la ventilation pulmonaire. On peut en dire autant pour NATALIS GUILLOT (1846) qui a figuré dans le sac diaphragmatique antérieur trois orifices distincts (fig. 2, PI. IV). L'un de ces orifices est placé sur le bord latéral du poumon et répond certainement à notre orifice récurrent : il donne accès dans plusieurs orifices bronchiques voisins les uns des autres. Les deux autres situés sur le bord médial du sac diaphragmatique, au voisinage de sa rencontre avec sa paroi craniale, sont placés immédiatement l’un derrière l’autre ; ils semblent répondre à notre orifice direct et à l'embouchure du canal interclaviculaire qui en est si voisine. Du reste l’auteur ne donne aucun détail dans son texte (p. 56) sur ces trois orifices, et il est bien évident qu'il n’a point vu leur rôle différent et qu'il n’a pas su distinguer l’orifice direct du L'œt r3 POUMON DES OISEAUX 279 récurrent. D'ailleurs cela s'explique aisément raisons que nous allons mettre en lumière. par différentes Cet orifice est situé immédiatement en arrière de celui ou de ceux que l’on a décrit précédemment pour le sac interclaviculaire. Il n’en est séparé que par la lame verticale, formée par la paroi caudale du sac thoracique et la paroi craniale du sac diaphragmatique antérieur accolées. Or, le plus souvent lorsqu’on prépare le poumon pour montrer sa face ventrale, on à soin de couper cette lame aussi bien que les autres formations analogues au ras du tissu pulmonaire, et dans cette opération les deux orifices se trouvent réunis en un seul et confondus. Et du reste, un autre point méconnu de la structure à propos de la même région du poumon, vient encore expliquer la confusion qui à pu se produire pour les orifices dont nous venons de parler. Les bronches récurrentes du sac diaphragmatique antérieur s’ouvrent en effet tout près du bord latéral de la pente caudale du poumon, au voisinage immédiat de la crête qui sépare cette pente caudale de la pente craniale; si l’on se souvient que cette division de la face ventrale du poumon, si nette sur les moulages obtenus à l’aide de la masse de Wood, aussi facile à constater ensuite sur le poumon frais, quand l’attention à été attirée sur elle, a totalement échappé aux auteurs, on comprend aisément que les dispositions anatomiques existant précisément au point de passage de ces deux régions et sur la crête qui les sépare, leur aient complètement échappé. Enfin CAMPANA (1875) qui a bien décrit les différents orifices des sacs aériens fait remarquer (p. 212), que l’orifice externe du sac diaphragmatique antérieur « confine à celui du sac claviculaire. » SAC DIAPHRAGMATIQUE POSTÉRIEUR. — Chez le Poulet ce sac ne contracte avec le poumon que des rapports peu étendus et ne recouvre seulement qu’un petit espace triangulaire, situé sur la partie latérale de la pente caudale du poumon, et la partie tout à fait caudale de cette pente (fig, 1v). Ces rapports 280 A. JUILLET avec la surface pulmonaire sont d’ailleurs différents chez d’autres Oiseaux, comme on le verra p.340. Cet espace triangulaire est à peine plus étendu que le trou par lequel ce sac communique avec le tissu pulmonaire. L'orifice que l’on observe pour ce sac, et qui à été bien figuré par BERTELLI (1905) et par SAPPEY (1847), est, avec l’orifice du sac abdominal, le plus large que l’on puisse observer. Il est ovale, avec son grand axe oblique de dedans en dehors et d'avant en arrière. Il est situé tout à fait sur le bord latéral du poumon et son tiers externe s’étend contre la paroi thoracique. Lorsqu'on le regarde par la face ventrale du poumon encore en place dans l’animal couché sur le dos, on aperçoit dans son fond des orifices bronchiques nombreux. On considère habituellement cet orifice comme l’entrée du sac diaphragmatique postérieur ; mais cette manière de voir n’est pas absolument exacte, et il faut préciser quelles sont les communications que le sac diaphragmatique postérieur peut contracter avec le poumon au niveau de cet orifice. Nous savons déjà que CAMPANA (1875) l’a décrit comme un polybronchique et plus exactement comme un polybronchique simple, bien qu’il ait déjà reconnu que, sous le rapport de ses communications, il est plus compliqué que les orifices polybronchiques simples sus décrits (récurrents du sac interclaviculaire et du sac diaphragmatique antérieur). « Il reçoit en effet, outre un assez grand nombre de tertiaires émanant de secondaires externes et aussi de la quatrième divergente (quatrième entobronche), la plus importante des bronches secondaires externes, c’est-à-dire la deuxième. » Mais la présence de cette dernière bronche parmi celles qui aboutissent au sac diaphragmatique postérieur fait justement de cetorifice, quelque chose de très différent des orifices polybronchiques simples jusqu'ici décrits, puisque on trouve à son niveau deux ordres de conduits bronchiques aussi différents que des secondaires et des tertiaires. Voici à notre avis comment on doit comprendre cette structure. POUMON DES OISEAUX 281 Le sac diaphragmatique postérieur présente, comme les deux sacs qui le précèdent, deux sortes d’orifices de communication avec le poumon : parmi ces orifices l’un est direct O, d, p, les autres À, d, p, sont récurrents (fig. 1v). Seulement, à cause de l'étendue très réduite de la partie de ce sac en contact avec le poumon, ces orifices se sont rapprochés les uns des autres jusqu’à se confondre, comme on le verra plus loin en étudiant leur embryogenèse. Cependant, l’examen même superficiel des moulages métalliques permet de comprendre facilement la disposition du tronc direct et des troncs récurrents, et l’orifice du sac diaphragmatique postérieur est l’un de ceux où l’existence des troncs récurrents se montre avec le plus d’évidence. Etudions séparément sur des injections métalliques les deux sortes de conduits directs et récurrents qui aboutissent à cet orifice. Le tronc d’origine ou le conduit direct du sac diaphragmatique postérieur (O, d, p, fig. 1 et 2), naît à peu près sur le milieu de la mésobronche assez profondément dans l’épaisseur du tissu pulmonaire. Ce conduit, qui fait partie du groupe des bronches secondaires externes de CAMPANA, se dirige latéralement et un peu ventralement, si bien que sa portion terminale est placée sur la face ventrale du poumon, tout contre le diaphragme ornithique. Dans cette partie de son trajet, il s’élargit en allant d'avant en arrière, de sorte qu’au voisinage de son embouchure dans l’orifice du sac il est beaucoup plus large qu’à son insertion sur la mésobronche. Par cette disposition, par son évasement de plus en plus prononcé à mesure qu’il s'éloigne de la mésobronche, il offre les caractères d’une bronche récurrente. Mais on ne peut oublier que par son insertion directe sur la mésobronche, par ce fait qu’il se continue d’une manière tout à fait rectiligne à partir de ce point Jjusqu’à son ouverture dans le sac, il constitue bien véritablement le conduit direct du sac diaphragmatique postérieur : d’ailleurs les troncs récurrents sont faciles à trouver. Ils sont placés latéralement à lui, sur la face ventrale et sur la face dorsale du poumon (À. d. p. fig. 1 et 2), et se dirigent d’arrière en avant 282 A. JUILLET en se dichotomisant pour s'unir avec les conduits aériens qui parcourent ces surfaces. Sur les pièces métalliques on voit que ces troncs directs et récurrents sont réunis à leur base en une masse commune qu'il faut sectionner lorsqu'on veut détacher le sac diaphragmatique postérieur. Au point de vue physiologique, il y a lieu de faire quelques remarques suggérées par la forme du conduit direct du sac. Il est évident que dans l’inspiration, l’air extérieur arrive dans le sac diaphragmatique postérieur par le tronc direct inséré sur la mésobronche, mais à cause de l’évasement de ce tronc et des innombrables orifices parabronchiques qui le criblent. il est bien évident que dans l’expiration, le tronc d’origine lui-même, tout comme les bronches récurrentes placées à côté de lui, sert de voie récurrente, c’est-à-dire dissémine dans le parenchyme pulmonaire l’air contenu dans le sac. SAC ABDOMINAL. — Ce sac en rapport seulement avec le bord caudal du poumon (fig. IV) est pourvu d’un orifice unique, large, ovalaire, qui par son bord dorsal s'attache à la septième côte et dont le bord ventral est formé par une arcade fibreuse développée dans le bord caudal du diaphragme ornithique. Le grand axe de cet orifice ovalaire est à peu près transversal, bien qu’un peu oblique d’avant en arrière et de dedans en dehors. Chez le Poulet, les orifices des deux sacs abdominaux ne sont pas absolument symétriques par rapport au plan sagittal le bord médial de l’orifice droit étant à 22 millimètres du plan sagittal, tandis que le gauche est à 25 millimètres. Cette asymétrie observée chez la femelle tient sans doute à la présence de l’ovaire développé du côté gauche et qui aurait repoussé latéralement l’orifice du sac correspondant. Comme pour le sac diaphragmatique postérieur, cet orifice n’est unique qu’en apparence, car il répond à deux sortes de canaux qui convergent vers lui : un canal direct et des canaux récurrents. Le canal direct est formé par l’extrémité distale de la mésobronche qui vient s’ouvrir directement dans le sac comme l’ont constaté tous les auteurs (0. ab. fig. 1v et fig. 1 et 2) ; les conduits récur- a 1 SU POUMON DES OISEAUX 283 rents sont formés par un certain nombre de bronches qui naissent tout autour de l’orifice direct, mais en plus grande abondance sur son bord latéral et sur sa face dorsale (R. ab. fig. 1v et fig. 1 et 2). Ces bronches se placent immédiatement sous la face dorsale du poumon et se dirigent cranialement en conservant, malgré quelques anastomoses transversales et obliques, une individualité très nette, jusqu’un peu en arrière du sillon de la cinquième côte. À partir de ce dernier elles se perdent dans le réseau inextricable des bronches qui occupent toute la moitié latérale des faces dorsale et ventrale du poumon. D’habitude deux des bronches récurrentes les plus latérales, appartenant à l’orifice du sac abdominal naissent sur un trone commun court et constituent grâce à cela, un des exemples les plus nets qui se puissent voir de bronches récurrentes. C’est un trone semblable, bien qu’un peu différent qui a été représenté dans diverses photographies de G. FISCHER (1905), notamment dans la figure 5Taf. IV chez le Canard, dans la figure 15 Taf. V chez le Poulet, dans la figure 16 Taf. V chez le Pigeon. Il est évident que ces bronches récurrentes fournissent une grande partie de l’air qui remplit les conduits aériens de la moitié latérale du poumon où il se rencontre avec l’air inspiré apporté par les rameaux latéraux des dernières entobronches, et ainsi le territoire anas- tomotique de la moitié latérale du poumon est un des points où se ferme le circuit aérien entre le trajet de l’air inspiré et de l’air expiré. L’orifice du sac abdominal est considéré par CAMPANA (1875) comme un orifice polybronchique mixte. Il comprend sous ce nom les orifices : « dont la caractéristique est d'établir des * communications directes entre deux réceptacles (sacs) distincts, ou bien entre un réceptacle et l’air extérieur par la bronche trachéale, sans préjudice d’ailleurs des communications ordinaires avec les bronches » (p. 213). Cette dernière partie de la description s’applique justement à l’orifice du sac abdominal qui comprend à la fois l'extrémité de la mésobronche et les orifices bronchiques qui l’entourent. 284 À. JUILLET Nous voyons donc bien maintenant l’idée que se faisait CaAmPANA (1875), de la constitution des orifices pulmonaires des sacs aériens. Comme je l’ai dit, il en distinguait trois sortes chez le Poulet: 10 les orifices monobronchiques conduisant directement d'une grosse bronche dans un sac aérien, orifices toujours simples et placées près du: bord médial du poumon comme le sont les grosses bronches elles-mêmes (orifice du sac cervical et orifice interne ou direct du sac diaphragmatique antérieur) ; 20 Les orifices polybronchiques simples, situés sur le bord latéral du poumon et qui sont caractérisés par ce fait qu’ils reçoivent des bronches tertiaires ou parabronches (orifice latéral ou récurrent du sac interclaviculaire, orifice latéral ou récurrent du sac diaphragmatique antérieur, orifice du sac diaphragmatique postérieur). 30 les orifices polybronchiques mixtes, définis par CAMPANA comme nous l’avons dit précédemment (p. 283) c’est-à-dire, reliant tantôt deux réceptacles voisins (canal direct du sac interclaviculaire), tantôt mettant en communication directe un réceptacle avec la bronche trachéale et les bronches (sac abdominal). Il résulte de toute notre description, que le sac diaphragmatique postérieur doit aussi être placé dans cette catégorie. Or, CAMPANA est obligé pour le sac diaphragmatique postérieur, de faire un accroc au principe de sa classification, puisqu'il reconnaît que son orifice reçoit non seulement des bronches tertiaires, mais aussi une bronche secondaire, la plus grosse des bronches du groupe des secondaires externes. Cette inadvertance de l’auteur indique bien qu’il n’a pas compris lui-même le rôle des différentes bronches aboutissant à un de ces orifices polybronchiques, sans cela il aurait bien vu que l’orifice du sac diaphragmatique postérieur, chez le Poulet, se trouve exactement dans les mêmes conditions que lorifice polybronchique mixte du sac abdominal. En effet on voit aboutir à ces deux orifices, un gros tronc qui vient directement de la bronche POUMON DES OISEAUX 285 souche, et qui n’est autre chose que la terminaison même de cette bronche, c’est-à-dire un canal qui est incontestablement la voie la plus directe pour l’apport de l’air extérieur dans le sac. Tout autour, sont disposés des canaux bronchiques qui ne sont plus en relation directe avec les premières voies aériennes intra-pulmonaires, mais vont se perdre au contraire, en se ramifiant, dans le réseau des fines bronches tertiaires ou parabronches circulant dans le parenchyme pulmonaire. De cette discussion, il apparaît clairement que CAMPANA (1875), n'a pas eu la notion des bronches récurrentes. Sans doute, il a vu et figuré les principales d’entre elles, c’est-à-dire, celles du sac diaphragmatique antérieur (c-d, fig. 11), celles du sac diaphragmatique postérieur dans cette même figure du texte (f) et dans la photographie 11, PL IV, celles du sac abdominal dans les figures 11 et 13 du texte et dans la photographie 13, PI. IV. Mais il les considère comme résultant « de la reconsti- tution en un seul tronc de plusieurs tertiaires proprement dites, convergeant pour venir s’unir les unes avec les autres » (p. 45). « Elles sont placées, dit-il à la même page, entre les circuits bronchiques et les réceptacles dont elles sont les troncs collecteurs. » Il semblerait par cette phrase que CAMPANA ait saisi une partie de la nature des bronches récurrentes à savoir leur rôle physiologique exprimé par ces termes que les bronches plus volumineuses que les tertiaires et que l’on trouve à la périphérie du poumon sur son bord latéral, sont des troncs collecteurs de l’air contenu dans les sacs aériens ; maïs faute de connaissance sur le développement des sacs et en particulier, sur le développement des bronches récurrentes de ces sacs, que je crois être le premier à mettre en lumière, il n’a pas saisi leur véritable nature anatomique et n’a pas su en donner une compréhension complète. Il comprenait leur origine d’une manière exactement inverse de ce qu’elle est en réalité, les faisant naître de tertiaires qui, après s'être fusionnées, allaient rejoindre les sacs, tandis que lembryologie nous les montre naissant des sacs pour aller se perdre dans le parenchyme 286 A. JUILLET pulmonaire. D'ailleurs, ces notions, passablement obscures dans l’ouvrage de CAMPANA, n’ont point trouvé asile dans les descriptions classiques du poumon des Oiseaux, et nous voyons G. FiscHer (1905) ignorer que la grosse bronche qu’il mentionne comme non décrite, à propos de la figure 5 Taf. IV du Canard, avait été signalée en détails par CAMPANA. Nous substituerons donc à la description que cet auteur donne pour les orifices des sacs, ce court exposé qui résume bien le résultat de nos recherches sur ce point : Les sacs aériens peuvent communiquer avec le poumon par deux espèces d’orifices : les orifices directs et les orifices récurrents. Les premiers relient un sac au vestibule ou à la mésobronche par l'intermédiaire d’une bronche toujours de fort calibre, de telle sorte que l’air inspiré est directement conduit dans le sac, par l’intermédiaire de ce canal, qui constitue la voie la plus largement ouverte aux gaz inspirés. Les autres, orifices récurrents, conduisent l’air du sac aérien lui-même, dans les bronches superficielles qui se ramifient et vont dans le parenchyme pulmonaire. Certains sacs ne possèdent qu’un orifice : l’orifice direct (sac cervical) ; tous les autres présentent les deux sortes d’orifices, directs et récurrents, mais ces orifices peuvent être distincts ou réunis sur un même territoire. Les orifices directs sont distincts des orifices récurrents dans le sac interclaviculaire et dans le sac diaphragmatique antérieur, parfois même dans le sac diaphragmatique postérieur. Dans tous ces sacs les orifices directs sont toujours monobronchiques, les orifices récurrents sont au contraire polybronchiques simples parce qu’ils renferment plusieurs bronches de la même catégorie (récurrentes) ; ils sont d’autre part situés plus latéralement que les orifices directs. Dans d’autres cas, orifices directs et orifices récurrents sont réunis sur une même aire ovale, au milieu de laquelle, en général, se trouve l’orifice direct envi- ronné des récurrents. C’est le cas pour le sac diaphragmatique postérieur (Poulet, Pigeon, Canard), et pour le sac abdominal. On peut donner le nom d’orifices polybronchiques mixtes aux d POUMON DES OISEAUX 287 orifices ainsi constitués puisqu'ils renferment deux catégories différentes, une bronche bronches récurrentes. des bronches de directe et des Rôle physiologique des bronches récurrentes La question des bronches récurrentes joue un grand rôle dans la compréhension de la physiologie du poumon chez les Oiseaux, et elle permet de se rendre compte des divergences qui se sont exprimées parmi les auteurs qui ont étudié cet organe. La distribution des bronches de divers ordres, l'insertion des conduits directs sur les grosses bronches, celle des conduits récurrents sur les bronches de troisième ordre et sur des points de leur parcours où elles sont largement et fortement anastomosées entre elles, montrent à l'évidence que les sacs aériens jouent un rôle considérable dans la ventilation pulmonaire. Ils reçoivent de l’air presque pur pendant l’inspiration et le refoulent dans le poumon au moment de leur resserrement, lui fournissant ainsi une grande partie de l’air nécessaire à l’hématose. Les expériences de Soum (1896), montrant que l’on peut ouvrir les sacs pulmonaires sans tuer l’animal, ne prouvent point du tout que les sacs ne servent pas à injecter l’air destiné à la respiration, mais elles montrent plutôt que l’air peut passer à travers le parenchyme pulmonaire sans l’intervention de leur resserrement ou deleur dilatation alternatifs. Ce passage de l’air se comprend aisément, si l’on réfléchit que les voies aériennes du poumon forment des circuits complets dont chacun présente au moins deux ouvertures, une directe et une récurrente, donnant sur l’air extérieur (si l’on met la cavité du sac en communication avec ce dernier par un trou fait à sa paroi), la circulation aérienne est tout à fait facile et se produit comme l’a vu Soum, par la dilatation rythmée des bronches, qui accompagne les efforts violents faits par l'animal pour respirer. ROCH, DE ZOOL. EXP. ET GÉN. — 5° SÉRIE. — T, IX. — (II), 19 288 A. JUILLET SIEFERT qui à fait, à la même époque que MAx BAER et que Soum (1896), des recherches sur la respiration des Oiseaux admet comme nous que les ouvertures bronchiques de la face médioventrale des poumons, mises en communication avec l'air extérieur, peuvent remplacer d’une manière parfaite la trachée dans l’apport de l’air et sont susceptibles d’inspirer et d’expirer l’air atmosphérique (1896, p. 447). Mais cet auteur a tort de conclure de ses expériences que (p. 474) : « ni les sacs aériens, ni le prétendu diaphragme ne sont nécessaires pour assurer le renouvellement de l’air dans les poumons et qu’il ne reste rien autre que de voir dans l’action directe des mouvements des côtes sur les poumons, la cause mécanique du renouvellement de l’air respiratoire. » Les expériences de MAx BAER sur l’apnée obtenue par insufflation du sac claviculaire ou même de l’un quelconque des autres sacs, comme il l’indique d’une manière précise (1896, p. 489-490), justifient au contraire le rôle que nous attribuons à ces sacs dans la ventilation pulmonaire au cours de la vie normale de lOiseau. Si la nature des bronches récurrentes avait été comprise par CAMPANA et si les détails de cette structure pulmonaire n'avaient pas échappé aux physiologistes qui ont traité la question après lui, il n’y aurait pas tant de contradictions dans leur manière de voir. CAMPANA était dominé par l’idée de l’antagonisme des sacs aériens qui à régné pendant si longtemps. Pour lui, comme pour les auteurs qui l’avaient précédé, avec des différences de détail, les sacs diaphragmatiques s’opposaient à tous les autres en ce qu’ils étaient inspirateurs, tandis que les sacs extrèmes, cervical et interclaviculaire, puis abdominal, étaient expirateurs. D’après ces auteurs, l’air extérieur qui est introduit par la trachée ne pénètre que peu ou point dans le parenchyme pulmonaire et se rend dans les réceptacles diaphragmatiques où il se mélange à de l’air venu des réceptacles expirateurs, soit directement par l'intermédiaire du canal interclaviculaire, soit POUMON DES OISEAUX 289 indirectement par des circuits bronchiques qui aboutissent aux orifices polybronchiques. La composition de l'air contenu dans les sacs expirateurs est à peu près la même, sauf que l’air extérieur ne parvenant aux réceptacles extrêmes que par l’intermédiaire des réceptacles voisins, l’air pur s’y trouve naturellement en proportion un peu moindre que dans ces derniers. L'air respirable est donc en première ligne, celui des réceptacles moyens, en seconde ligne et avec une faible différence, celui des réceptacles expirateurs. Le premier est injecté dans le poumon pendant l’expiration trachéale, le second y est injecté au contraire pendant l'inspiration. « Le poumon ne reçoit pas probablement l’air extérieur à l’état de pureté; mais il est parcouru sans intermission et en sens alternatif par un courant d'air de composition faiblement variable, pas très différente de l’air expiré par la trachée, et venant tour à tour des réceptacles expirateurs et des réceptacles inspirateurs » (1875, p. 231). Ainsi CAMPANA a bien montré le rôle des sacs dans la ventilation pulmonaire. Il est indiscutable que le parenchyme pulmonaire ne reçoit que de Pair ayant déjà séjourné dans les sacs, s’y étant réchauffé et humecté. Les sacs sont avant tout des ventilateurs pulmonaires. Mais l’antagonisme des différents groupes de sacs aériens est-il aussi marqué que le croyaient CAMPANA et ses prédécesseurs ? Cela n’est pas absolument certain, et SouM s’est efforcé de montrer qu’il y avait non pas antagonisme entre les sacs, mais bien plutôt synergie ou même, pour exprimer ce fait avec plus de force, synagonisme comme il le dit lui-même. Il n'entre point dans le cadre de cette étude de discuter les expériences de SouM, mais il semble nécessaire de relever certaines opinions de l’exposition de cet auteur qui repoussées par lui, faute de connaissances anatomiques suffisantes, sont fort bien expliquées par ma description du système bronchique. Soum écrit (1896, p. 95) : « L’air n’est aspiré ou injecté dans le parenchyme pulmonaire par le jeu des sacs aériens à aucun moment. Etant donné le nombre et la largeur des bronches 290 A. JUILLET par lesquelles les différents sacs sont mis en rapport avec la trachée et la facilité avec laquelle l’air y circule, soit à l’aller, soit au retour, il est permis de supposer que cet air, sous l'influence des réceptacles, subit seulement un va et vient dans ces grosses bronches sans pénétrer dans la substance pulmonaire dont les derniers capillaires aériens ont seulement un diamètre de 0 mm. 012 (en raison de la grande résistance qu’ils rencontreraient dans ces derniers conduits) ». Cela est vrai pour quelqu'un qui ne connaît pas l’existence des bronches récurrentes ou la disposition physiologiquement récurrente des conduits directs des sacs diaphragmatique postérieur et abdominal. Mais ilest bien évident que dans les mouvements d'expiration et de resserrement plus complets des sacs, comme il s’en produit à intervalles réguliers par la volonté de l'oiseau, une grande partie de l’air expiré est forcée de traverser le parenchyme pulmonaire, comme le montre en particulier la disposition infundibuliforme du conduit direct du sac diaphragmatique postérieur, si évidemment favorable à la dispersion dans le parenchyme pulmonaire de l'air inspiré par ce sac. La phrase qui résume l’opinion de Soum (1896, p. 86) : « Les sacs aériens assurent la ventilation de la trachée, des grosses bronches et peut-être du parenchyme qui entoure immédiatement ces dernières », n’exprime donc pas suffisamment le rôle des sacs, et il est indubitable que le parenchyme pulmonaire est parfaitement bien ventilé par eux. Toutefois, la question de l’antagonisme est probablement résolue dans le sens de Soum, et, comme le disait déjà COLLIN, il est bien probable que « lors de l’inspiration, l'air extérieur doit être appelé à la fois dans les sacs thoraciques et les abdominaux seulement, beaucoup plus dans les premiers que dans les seconds, puisque le thorax s’agrandit largement, tandis que l’abdomen éprouve un simple relâchement » (référence de Soum (1896, p. 69). En même temps que Soum et tout à fait indépendamment de lui, MAx BAER (1896) arrivait à la même conclusion que POUMON DES OISEAUX 291 tous les sacs à la fois sont dilatés dans l'inspiration et resserrés dans l’expiration, qu’ils se comportent done de même et qu’il n’y a point d’antagonisme entre eux; mais il a parfaitement compris le rôle énorme de ces sacs dans la ventilation pulmonaire, comme le prouve cette phrase que, pendant le rétrécissement du thorax « tous les sacs sont comprimés et leur contenu est versé dans les ouvertures bronchiques ; mais il ne peut pas arriver au dehors sans passer par le poumon, d’où il sert ainsi à l’hématose et balaye en même temps au dehors l’air déjà utilisé » (1896, p. 79). Les descriptions des bronches récurrentes et l'opposition qu’il y a entre elles et les conduits directs au point de vue des rapports immédiats avec la trachée, expliquent très aisément l’idée physiologique de Max BAER. Lorsque CAMPANA établissait d’une façon si précise l’antagonisme des sacs diaphragmatiques avec les sacs extrêmes, il s’appuyait surtout sur une disposition anatomique qu’il avait découverte et qui lui faisait croire à une communication indispensable entre des sacs qu'il regardait comme antagonistes. Cette disposition était le canal interclaviculaire reliant le sac interclaviculaire au sac diaphragmatique antérieur. Il imaginait que d’autres communications moins directes s’éta- blissaient entre l’abdominal et le diaphragmatique antérieur par les bronches tertiaires qui s’étendent à la face ventrale du poumon entre les orifices de ces sacs. Ces dernières communi- cations indirectes ne sont vraiment point à comparer avec le canal du sac interclaviculaire et des moulages supérieurs à ceux de CAMPANA montrent bien que les bronches placées autour de ces orifices conduisent bien plus directement dans le parenchyme pulmonaire que d’un sac à un autre. Mais en outre ces communications entre sacs ne sont point indispensables et l’on verra plus loin que le canal interclaviculaire peut manquer chez certains Oiseaux où le mécanisme de la ventilation pulmonaire est un peu différent. Ce que nous retiendrons donc de la description de CAMPANA, 292 A. JUILLET ce n’est pas tant sa théorie de l’antagonisme des sacs, qui a plutôt obscurci dans son esprit la structure pulmonaire, que sa description excellente des orifices des sacs. Nous verrons alors que la disposition de ces orifices.s’accorde très bien avec le rôle de ventilation attribué aux sacs : du reste, en étudiant attentivement cette distribution et celle des bronches intrapulmonaires, on constate que la théorie de l’antagonisme, dépouillée de labsolutisme que lui attribuait CAMPANA et prise d’une manière plus large, moins exclusive, est encore vraie, de sorte que l’on comprend parfaitement qu’elle ait été adoptée par la presque unanimité des physiologistes, y compris illustre PAUL BERT qui, tout en l’acceptant dans ses grandes lignes, ne lui attribuait point la valeur exclusive qui lui a été donnée par CAMPANA et par SAPPEY. Reprenons, en effet, les données acquises à propos de chaque sac. Nous voyons que le sac cervical présente un seul orifice monobronchique, situé assez près de l’origine de la première entobronche et par conséquent un orifice direct. Ce sac doit jouer un faible rôle dans la ventilation pulmonaire. Il n’est du reste pas compris dans une région subissant des alternatives de resserrement et de dilatation ; cependant, il est probable que dans certains mouvements, comme celui de rentrer le cou dans les épaules (pendant le vol ou la marche), l’air qu’il renferme est repoussé dans le poumon et peut servir à assurer en partie la ventilation pulmonaire. Cependant, à cause de sa situation sur une très grosse bronche voisine de la trachée, l'air expiré par lui doit certainement passer en partie dans une des grosses bronches voisines de cette dernière et delà au dehors. Le sac interclaviculaire présente toujours un orifice direct et le plus souvent aussi un orifice récurrent. Ce dernier peut toutefois manquer chez de bons voiliers comme le Pigeon; mais dans ce cas, la surface du sac est en rapport avec le poumon sur un espace très restreint et l’air expiré par le sac inter- l For in ns 1 POUMON DES OISEAUX 293 claviculaire est aisément reçu par les bronches récurrentes du sat diaphragmatique antérieur. Ce qui caractérise le plus souvent le sac interclaviculaire, c’est son canal de communication avec le sac diaphragmatique antérieur. Cette communication à une importance considérable, notamment pendant le vol où elle joue un grand rôle dans la ventilation pulmonaire. On ne peut douter en effet que le sac interclaviculaire puisse assurer à Lui seul, par les mouvements dont il est le siège, la ventilation pulmonaire. Max BAER a montré que l’on pouvait mettre un oiseau en apnée, lui fournir tout l’air nécessaire à la respiration, sans l’aide d'aucun mouvement respiratoire, si l’on insufflait l’air par un humérus en le laissant sortir par la trachée (1896, p. 489). Pendant le vol, les prolongements sous-alaires du sac claviculaire sont soumis à des alternatives de resserrement et de dilatation qui entraînent un mouvement alternatif de l’air contenu dans le sac interclaviculaire, mouve- ment qui à certainement pour résultat d'amener une large ventilation du territoire de ce sac lorsque les bronches récurrentes y existent, ou, lorsque ces dernières n'existent point de déterminer cette ventilation dans le territoire du sac diaphragmatique antérieur, grâce à la large communication du canal interclaviculaire. Il ne faut point oublier que dans ces cas les bronches récurrentes du diaphragmatique antérieur s’insérent en face du canal interclaviculaire lui-même, de façon à assurer un passage facile de l'air refoulé dans ces grosses bronches récurrentes (voy. fig. xv). Lorsqu'on envisage l’existence constante de ce canal (les Ansériformes — Oie, Canard, Sarcelle — exceptés), on se rend bien compte qu'il est destiné à solidariser les sacs interclaviculaires et diaphragmatiques antérieurs vis-à-vis de la respiration pendant le vol, et l’on com- prend toute l’importance apportée par CAMPANA à cette communication inter réceptaculaire. On comprend aussi comment cet auteur pouvait considérer les sacs diaphragmatiques comme les inspirateurs par excellence, puisque le canal inter- 294 A. JUILLET claviculaire reçoit lui-même son air par le conduit direct du sac diaphragmatique antérieur inséré sur la troisième entobronche. C’est par ce canal de la troisième entobronche que pendant le vol pénètre tout l’air inspiré par les mouvements alaires et que les sacs diaphragmatiques se montrent comme les principales voies d'apport de l'air extérieur dans le parenchyme pulmonaire. Enfin la constance des bronches récurrentes aux orifices des sacs diaphragmatiques postérieurs et abdominaux, leur puissance et leur disposition sur la pente caudale du poumon, montrent à l’évidence le rôle considérable que ces bronches doivent avoir dans la ventilation pulmonaire. Ce rôle n'avait pas échappé aux partisans de la théorie de l’antagonisme qui faisaient de ces sacs les expirateurs par excellence. Mais leurs fonctions se trouvent être considérablement amplifiées par la découverte de leurs bronches récurrentes qui, au cours de la contraction de ces sacs refoulent l’air qu’ils contenaient dans le parenchyme pulmonaire, l’y disséminent par les innombrables anastomoses qui parcourent la partie caudale du poumon, tant sur ses faces ventrale et dorsale que dans sa profondeur. Les bronches récurrentes assurent un balayage de l’air confiné par du fluide respiratoire, non seulement dans cette région caudale mais encore dans toutes les ectobronches et les entobronches, grâce aux anastomoses précitées et que CAMPANA avait lui-même si bien décrites. Loin d’être indépendant, le rôle des sacs diaphragmatiques postérieurs et abdominaux complète celui déjà décrit pour les sacs diaphragmatiques antérieurs et interclaviculaires et ils concourent eux aussi dans une très large mesure à la ventilation parfaite du poumon. Ont-ils comme les précédents un rôle primordial pendant le vol ? Nous ne pouvons rien affirmer à cet égard, et seules des expériences qui n’entrent pas dans le cadre de cette étude seraient susceptibles de le démontrer. La constance et le grand développement des bronches récurrentes de ces sacs chez de très bons voiliers (Mouette, Goéland, Canard sauvage, etc.), POUMON DES OISEAUX feraient penser à leur intervention probable 295 dans la venti- lation pulmonaire pendant le vol. A notre avis, cependant, ces sacs doivent avoir d’après leur situation anatomique et leurs rapports avec les viscères qui les entourent, une fonction mécanique concourant à diminuer le poids spécifique de l’animal, à maintenir son centre de gravité et à amplifier sa voix. Leur intervention dans la respiration serait surtout efficace pendant la marche ou pendant le repos de l'oiseau, les sacs interclaviculaires et diaphragmatiques antérieurs ayant alors un rôle moindre. CHAPITRE DÉVELOPPEMENT IV DU POUMON ET DES SACS Le poumon prend assez rapidement chez l’embryon une structure caractéristique qui permet de reconnaître de bonne heure les principales dispositions réalisées chez l'adulte. Au début du sixième jour il ne présente d’après Fanny Moser (1902) que la mésobronche qui le traverse longitudinalement et l’ébauche des quatre premières bronches diaphragmatiques ou entobronches. Sous cet état, il n'offre encore rien de bien caractéristique et sur les coupes transversales, l’ébauche pulmonaire tout entière avec les conduits épithéliaux qu’elle renferme et le mésenchyme qui les enveloppe ne diffère pas de l’ébauche pulmonaire des autres Amniotes d’une manière suffisante pour attirer l’attention. Mais il n’en est pas de même à la fin du sixième jour, et le poumon d’un embryon de cet âge dont la reconstruction plastique des conduits aériens est représentée dans la figure 4 est tout à fait caractéristique des Oiseaux. Nous commencerons par lui notre description embryologique qui est divisée en trois parties : 19 Développement des grandes voies aériennes (mésobronche, ento- et ectobronches) ; 20 Développement des bronches récurrentes ; 3° Développe- 296 A. JUILLET ment des parabronches et du parenchyme pulmonaire. Le résumé de nos observations embryologiques a déjà été publié (A. JUILLET, 1911, c). DÉVELOPPEMENT DES GRANDES VOIES AÉRIENNES. — Dans l'embryon de la fin du sixième jour la bronche extra-pulmonaire entre dans le poumon sous la forme d’un tube cylindrique étroit, à peine arrivée dans l’ébauche pulmonaire elle se continue par un tronc qui ne tarde pas à devenir beaucoup plus volumineux et de calibre irrégulier et qui répond au vestibule et à la mésobronche. Ce tronc parcourt toute la longueur de l’ébauche pulmonaire en décrivant un $ allongé dont la partie moyenne, plus étendue que le reste, est contiguë à la face latéro-dorsale de l’ébauche pulmonaire. Sur cette bronche souche apparaissent à l’état d’ébauches plus ou moins avancées les principaux troncs bronchiques. La première entobronche E1 implantée sur la partie initiale même du tronc intra-pulmonaire se présente sous la forme d’un canal cylindrique légèrement courbé du côté ventral et qui se dirige médialement pour se renfler, sur le bord médial de l’ébauche, en une vaste poche irrégulière présentant des bourgeons au nombre de cinq dont deux sont bien visibles sur la figure 4. L’un répond au sac cervical, l’autre au rameau transverse de la première entobronche de l’adulte. Les autres bourgeons, non visibles sur la figure, donneront naïssance aux bronches et aux parabronches situées sur le bord médial de la pente craniale. La deuxième entobronche Ez naît immédiatement en arrière de la précédente et, comme celle-ci, par un court conduit qui se bifurque bientôt en un rameau cylindrique Ez r.d., se recourbant dorsalement par rapport à la trachée et formant le rameau dorsal de l’adulte, puis en un deuxième rameau également cylindrique à son origine, qui décrit un S vers la face venrale du poumon et se termine du côté médial par un renflement aplati dans le sens dorso-ventral et portant déjà quelques extroflexions peu marquées, premiers indices des POUMON DES OISEAUX 297 bourgeons parabronchiques qui ne tarderont pas à paraître. La troisième entobronche E; succède immédiatement à la précédente : elle comprend un tronc d’origine à peu près cylindrique qui se dirige vers la face ventrale du poumon, l’atteint et là se divise en deux renflements, l’un qui s’étend dans la partie médiale de l’ébauche pulmonaire est large, aplati et présente une série de petites bosselures, bourgeons de futures parabronches ; l’autre $.d.a vient faire une légère saillie sur la face ventrale et correspond à l’ébauche du sac diaphragmatique antérieur. La quatrième entobronche E4 succède aux précédentes et se dirige sur la face ventrale du poumon ; après s’être un peu élargie, elle se divise en deux prolongements, l’un longitudinal constitue l’ébauche de son tronc principal : l’autre transversal représente le tronc correspondant de l’adulte. ‘Les quatre entobronches se succèdent sans intervalle sur la portion initiale du tronc souche qui deviendra plus tard le vestibule mais qui, pour le moment, ne se distingue point du reste, bien que FaAnNNY Moser croie ie reconnaître dès ce stade. Cette portion du tronc souche forme une zone de transition graduelle entre la bronche extra-pulmonaire régulièrement cylindrique et la mésobronche beaucoup plus renflée et à section plus nettement ovale. Il faut remarquer que les troncs originels des entobronches, nés assez profondément dans l’épaisseur de l’ébauche pulmonaire se dirigent d’abord vers la face ventrale pour s’y étaler et atteindre ensuite la face médiale ; de cette façon, le lieu d’origine des parabronches qui vont en naître est toujours très périphérique, très écarté du centre de l’ébauche pulmo- naire (fig. 4) (1). La portion originelle des entobronches È est chez l'embryon relativement beaucoup plus étendue que chez l’adulte : elle (1) En arrière de la quatrième entobronche on voit parfois un petit bourgeon cylindrique légèrement recourbé qui suit la direction de la quatrième entobronche. C’est sans doute l’ébauche d'une cinquième entobronche que l’on observe dans quelques cas chez l’adulte. 298 A. JUILLET occupe en effet toute la partie supérieure de l’S renversé, représenté par le tronc souche. En arrière de la dernière entobronche, la courbure de PS s’enfonce profondément, de manière à amener la nrésobronche jusque sous la face dorsale de l’ébauche pulmonaire. Dans cette portion de son trajet la mésobronche est élargie, un peu aplatie et donne naissance à différents troncs bronchiques. De ces troncs, cinq naissent du côté médial, et cinq du côté latéral. Ceux du côté médial sont plus volumineux, ils se dirigent sur la face médiale du poumon et répondent manifestement aux ectobronches. La première ectobronche dont l’origine ne se voit pas sur la figure parce qu’elle est placée du côté médial, est séparée de la dernière entobronche par un intervalle assez marqué. Elle offre à son extrémité distale et surtout du côté dorsal, un élar- gissement analogue à ceux aue nous avons trouvés jusqu'ici sur les gros troncs bronchiques. La deuxième et la troisième ectobronches succèdent à la première et leur insertion sur la mésobronche se rapproche du bord médial comme on l’a vu chez l'adulte. La quatrième ectobronche est un peu plus éloignée du côté caudal. Enfin, plus loin encore, se trouve la cinquième ectobronche Ect. % tout à fait sur la convexité de la courbure extrême de PS. Les bourgeons latéraux qui naissent sur la face dorso-latérale du tronc souche dans la portion moyenne de son trajet, sont toujours plus petits que les bourgeons des ectobronches : ils sont au nombre de cinq. L’antérieur est placé dorsalement sur la mésobronche à côté de la première ectobronche : trois autres b.s.{. suivent le premier dont ils ne sont séparés que par un léger intervalle et se dirigent sur le bord latéral en formant des tubes étroits à peine renflés à leur extrémité. Le cinquième est un peu aplati et à peine saillant sur la figure 4. Ces bourgeons répondent au groupe des bronches secondaires latérales de CAMPANA, lesquelles restent toujours plus petites que les ectobronches et les entobronches, et se confondent bientôt POUMON DES OISEAUX 299 dans la profondeur du poumon avec les parabronches issues des autres gros troncs bronchiques. On les distingue très bien sur les reconstructions de poumon d’embryons plus avancés, où elles ont atteint un développement un peu plus grand mais qui contraste toujours par sa faiblesse avec le développement des ectobronches. L’un des bourgeons représentés dans la figure 4, le troisième, répond au canal direct du sac diaphragmatique postérieur. Les bronches qui prennent naissance sur le tronc souche ne sont point disposées irrégulièrement sur tout le pourtour de ce tronc, mais elles forment en somme deux rangées principales : l’une qui décrit une sorte de spire très allongée, est formée par les entobronches et les ectobronches qui peuvent en effet être reliées par une ligne spirale continue commençant sur le bord ventral du tronc souche à la première entobronche, pour se terminer sur le bord dorsal de a mésobronche, au niveau de la dernière ectobronche. La deuxième rangée est constituée par les bronches du groupe latéral de CAMPANA, par conséquent nous trouvons là aussi comme d’HARDIVILLIER (1897) l’a indiqué chez les Mammifères, la sériation linéaire des premières bronches. La terminaison de la mésobronche dans le poumon de six jours mérite d’attirer l’attention (Més. fig. 4). Elle consiste en un tube cylindrique de calibre assez régulier qui décrit une courbure à concavité dorsale très fortement marquée, formant la courbure extrême de LS dont nous avons déjà parlé etque FANNY Moser (1902) avait représentée dans son schéma figure 9, sans insister du reste sur l'importance de cette disposition. Cette importance n’en est pas moins réelle cependant, car les courbures si marquées de la mésobronche et le fait que les principaux troncs bronchiques ne se suivent pas sur ce tronc souche, mais sont disposés en deux groupes distincts, entobronches et ectobronches, placés chacun sur les courbures de l’S, a pour conséquence que les deux groupes de ces grosses bronches se trouvent par là même portés vers 300 A. JUILLET la surface de l’ébauche pulmonaire ; en même temps ils s’écartent largement l’un de l’autre laissant entre eux une partie importante de mésenchyme compact et dépourvu de tout conduit aérien. Cette disposition sera encore plus marquée dans les stades postérieurs comme on le verra dans le poumon d’un embryon de huit jours. Dans la reconstruction d’un poumon de huit jours, effectuée au même grossissement que celle d’un poumon de six jours, ce qui frappe d’abord, c’est l’énorme développement qu’a pris dans le sens dorso-ventral, l’ébauche pulmonaire et que l’on peut exprimer rapidement en disant que le rapport entre la longueur de la reconstruction et son épaisseur qui était de 2 au sixième jour, est maintenant 1,70. Mais en dehors de ce grand changement intervenu dans l’épaisseur, les autres modifications sont peu profondes. Elles se bornent à développer certains détails indiqués dans le modèle précédent et à lui donner une forme plus voisine encore du type de l’adulte, mais néanmoins dans un état intermédiaire très propre à faire comprendre le développement général. C’est pour cette raison qu’il m'a paru inutile de reconstruire des états intermédiaires qui n'auraient certainement point donné de résultat valant la peine et le temps qu’ils m’auraient coûtés. * Dans le poumon de huit jours, le tronc souche garde la forme en S qu'il avait au stade précédent et la partie moyenne de l’'S est toujours voisine de la face dorsale. A ce stade comme au précédent, il est impossible de distinguer, soit par un changement de calibre, soit par tout autre caractère, le vestibule de la mésobronche. Celle-ci est toujours large, sauf dans sa partie caudale, et aplatie dorso-ventralement. Le plus grand changement qui se manifeste dans son étendue porte dans sa partie terminale, sur la courbure extrême de l’S qui se ferme beaucoup, de telle sorte que l’extrémité de la mésobronche est presque pliée à angle droit sur le reste du tronc (caractère qui s'éloigne passablement de la disposition qu’elle montre chez l'adulte). POUMON DES OISEAUX 501 Sur cette extrémité de la mésobronche, est inséré le sac abdo- minal qui forme à ce moment un tube variqueux dirigé directement en arrière (S. ab. fig. 5). Ce tube ne paraît pas prolonger directement la mésobronche comme il le fait chez l'adulte; mais cette disposition tient sans doute à la forte courbure de extrémité mésobronchique dont il vient d’être question. Elle s’effacera peu à peu au fur et à mesure de l'allongement général des parties et du poumon tout entier ; la mésobronche perdra les courbures successives qui, chez les embryons des premiers jours, lui donnent la forme d’un $, pour prendre la direction à peu près rectiligne qu’elle garde chez l’adulte. Reprenons maintenant le détail des diverses bronches dans le poumon de huit jours. La première entobronche (Z:1 fig. 5) se dirige un peu plus cranialement qu’au stade antérieur, et son tronc au lieu d’être transversal est devenu oblique. Son extrémité élargie s’est fendue en trois digitations, élargies elles-mêmes distalement, et présentant à ce niveau des sillons séparant des saillies qui s’indiquent déjà comme des divisions futures. Ces trois grandes digitations répondent aux deux premières, décrites dans l’embryon de six jours. Le petit bourgeon cylindrique désigné comme ébauche du sac cervical s’est allongé et fait une saillie spéciale à la surface du poumon S.c. Des deux bourgeons bronchiques appartenant à cette même entobronche, l’un situé plus médialement, a donné naissance à un rameau bifurqué qui correspond indubitablement au rameau cranial, l’autre, situé plus en dehors et du côté dorsal, forme les branches du rameau latéral de l’entobronche. La deuxième entobronche Æ2 s’est développée dans la même proportion que la première. L’extrémité distale de son rameau médial s’est divisée en trois digitations qui montrent une tendance à se subdiviser elles-mêmes par un plan horizontal et par conséquent à se dédoubler. Son rameau dorsal Æ2 r, d, qui contourne la trachée est très variqueux et présente déjà quelques bourgeons distincts, origines de parabronches futures. 302 A. JUILLET La troisième entobronche ÆZ3 a son extrémité distale largement étalée et divisée en trois digitations principales par des sillons très profonds qui entament toute l’épaisseur de l’élargissement de la bronche. Son extrémité caudale présente aussi une digitation moins marquée, formant l’ébauche du quatrième rameau issu de cette extrémité bronchique. Comme pour la bronche précédente, les bourgeons formés par les incisures qui ont divisé l’élargissement distal, paraissent s’être dédoublés par un plan frontal et forment deux séries superposées de rameaux bronchiques, l’une superficielle, l’autre profonde. La portion latérale de cette troisième entobronche non visible sur la figure 5 s’est beaucoup développée : elle forme une vaste poche placée à la face ventrale du poumon et un peu du côté latéral ; c’est le sac diaphragmatique antérieur qui donne, par son extrémité craniale, un bourgeon très net s’enfonçant dans le tissu pulmonaire et qui constitue l’origine des bronches récurrentes de ce sac. Sur le conduit qui rattache le sac diaphragmatique antérieur à la troisième entobronche est inséré un tube variqueux irrégulier dirigé cranialement et en dehors pour aller se placer cranialement à l’entrée de la bronche dans le poumon et qui constitue l’ébauche du sac thoracique. Ces parties ne pouvaient être représentées sur la figure 5, vue par la face médiale. Pour comprendre leur disposition voir fig. X. La quatrième entobronche, £4 qui au stade précédent avait ses extrémités distales placées encore profondément dans le mésenchyme et assez éloignées de la périphérie de l’ébauche pulmonaire, a atteint maintenant cette dernière, au moins pour ce qui est de son rameau médial qui se comporte à son extrémité distale comme ceux des précédentes entobronches. II se termine en effet, par un élargissement en patte d’oie, aplati contre la périphérie de l’ébauche pulmonaire et qui tend à se diviser par des sillons, en rameaux bronchiques tant superficiels que profonds. Le rameau latéral à beaucoup grandi et s’est porté très nettement vers le bord latéral de l’ébauche. dune POUMON DES OISEAUX 303 Il existe parfois dans cet embryon, une cinquième entobronche dont le tronc irrégulier présente une série de bosselures, origine d’autant de futurs rameaux, mais qui n’est pas vue sur la face représentée de la reconstruction. Les ectobronches ont beaucoup grandi depuis le sixième jour, elles se déploient en un éventail dont les branches partant du bord médial de la mésobronche se dirigent d’abord en dedans puis se recourbent de haut en bas, dans la direction dorsoventrale pour se porter vers les extrémités correspondantes des entobronches qui commencent à s’incliner vers elles (fig. 5). L’extrémité distale des ectobronches qui se replie ainsi le long de la face médiale du poumon et va au-devant des bourgeons entobronchiques présente la même forme que ces derniers, c’est-à-dire un élargissement distal en patte d’oie avec des bronches superficielles et des bronches profondes, indiquées seulement à ce moment par quelques renflements peu distincts. Le groupe des bronches secondaires latérales que nous avons vu exister au stade de six jours, présente maintenant un développement plus considérable, chacune de ces bronches s'étant fortement allongée dans le sens latéral et munie en outre de bronches tertiaires. L’une d'elle, la troisième, est plus longue que les autres et présente à son extrémité distale, un bourgeon spécial qui s'enfonce dans le mésenchyme du diaphragme primaire et qui va devenir le sac diaphragmatique postérieur. Si on examine la face médiale de la reconstruction (fig. 5), on observe une série de dispositions très importantes parce qu’elles font encore ressortir la caractérisation précoce du poumon des Oiseaux, par certains traits particuliers de son ébauche et par le mode de formation des ramifications bronchiques. Sur cette figure, on voit en effet que les ramifications bronchiques sont groupées sur les deux faces dorsale et ventrale du poumon d’une manière exclusive, laissant entre elles un large espace vide occupé par le mésenchyme. La face ventrale nous présente les quatre entobronches ARCH. DE Z00Z. EXP. ET GÉN. — 5° SÉRIE. — T. IX. — (Ill). 20 304 A. JUILLET dont le bord médial élargi offre maintenant une série de digitations correspondant aux bourgeons des parabronches. €es digitations s’infléchissent du côté dorsal, pour passer de la face ventrale du poumon, où elles sont nées, sur la face médiale : . elles semblent ainsi aller au-devant des digitations venues des ectobronches. Dans certains points et notamment sur la troisième entobronche, on distingue nettement deux rangs de digitations superposées, les unes plus externes contiguës à la face médiale de l’ébauche, les autres plus profondes et situées dans l’épaisseur même du mésenchyme. Cette subdivision des extrémités bronchiques, répondant comme il a été dit à la formation de parabronches internes, paraît se faire, comme la formation des digitations elles-mêmes, par une sorte d’étranglement et de pincement des extrémités élargies des entobronches, telles qu’on les a décrites au stade précédent. Par suite, ces extrémités d’abord continues présentent des formations digitées ou pennées, subdivisées même en deux plans, ou tout au moins pourvues de digitations pennées à leur face profonde. Les extrémités médiales des ectobronches se comportent de même et se dirigent vers celles des entobronches en suivant la face médiale du poumon : on voit ainsi dans la figure 5 ces extrémités en regard les unes des autres, faire présager la liaison qui s’effectuera plus tard entre elles et qui viendra fermer le cercle constitué par chaque conduit aérien. Ainsi les premières ramifications des gros troncs bronchiques sont d’abord exclusivement superficielles et, rampant sur les faces opposées de l’ébauche pulmonaire, s’avancent les unes vers les autres, laissant entre elles comme aux stades précédents une partie de ébauche pulmonaire dépourvue de ramifications bronchiques et uniquement occupée par le mésenchyme. Cette disposition assez facile à voir sur la reconstruction se retrouve de même sur les coupes et notamment sur le bord médial où les parabronches Pb vont nettement les unes au devant des autres (fig. v). POUMON DES OISEAUX 305 Toutes les ébauches bronchiques contenues dans le poumon de huit jours étant ainsi décrites, il convient de signaler les ressemblances et les différences que ce poumon présente avec celui des autres animaux. . La distribution superficielle des principaux troncs bronDorsal Pb. V- A. Latérl\ o ge © © Pb. aa AU] Media E:1 entral FIG. V. — Coupe transversale dans le poumon droit d’un embryon de Poulet de 9 jours. Développement des parabronches dans le mésenchyme pulmonaire. Gr. = 1 x 50 — B., bronche extra-pulmonaire ; Æ,., première entobronche ; Z,., deuxième entobronche ; P. b., parabronches ; S À, sac interclaviculaire ; »., vaisseaux sanguins. chiques indique déjà un des caractères les plus frappants du poumon adulte des Oiseaux ; mais le poumon de huit jours n'offre pas encore la caractéristique dominante, la continuation directe des tubes bronchiques les uns dans les autres par confluence de leurs extrémités distales. Sous ce rapport cette ébauche pulmonaire rappelle donc encore les caractères de l'arbre bronchique des autres Amniotes chez lesquels cette fusion ne s’opère jamais. Il est vrai que l’ébauche des sacs aériens déjà visibles à 306 A. JUILLET ce stade est encore un trait caractéristique ; maïs à ce moment les sacs aériens ne sont point encore assez dilatés pour attirer l'attention d’une manière trop forte, et à part l’un deux (le sac diaphragmatique antérieur) dont les dimensions relativement considérables et la forme très différente des extrémités distales des bronches, constituent des traits distinctifs, les autres n’offrent pas encore à ce moment de caractères bien particuliers et ne s’opposent pas suffisamment à de simples bronches pour qu’on les distingue sans autre considération. En effet, sur les reconstructions de l’arbre bronchique seules reconstructions que l’on puisse faire de l’ébauche pulmonaire, les ébauches des sacs cervical, interclaviculaire, diaphragmatique postérieur et abdominal se montrent comme des conduits plus ou moins cylindriques. On a déjà signalé celles du sac cervical, du sac interclaviculaire, du sac abdominal : celle du sac diaphragmatique postérieur est constituée par la troisième bronche latérale, née sur la mésobronche et que l’on a signalée dans l’embryon de six jours. Mais sur les coupes totales où se voit la périphérie de l’ébauche pulmonaire, les rapports particuliers que les ébauches des sacs contractent avec elle, permettent de les distinguer nettement : en effet, comme l’a fait remarquer BERTELLI (1905), ces ébauches s’enfoncent plus ou moins profondément dans la lame mésenchymateuse qui constitue le diaphragme primaire et s’y développent ultérieurement d’une manière prépondérante. Dans ce développement la partie cavitaire des sacs repousse au-devant d’elle une partie du diaphragme primaire, tandis qu’elle laisse entre elle-même et le tissu du poumon, l’autre moitié du diaphragme primaire qui va constituer le diaphragme définitif. Nous ne occuperons pas du développement ultérieur de la partie cavitaire des sacs renvoyant pour cela au travail très détaillé de l’auteur italien. Nous ferons remarquer seulement que les sacs aériens se comportent dans leurs ébauches primitives exactement comme des bronches. Ce qui le prouve bien, c’est qu’ils participent de ce caractère si particulier des LIEN POUMON DES OISEAUX bronches des Oiseaux, à savoir d'entrer à nouveau 307 en contact avec les bronches venues d’un point opposé et de se fusionner avec elles. C’est ainsi en effet, qu’il faut expliquer la formation des bronches récurrentes dont nous allons examiner maintenant le développement. KE Ventral F1G. VI. — Coupe transversale dans le poumon droit d’un embryon de Poulet de 10 jours. Développement des bronches récurrentes du sac interclaviculaire, Gr, = 1 x 38. — B., bronche extra-pulmonaire ; B, r., bronche récurrente du sac interclaviculaire ; E,, première entobronche; Æ,, deuxième entobronche; ÆZef,., première ectobronche ; Pb., parabronches ; $, à., sac interclaviculaire ; v., vaisseaux sanguins. DÉVELOPPEMENT DES BRONCHES RÉCURRENTES. — (Ces bronches ne sont autre chose que la continuation de l'extrémité distale des sacs, elles établissent une anastomose entre un sac aérien comparable à une bronche et une autre bronche. En se basant sur cettemanière de voir, on peut donc décrire dans les sacs aériens trois parties qui sont : 1° un conduit direct représenté par un tronc bronchique, naissant sur une grosse bronche (entobronche ou mésobronche) ; 2° une partie cavitaire cons- 308 A. JUILLET tituant le sac lui-même et se développant en dehors du poumon; 3° une partie récurrente qui représente l'extrémité distale des bronches ordinaires et qui, comme celles-ci, sert à établir la communication entre deux territoires bronchiques d’origine distincte. La description du développement des bronches récurrentes mérite d'autant plus de nous retenir un instant, qu’elle est un point absolument inconnu encore de la structure pulmonaire des Oiseaux et qu’elle confirme pleinement linterprétation que nous avons donnée de ces bronches. Nous les examinerons à propos de chaque sac. Le sac cervical ne présente jamais chez l’adulte de bronche récurrente. Chez l’embryon son ébauche est toujours très simple et a la forme d’un canal plus ou moins renflé à son extrémité distale, saillant hors de l’ébauche pulmonaire, mais dépourvu de bourgeons ‘latéraux. Le sac interclaviculaire présente des bronches récurrentes très visibles sur les coupes transversales de l’embryon au dixième jour. Comme le montre la figure vi, le sac interclaviculaire offre à ce moment la forme d’une cavité aplatie S. 2. fig. vi, situé ventralement à la bronche extra-pulmonaire dans l’épaisseur du diaphragme primaire ; mais cette cavité n’est pas simple, elle se prolonge par des diverticules distincts : deux sont représentés dans la figure vi, l’un plus médial se place au voisinage de la bronche et formera plus tard la partie du sac qui enveloppe cette dernière. L'autre placé du côté latéral, comprend deux bourgeons qui s’enfoncent dans le mésenchyme de l’ébauche pulmonaire et qui répondent manifestement aux bronches récurrentes. L’un d’eux dirigé vers la bronche intra-pulmonaire, présente à son extrémité distale un épaississement de son épithélium qui indique le bour- geonnement de cette extrémité bronchique. Ce caractère montre que l’on a bien à faire à un rameau né sur ce sac et non pas à un tube qui viendrait s’y ouvrir en partant d’ailleurs. L'examen attentif de la série confirme entièrement que cette POUMON DES OISEAUX 309 bronche est un diverticule du sac, car elle ne se rattache qu’à lui et ne communique encore avec aucune autre cavité aérienne. En outre, l'étude comparative d’autres embryons du même âge ne laisse pas douter du bourgeonnement intrapulmonaire, de interclaviculaire. l'extrémité Comme latérale de l’ébauche d'autre part, ces du sac bourgeons sont Dorsal Ect2 Latéral \| # .£ Ventral F1G. VII. — Coupe transversale dans le poumon droit d’un embryon de Poulet de 10 jours : cette coupe passe un peu en arrière de celle représentée dans la figure VI. Développement des bronches récurrentes du sac diaphragmatique antérieur. Gr. = 1 x 38. — B r., bronches récurrentes du sac diaphragmatique antérieur; Æ,., deuxième, entobronche ; Æct,., première ectobronche; Æct., deuxième ectobronche; P. b. parabronches ; S. d. «., sac diaphragmatique antérieur; $, à., sac interclaviculaire ; V, vestibule; ». p., veine pulmonaire. situés latéralement à la bronche, il ne peut être mis en doute qu’ils répondent aux bronches récurrentes, décrites dans le sac interclaviculaire de l’adulte. Les bronches récurrentes du sac diaphragmatique antérieur, se distinguent de même très aisément sur l'embryon de 10 jours. La figure vit représente ce sac dont la cavité, située dans le diaphragme primaire, est fort irrégulière S. d. a. fig. VII. et de même que pour le sac précédent, on distingue sur lex- 310 A. JUILLET trémité latérale une série de bourgeons ou de conduits répondant indubitablement aux bronches récurrentes. Ces conduits sont en effet très nettement intra-pulmonaires. Si l’on fait passer une ligne droite par les extrémités de la cavité pleurale, "Cranial Ventral S.d.p Caudal FIG. VIIT, — Coupe sagittale dans le poumon droit d’un embryon de Poulet de 9 jours. Développement des bronches récurrentes du sac diaphragmatique postérieur. Gr.= 1 x 38. — B. r., bronches récurrentes du sac diaphragmatique postérieur ; C! d., Canal direct de ce sac; Æ,, première entobronche ; Pb., parabronches ; $, d. &., sac diaphragmatique antérieur ; S. d. p., sac diaphragmatique postérieur ; $. à, sac interclaviculaire ; W, vestibule, cette ligne, répondant à une coupe du hile pulmonaire, rencontre le bord ventral de la bronche extra-pulmonaire et laisse en dehors du poumon la plus grande partie du sac diaphragmatique antérieur, tandis que le reste de ce sac, et en particulier les bourgeons et les conduits qui lui sont annexés, sont nettement intra-pulmonaires, Comme pour le 14 POUMON DES OISEAUX 311 sac interclaviculaire ces bronches récurrentes sont situées du côté latéral, c’est-à-dire en dehors de la bronche et par consé- quent dans une position Cranial tout à fait dis- tincte de celle du conduit direct de ce sac. Les bronches récur- \| Pb. 15 rentes du sac diaphragmatique postérieur, sont particulièrement bien vi- sibles sur série j F. É f j j Ect.2 Veniral Dorsal E3 ; Més. la figure VIII qui appartient à une E2 Es Ect.s de coupes faites parallèlement Br à la face ventrale de lé- bauche pulmonaire. Le S.ab. sac se montre rattaché au poumon par un canal et il s’y prolonge par trois conduits : deux dirigés à peu près parallèle- FIG. IX. — Coupe sagittale dans le poumon droit d’un embryon de Poulet de 9 jours. Développement des bronches récurrentes du sac abdominal. Gr. — 1 x 38. — B, r., bronches récurrentes du sac abdominal ; Æ,, E,, E., E,, première, deuxième, troisième, et quatrième entobronches; Zet., Ect,, deuxième et cinquième ectobronches ; Mes., mésobronche ; Pb., parabronches: S. ab., sac abdominal, 312 A. JUILLET ment l’un à l’autre vers l’extrémité craniale du poumon représentent les bronches récurrentes B. r. fig. vitr, l’autre dirigé médialement et d’arrière en avant est beaucoup plus court. Il représente l’extrémité du conduit direct du sac comme il est facile de le voir en suivant la série. Un peu en dedans de lui, se voient deux parabronches coupées transversalement. Enfin, dans la même figure, on distingue le sac diaphragmatique antérieur S. d. a. et une partie de son canal direct, situé sur le bord médial, enfin une partie du canal du sac interclaviculaire 4. 1. Le sac abdominal se comporte chez l'embryon d’une façon assez particulière, à cause de la courbure très prononcée de l'extrémité de la mésobronche qui le porte. Cette courbure se ferme presque sur l'embryon de huit jours, de sorte que le sac abdominal forme presque un angle droit avec l’extrémité de la mésobronche. Cette disposition est encore conservée dans l’embryon de dix jours où nous voyons ce sac appendu par un canal étroit à une sorte de carrefour arrondi, sur lequel s’implantent quelques bronches. Ce carrefour est formé par l’extrémité terminale de la mésobronche, sur laquelle s’insérent deux canaux récurrents à direction caudo-craniale B.r. qui pénètrent dans le poumon et dont l’un plus médial présente une bifurcation à son extrémité, laquelle est en voie de bourgeonnement comme le montre l’épaisseur de l’épithélium (fig. 1x). La figure x schématise bien le développement des sacs aériens et de leurs bronches récurrentes. Elle représente la face ventrale du poumon d’un embryon de Poulet sur laquelle ne sont figurés que les entobronches indiquées par un pointillé, la mésobronche et les sacs aériens. Ces derniers sont encore tout petits et ne recouvrent qu’une partie limitée de l’ébauche pulmonaire. . Le sac cervical S. c. inséré sur le rameau cranial de la première entobronche E1 forme un prolongement en massue sur l'extrémité craniale de l’ébauche pulmonaire. Le sac interclaviculaire #. 4. né sur le conduit d’origine du sac diaphragma- POUMON DES OISEAUX 313 tique antérieur, qui se rattache lui-même à la troisième entobronche, est relié à ce canal direct par un conduit cylindrique, le canal interclaviculaire précédemment décrit ©, 1. Il s’étale Lune Ci. FIG, X. — Schéma pour montrer les rapports des sacs et de leurs bronches récurrentes avee la face ventrale du poumon droit (embryon de Poulet au dixième jour). — 8., bronche extra-pulmonaire ; C. d. p., canal direct du sac diaphragmatique postérieur ; C!. à., canal du sac interclaviculaire ; Æ,, E,, E,, E,, première, deuxième, troisième et quatrième entobronches ; Mes., mésobronche ; O. «b., orifice direct du sac abdominal; ©. d. p., orifice direct du sac diaphragmatique postérieur; R. ab., bronches récurrentes du sac abdominal; Æ. d. a., bronches récurrentes du sac diaphragmatique antérieur ; R. d. p., bronches récurrentes du sac diaphragmatique postérieur ; À, ., bronches récurrentes du sac interclaviculaire ; S. ab. sac abdominal; S. c., sac cervical; S. d. a., sac diaphragmatique antérieur ; S. d. p. sac diaphragmatique postérieur ; S. à. sac interclaviculaire. sur la face ventrale du poumon donnant à son extrémité latérale deux ou trois bourgeons représentant ses bronches récurrentes À. à. et il émet du côté cranial un diverticule $. 2. qui se place dorsalement à la bronche extrapulmonaire B 314 A. JUILLET Le sac diaphragmatique antérieur #. d.a., né avec le précé- dent sur la troisième entobronche, s'étale comme lui à la face ventrale du poumon et émet également par son extrémité latérale des diverticules bronchiques qui rentrent dans le poumon pour former des bronches récurrentes R. d. a. Le sac diaphragmatique postérieur $.d. p., qui se développe en grande partie en dehors du poumon, se rattache par une grosse bronche latérale secondaire à la mésobranche Cd. p. Il émet des diverticules récurrents À. d.p. sur tout le pourtour de son orifice direct O. d. p. Il en est de même pour le sac abdominal S. ab. situé à l’extrémité caudale de la mésobronche. DÉVELOPPEMENT DES PARABRONCHES ET DU PARENCHYME. — Les parabronches se sont, depuis le huitième jour, considéra- blement développées. Dès le neuvième jour, les parabronches venues de la face profonde des ectobronches et des entobronches ont envahi le mésenchyme qui occupe le milieu de l’ébauche pulmonaire qui est alors traversée de toute part de conduits aériens. Dès le dixième jour le mésenchyme jusque-là continu et homogène, commence à se diviser en territoires distincts autour des parabronches grâce à l’apparition des vaisseaux sanguins qui décrivent, sur les coupes transversales, des polygones réguliers autour de chaque parabronche prise comme centre. Cette disposition qui s'aperçoit déjà sur le côté latéral de la coupe VIT est figurée en détail dans les planches (fig. 6). Les vaisseaux qui forment ce polygone sont d’abord à l’état de pointes d’accroissement pleines, puis ils se creusent, çà et là, de lumières dans lesquelles on aperçoit des hématies. L'apparition des vaisseaux divise le mésenchyme en une série de prismes polygonaux ayant chacun pour axe une parabronche et indique ainsi de bonne heure une des dispositions fondamentales du poumon des Oiseaux, à savoir la distribution de leur parenchyme pulmonaire en colonnes prismatiques, qui à été observée depuis longtemps et figurée par RAINEY (1849). Les différents conduits qui traversent le poumon à peu près tous à ce moment à! la même ont structure, et ils sont POUMON DES, OISEAUX 315 _ constitués par des tubes épithéliaux, formés de cellules eylindriques hautes dimensions, et présentant à peu près partout les mêmes sauf à l’extrémité végétante des parabronches où l’épithélium est un peu plus élevé. Autour de ces tubes épithéliaux le mésenchyme forme une couche plus dense dans le pourtour des gros troncs bronchiques ou plus exactement autour du vestibule, de la mésobronche et de la partie tout à fait initiale des entobronches. Ce revêtement comprend: 19° une couche de fibres musculaires lisses, situées immédiatement en dehors de l’épithélium ; 2° un strate de cellules mésenchymateuses serrées les unes contre les autres et qui double extérieurement cette couche musculaire. Au delà des gros troncs dont il vient d’être question, ce strate mésenchymateux fait complètement défaut et l’on observe seulement une couche musculaire lisse ; mais cette dernière s’étend d’une manière absolument continue sur toutes les ramifications de l’arbre bronchique ne s’arrêtant qu’à une courte distance de l’extrémité bourgeonnante des parabronches qu’elle n’atteint point, tant que dure leur accroissement. Cet accroissement s’effectue principalement du neuvième au treizième jour, et c’est pendant cette période que la masse mésenchymateuse centrale de l’ébauche pulmonaire est envahie par les parabronches marchant à la rencontre les unes des autres, comme on le voit bien sur la figure 5 ou sur la coupe V, E2 et Pb. La rencontre des parabronches opposées s'effectue à la fin de cette période et la figure 7 en montre un exemple très net, observé au treizième jour. Comme on pouvait déjà le deviner par la disposition des parabronches chez l’adulte, une parabronche avant de se fusionner avec celle qui vient du côté opposé, se divise en Ÿ ; puis chacun des bourgeons formant les bronches divergeantes de l’Y entre en contact avec un bourgeon venu d’une autre parabronche. Le tissu mésenchymateux qui les séparait, à entièrement disparu et les deux épithéliums s’acco- lent l’un à l’autre en s’engrènant profondément (fig. 7). Ils for- 316 A. JUILLET ment ainsi une cloison épithéliale double qui ne tardera pas à disparaître, faisant place à la continuité parfaite des deux parabronches. La disparition de la cloison épithéliale formée par la rencontre des parabronches opposées, doit avoir une durée assez limitée car on n’en n’observe pas un grand nombre dans une même coupe ; le plus souvent, on trouve les parabronches encore légèrement distantes ou au contraire en communication parfaite entre elles, sans laisser de trace de la manière dont cette communication s’est effectuée ; mais l’observation rapportée dans la figure 7 ne peut laisser aucun doute sur ce point. Cette communication s’établit constamment au niveau des extrémités les plus reculées des parabronches, par abouchement direct et non point par des communications latérales entre bronches voisines comme le pensait MILLER (1893) lorsqu'il comparait les communications bronchiques des Oiseaux aux perforations latérales que l’on peut observer dans les cloisons de sacs respiratoires voisins chez les Reptiles. Dans le même temps que les communications s’établissent entre parabronches, celles-ci commencent à engendrer par toute leur périphérie le parenchyme pulmonaire (fig. 8 et 9). Pour cela, elles émettent sur toute leur surface externe une série de bourgeons d’abord pleins qui se dirigent radialement en s’enfonçant peu à peu dansle mésenchyme. Ces bourgeons pleins, pour végéter en dehors, écartent les cellules musculaires lisses dont il a déjà été question et passent entre ces dernières. Les muscles restent au pourtour immédiat de la parabronche où ils forment non plus un revêtement continu, mais un réticulum dont les travées sont contenues entre les bourgeons radiés de la parabronche. La lumière de la parabronche, bien qu’elle ne se prolonge pas encore jusque dans l’épaisseur des bourgeons qui au treizième jour sont pleins, forme cependant des diverticules irréguliers et se montre sur les coupes transversales irrégulièrement étoilée. Le fond des bourgeons saillants dans le mésenchyme est formé de cellules plus claires que celles de l’épithélium parabronchique. Ces bourgeons se POUMON DES OISEAUX 317 produisent avec une grande rapidité et en grand nombre. Ils ne tardent pas non plus à devenir creux et au quinzième jour (fig. 9), autour de chaque parabronche, on distingue une série de petits diverticules épithéliaux constitués par un épithélium unistratifié qui rayonnent tout autour de la parabronche et dont on voit les lumières s’ouvrir directement dans celle de cette dernière, lorsque la coupe les à rencontrées suivant leur axe. L'ensemble de ces diverticules radiés forme maintenant dans chaque prisme parabronchique une petite masse centrale plus fortement colorée que le mésenchyme ambiant qui la limite, et cette opposition s’accuse d'autant plus que le mésenchyme lui-même devient plus transparent et plus clair par suite du développement de sa substance fondamentale qui écarte d’autant-plus ses cellules, et aussi par la formation de fentes dans son épaisseur. Les diverticules radiés engendrés autour de chaque parabronche sont d’abord simples, mais ils ne tardent pas à se bifurquer ou à se ramifier plus abondamment par leur extrémité distale, comme on le voit très bien à partir du seizième jour. Il résulte de ce bourgeonnement (fig. 10), que l’on peut décrire dans les diverticules des parabronches deux parties: 1° une partie centrale plus large qui débouche directement dans la lumière de la parabronche que nous appellerons vestibule ; 20 des conduits périphériques plus fins naissant à peu près à la même hauteur dans le vestibule et qui sont les capillaires aériens. Les vestibules sont séparés les uns des autres, par des lames étroites formées par les restes du tissu compris entre deux diverticules voisins. La partie la plus interne de ces lames qui répond au pourtour de la parabronche primitive forme comme un éperon saillant dans la lumière de cette dernière, éperon qui renferme près de son bord libre la bande musculaire lisse, résultant de l’écartement de la lame musculaire primitivement continue de la parabronche embryonnaire. Malgré les recherches multipliées faites sur des embryons 318 A. JUILLET recueillis entre le seizième jour et l’éclosion, il ne m’a pas été possible de suivre le développement du parenchyme pulmonaire. On peut observer aisément des figures un peu plus compliquées que celles du seizième jour. On voit en particulier les vestibules et les capillaires aériens qui leur font suite se différencier et s’opposer davantage, mais on ne saisit ni la formation ni le progrès des anastomoses qui s’établissent entre les différents capillaires d’un même parabronche ou de parabronches voisines et qui caractérisent le poumon achevé. On peut en effet, observer déjà des anastomoses parfaitement établies dans un petit Poulet encore dans la coquille, aussi bien que chez un Poulet éclos : mais le moment précis où s’établissent ces anastomoses m’a complètement échappé, bien que je me sois efforcé d'étudier des embryons de différents âges. CHAPITRE LE PARENCHYME PULMONAIRE V : ÉTUDE HISTOLOGIQUE Comme on l’a vu dans la description embryologique, les bronches des Oiseaux présentent une structure très uniforme pendant tout le cours du développement. Toutes consistent en un tube épithélial dont la paroi, formée de cellules cylindriques hautes, est revêtue extérieurement par une couche, d’abord continue, de fibres musculaires lisses. La partie initiale de l'arbre bronchique qui répond au vestibule de l’adulte présente seule en dehors de cette couche musculaire une couche plus dense de mésenchyme qui la distingue des autres bronches. Chez l’adulte, on peut distinguer dans l’arbre bronchique les parties suivantes: 19 La bronche extra-pulmonaire ; 20 le vestibule ; 39 la méso- bronche avec la partie initiale des entobronches jusqu’à la terminaison du canal direct des sacs aériens correspondants, POUMON DES OISEAUX 319 le canal direct du sac diaphragmatique postérieur, et enfin la partie initiale des bronches récurrentes, parties qui ont toutes la structure de la mésobronche et forment avec elle la troisième catégorie des voies aériennes ; 4° les gros troncs bronchiques (entobronches ou ectobronches) considérés un peu au delà de leur origine qui appartient à la catégorie précédente et qui ne portant de parenchyme pulmonaire que sur une partie de leur étendue ont reçu de F. E. Scxurze le nom d’hémiparabronches ; 5° toutes Les parabronches quelle que soit leur situation et leur diamètre. Chacune de ces catégories offre une structure différente et mérite d’être étudiée à part. BRONCHE EXTRA-PULMONAIRE. — La bronche extra-pulmonaire comprend une tunique muqueuse, des anneaux cartilagineux, et en dehors d’eux une mince couche de tissu conjonctif lâche, revêtue par l’épithélium du sac interclaviculaire qui l'entoure. La tunique muqueuse comprend un épithélium cylindrique haut, à cils vibratiles et présente des plis longitudinaux peu marqués dont le fond est généralement occupé par des cellules à mucus. Le chorion de la muqueuse est mince et peu développé. Les anneaux cartilagineux, toujours complets, sont souvent revêtus d’une couche mince de tissu osseux, d’origine périchondrale, et leurs cellules cartilagineuses sont fréquemment bourrées de granulations graisseuses, comme cela s’observe du reste dans le cartilage de l’arbre bronchique des Mammifères adultes. Ces anneaux sont unis entre eux par du tissu fibreux et par une couche épaisse de fibres musculaires lisses annulaires, en dehors de laquelle on observe le tissu conjonctif lâche dont il a été question et qui renferme, çà et là, de petits groupes de vésicules adipeuses. Ces trois couches : muqueuse, squeletto-musculaire et conjonctive sont les seules qui constituent la bronche à son entrée dans le poumon. Le revêtement externe d’épithélium pavimenteux unistratifié qui lui est fourni par le sac aérien interclaviculaire dans sa ARCH. DE Z0OL. EXP, ET GÉN, — 5° SÉRIE. — T. IX. — (II). 21 320 A. JUILLET partie extra-pulmonaire, fait défaut dès qu’elle aborde le poumon. VESTIBULE. — Le vestibule ne diffère de la bronche que par son diamètre et par la disposition des cartilages qui sont représentés ici seulement par des pièces semi-lunaires, siégeant entré les orifices des entobronches, dans l’éperon formé par ces dernières. Ces lames cartilagineuses sont les derniers représentants de ce tissu dans le poumon des Oiseaux et ne se retrouvent point au delà du vestibule. Comme les anneaux bronchiques, elles peuvent présenter sur leur surface une mince couche d’os (Poulet). En dehors de l’absence d’anneaux cartilagineux complets, le vestibule ne diffère que très peu de la bronche ; même tunique muqueuse avec épithélium cilié haut, même couche musculaire. Il y à lieu toutefois de signaler dans l’épaisseur du chorion muqueux de petits amas lymphoïdes en forme de nodules aplatis et plus ou moins discrets. En dehors de la tunique musculaire on observe chez les animaux adultes quelques petits groupes de vésicules adipeuses. BRONCHES DE LA TROISIÈME CATÉGORIE. — Les différents conduits que nous rangeons dans ce groupe (mésobronche, origines des entobronches jusqu’à la terminaison du canal direct des sacs aériens correspondants canal direct du sac diaphragmatique postérieur et origine des bronches récurrentes) possèdent tous la même structure. Ils sont caractérisés par ce fait qu’ils n’'émettent jamais sur leur parcours de fins canaux entrant dans la constitution du parenchyme pulmonaire et ne donnent naissance qu’à des parabronches. Ce ne sont donc point, à proprement parler, des voies respiratoires, puisque le parenchyme pulmonaire ne se développe point sur leurs parois, mais elles conduisent dans des bronches respiratoires qui, elles, naissent sur tout leur pourtour. En plus de ce caractère elles offrent une structure commune qui per- met de les reconnaître aisément sur les coupes, elles possèdent toutes une unique muqueuse comprenant un épithélium cilié analogue à celui des voies précédentes, des plis longitudinaux POUMON DES OISEAUX 321 peu saillants avec cellules à mucus, une couche musculaire lisse et en dehors de cette deruière une atmosphère de tissu conjonctif lâche qui les relie aux organes voisins et qui, plus ou moins développée suivant les connections anatomiques, peut enfermer des cellules adipeuses en petit nombre. Dans le chorion muqueux on observe fréquemment des nodules Iymphoïdes qui paraissent de préférence disposés au pourtour des orifices parabronchiques percés dans la paroi de ces conduits. La présence d’un épithélium cilié est absolument caractéristique de toutes ces bronches, d'autant plus que cet épithélium ne se rencontre jamais à partir des conduits de la catégorie suivante quelle que soit leur grosseur. La présence des cils vibratiles semble donc caractériser exclusivement dans le poumon des Oiseaux la portion des voies aériennes destinées uniquement à la conduction de l’air et dont la paroi ne joue aucun rôle direct dans la respiration proprement dite. Ces conduits purement vecteurs de l’air peuvent d’ailleurs tirer leur origine plus ou moins directement de la trachée ou au contraire des sacs aériens comme on le voit pour les bronches récurrentes. Mais dans ce cas, il faut se rappeler que ces conduits sont toujours extrêmement courts et ne tardent pas à se transformer en parabronches. Il faut signaler aussi que dans les bronches récurrentes et particulièrement au niveau des sacs diaphragmatique postérieur et abdominal la couche musculaire s’épaissit fortement autour de l’orifice du sac en constituant une espèce de sphincter. HEMIPARABRONCHES. — Ce terme récemment proposé par F. E. ScauLze (1908) qualifie heureusement les bronches dont nous avons à parler maintenant et qui se distinguent par ce fait qu’elles émettent les voies directement affectées à la respiration comme le font les parabronches, mais que contrairement à ces dernières, elles n’engendrent du parenchyme respiratoire que sur une partie de leur pourtour, soit la moitié ou un peu davantage. Elles sont représentées dans le poumon des Oiseaux par la plus grande partie des entobronches et des 322 A. JUILLET ectobronches (E1 fig. XI), en un mot par les gros troncs superficiels qui viennent en contact immédiat avec la surface pulmonaire et qui ne donnent point de parenchyme dans toute la partie de leur surface où ce contact est trop immédiat pour laisser place à la production de ce parenchyme. Comme l’avait déjà fort bien vu CAMPANA (1875, p. 215 et suivantes), la paroi des hémiparabronches est parcourue par un réseau de petits plis saillants déterminant des alvéoles de grandeur différente. Ce réseau avait, il est vrai, été signalé antérieurement à CAMPANA, et sa nature principalement musculaire avait été bien montrée par EBERTH (1863), mais CAMPANA a mieux précisé les rapports de ces différentes alvéoles, il a montré que les plus grandes renfermaient l’origine des parabronches, tandis que les plus petites interposées aux précédentes et les séparant les unes des autres sur des intervalles de 1 mm. à 1 mm. 5, répondaient aux orifices du parenchyme pulmonaire. Les hémiparabronches présentent donc en somme des conduits de diamètre variable dont une portion, contiguë à la surface pulmonaire est formée par une simple paroi mince et dont le reste présente à la fois des orifices parabronchiques et des orifices plus fins conduisant dans le parenchyme pulmonaire. La partie mince des hémiparabronches permet de voir aisément la structure originelle de ces conduits et les différences qu’elle présente avec les autres conduits précédemment décrits. Elle consiste uniquement en une muqueuse mince pourvue de fibres musculaires lisses disposées en un réticulum qui fait une saillie plus ou moins marquée dans la lumière des hémiparabronches. La muqueuse comprend un épithélium pavimenteux plat, bien différent de l’épithélium cilié qui a été décrit dans les précédents canaux. Le chorion est très mince, il ne renferme jamais de formations lymphoïdes mais il est parcouru par des fibres musculaires lisses qui se groupent en travées plus ou moins épaisses anastomosées en réseaux. Les travées les plus épaisses forment des mailles larges, bien saillantes dans la lumière, et parcourues elles-mêmes par un réticulum plus m c POUMON DES OISEAUX 323 délicat, formé par des travées d'épaisseur moindre. Toutes ces travées sont du reste revêtues par l’épithélium pavimenteux qu’elles repoussent au-devant d'elles, et l’ensemble forme, sur la coupe, des éperons plus ou moins saillants selon l’état du développement des parties, et qui sont reliés par des transitions insensibles avec les éperons très saillants qui séparent entre eux les vestibules sur la face opposée de l’hémiparabronche. Il est très remarquable que même sur la portion la plus mince de la paroi de l’hémiparabronche, qui semble à lœil nu absolument lisse, on distingue aisément au microscope le réseau musculaire qui s’est disposé sur cette paroi, cependant dépourvue de parenchyme, exactement de la même façon que dans sa partie parenchymateuse. Cette sorte de tentative d'achèvement de la structure dans une partie qui cependant ne fonctionnera jamais s’observe non seulement pour les muscles, mais aussi pour le système vasculaire des hémiparabronches, et l’on voit sur les injections, dans la portion de leur paroi mince qui confine à celle dans laquelle le parenchyme se développe, de véritables nappes de capillaires sanguins, parallèles entre eux et tous au contact qui semblent être là en si grand nombre pour attendre les capillaires aériens qui ne sont point développés dans cette partie de la bronche. Cette disposition particulière des capillaires sanguins n'avait pas échappé aux anciens auteurs et DUvERNoy la signale dans la seconde édition de l'anatomie comparée de Cuvrer (t. VII, p. 151), G. FIscHEr l’a également représentée dans son travail (1905, Taf. I, fig. 10 et 11): je l’ai observée très facilement sur des injections au nitrate d'argent faites par l'artère pulmonaire chez le Pigeon. La structure de la portion mince des hémiparabronches permet de comprendre la constitution typique de leurs parois propres, car c’est à peu près le seul point de ces conduits ou cette paroi puisse s’observer à l’état d'isolement. Partout ailleurs elle est si bien interrompue par les innombrables orifices qui la criblent, tant ceux des parabronches que ceux des vestibules, qu’il est 324 A. JUILLET impossible de la suivre sur une longueur suffisante pour s’en faire une bonne idée. Toutefois, au voisinage des orifices parabronchiques, on n’observe point immédiatement d’orifices vestibalaires et 1l existe toujours sur une longueur répondant au moins à toute l’épaisseur du prisme formé par le parenchyme autour de la parabronche, une surface où la paroi propre de l’hémiparabronche peut être suivie et en quelque sorte isolée. C’est là un caractère qui permet de distinguer facilement au point de vue histologique les hémiparabronches des parabronches elles-mêmes. En effet, ces dernières n’ensgendrent point sur leur pourtour de nouvelles parabronches mais seulement des tubes aériens plus fins, les vestibules qui, plus uniformément répartis, ne laissent pour ainsi dire point de portion imperforée dans la paroi de la parabronche qui se trouve réduite par là même à un simple réseau formé par le bord des lames séparant les vestibules les uns des autres. Il en résulte que l’on ne peut ainsi décrire aux parabronches de véritable paroi propre, cette paroi étant pour ainsi dire réduite à l’état d’une simple dentelle extrêmement délicate. Il était donc très important de bien préciser la nature histologique de la paroi propre des hémiparabronches et de voir les ressemblances et les différences qu’elle présente avec des conduits précédemment décrits. Nous avons vu que cette paroi est formée d’un épithélium plat ne présentant jamais de cils vibratiles ni de plis longitudinaux, contrairement à ce que l’on observe dans les premières voies aériennes. CAMPANA avait déjà signalé les différences de la paroi de ces deux sortes de conduits et fait remarquer que la tunique muqueuse des bronches qui nous occupe est «différente de celle qui revêt la bronche trachéale, beaucoup plus mince, sèche, pâle et dépourvue des plis longitudinaux et onduleux, que nous avons signalés dans la muqueuse trachéo-bronchique ». PARABRONCHES. — Les parabronches se distinguent par une série de caractères macroscopiques et microscopiques. Tout d’abord leur calibre est à peu près uniforme pour une espèce POUMON DES OISEAUX 325 donnée et dans les points correspondants du poumon. Ce sont des tubes flexueux, plus fins d’une manière générale chez les Oiseaux bons voiliers, plus larges chez le Poulet, mais toujours d’un diamètre assez considérable, ce qui donne aux coupes du Dorsal pe Pb. Ap. le : Lateral #7 ) Medial FRE Ea Ao. Oe C1. Ventral F1G. XI. — Coupe transversale dans un poumon de Serin, Pyrrhula canaria. Gr. = 1 x 15. —Ao., aorte ; Ap., artère pulmonaire ; B., bronche extra-pulmonaire ; ©, à., canal interclaviculaire ; Æ,, première entobronche ; Oe, œsophage ; Pb., parabronche; p. p., parenchyme pulmonaire. poumon cet aspect perforé si caractéristique (fig. xt1). D'autre part, leur calibre reste toujours le même sur toute leur longueur et contrairement aux fines bronches des Mammifères, elles ne vont point en s’atténuant. De plus, elles ne donnent point naissance à des bronches plus fines, mais sont elles-mêmes les plus déliées de ces canaux et tout ce qui s’ouvre dans Îles parabronches appartient au parenchyme pulmonaire ; de là, résulte une opposition très nette entre elles et les hémipa- 326 A. JUILLET rabronches, parce que leur lumière ne conduit que dans une seule sorte de voies aériennes, les vestibules, tandis que dans les hémiparabronches on trouve à la fois les vestibules et les orifices des parabronches. Une autre conséquence de cette disposition, c’est que la paroi propre qui limite leur lumière et qui dans les bronches précédentes était anatomiquement isolable, n’est plus ici représentée que par le reticulum très fin, séparant les orifices vestibulaires les uns des autres et qui répond sur les coupes aux minces lames séparant les cavités vestibulaires voisines. Ces lames, constituées par l’épithélium et par le reste du mésenchyme qui sépare primitivement dans l'embryon les évaginations vestibulaires, se terminent dans la lumière de la méso- bronche par un léger épaississement, dû principalement à la présence à ce niveau d’une travée musculaire lisse, reste de la couche musculaire continue observable dès le dixième jour chez l'embryon. Ces petits renflements des extrémités internes des lames intervestibulaires forment le reticulum qui a été décrit par maints auteurs à la surface interne des parabronches et dont nous avons parlé plus haut. Les parabronches émettent de toutes parts des expansions radiées de leur lumière, dérivées des évaginations décrites à à propos de l’embryologie. Ces diverticules se distinguent par leur diamètre et par leur structure en deux catégories: 10 le vestibule ; 2° les capillaires aériens. Les vestibules sont des conduits cylindriques courts, c’est-àdire dont la longueur ne dépasse pas le tiers du rayon du parenchyme affecté à chaque parabronche. Leur diamètre est assez large et mesure : 0 mm. 10 à 0 mm. 14 chez le Poulet, 0 mm. 06 à 0 mm. 10 chez le Pigeon. La paroi de ces vestibules continue près de leur extrémité interne, se trouve au contraire perforée par les orifices de nombreux capillaires aériens dans leur partie externe. Elle est constituée par un épithélium aplati formé de cellules à grand axe transversal, c’est-à-dire perpendiculaire à celui du vestibule (fig. x11). En dehors de cet épithélium la paroi vestibulaire comprend POUMON une membrane laires OISEAUX 327 vitrée très mince et un riche réseau de capil- sanguins qui çà et là se rassemblent pour former les troncs plus DES d’origine interne de la veine du vestibule pulmonaire. la paroi épaisse, comprend, outre la travée question, une lame connective Dans la portion intervestibulaire, musculaire qui occupe la plus dont il a été la place réservée aux vaisseaux dans les parties les plus externes du vestibule. Les vaisseaux sont en effet ici réduits à un seul capillaire large qui suit exactement le parcours des cloisons et sant intervestibulaires forme en s’anastomoavec ses similaires un magnifique réseau vasculaire formé . . par un seul C F1G. XII. — Endothélium des capillaires aériens chez le Pigeon Columba livia dom. (L), impré- capillaire de grande taille gnation au nitrate d’argent. Gr. = 1 x 320. — et reproduit eXAaC- end., endothélium ; O. €. a, orifices laires aériens,313 ; vest., vestibule, qui des capil- tement le dessin des réticulum formés à la face interne de la parabronche. A partir du point où l’on voit apparaître dans la paroi des vestibules les orifices des capillaires aériens dont il a été parlé plus haut, le parenchyme pulmonaire prend une structure réticulée tout à fait caractéristique et à la place d’une surface respiratoire continue comme l’est celle de la partie interne des vestibules, on ne trouve plus qu’un véritable treillis de travées anastomosées et séparées les unes des autres par la lumière des capillaires aériens (fig. 11). Les capillaires aériens naïissent de toute la partie externe des vestibules aussi bien sur leur paroi que sur leur fond, du reste difficile à limiter exactement à cause de l’arrangement compliqué des capillaires aériens. Quoi qu’il en soit, on distingue toujours dans les points favorables un certain nombre de capillaires aériens qui s'ouvrent directement dans le vestibule dont 328 A. JUILLET ils continuent la direction. Mais, contrairement aux vestibules qui sont séparés les uns des autres dans leur portion la plus interne, les capillaires aériens ne sont point isolés les uns des autres par des cloisons d’une certaine étendue : ils communiquent latéralement entre eux un grand nombre de fois ; par suite, les cloisons qui les séparent prennent elles-mêmes la forme de travées cylindriques minces ou, plus exactement, celles de mailles d’un réseau arrondi. à peu près toutes égales entre elles, si bien que l’ancien schéma de RAINEY (1849), représentant le parenchyme pulmonaire sous la forme d’un réseau spongieux, est plus exact que celui de F. E. Scxurze (1871), le figurant comme des tubes radiés subdivisés en tubes secondaires, mais tous terminés en cul-de-sac. Cette forme en cul- de-sac ramifié qui pourrait répondre à la structure du parenchyme pulmonaire d’un embryon presque à terme, n’est plus celle du poumon achevé qui, au contraire, ne présente plus qu’un réticulum de tubes communiquant tous entre eux et ne se terminant nulle part en cul-de-sac. Il y a deux manières bien différentes de se comporter pour les capillaires aériens. Tantôt ils se limitent strictement au territoire d’une seule parabronche, et anastomosés entre eux dans toute l’étendue de la colonne parenchymateuse qui accompagne chaque parabronche, ils ne sortent point de cette dernière. G. FIscHer (1905) attribue ce type aux Oiseaux mauvais voiliers, tels que le Poulet. Dans ce cas les limites du parenchyme de chaque parabronche sont très nettement indiquées sur les coupes par une lame continue de tissu conjonctif (/. p. b. fig. 11) décrivant un polygone hexagonal autour de chaque parabronche. Çà et là aux angles de ce polygone, on observe un épaississement de tissu conjonctif renfermant côte à côte un rameau de l’artère et de la veine pulmonaires. Ces cloisons fibreuses interparabronchiques se voient très aisément sur les préparations traitées par les colorants spécifiques du tissu conjonctif (méthodes de Van Gieson et de Curtis). Dans d’autres cas (Oiseaux bons voiliers, G. Fiscxer 1905.) POUMON DES OISEAUX 329 il s'établit des communications entre les capillaires aériens des parabronches voisines. Le tissu conjonctif interparabronchique est alors fort réduit et ne se rencontre plus qu’au voisinage des troncs vasculaires, Quoi qu’il en soit, la constitution fondamentale du parenchyme reste la même, il est partout formé de minces travées entre lesquelles Pair circule. La constitution histologique de ces travées mérite d’attirer l’attention. Elles sont formées exclusivement par des vaisseaux sanguins et par l’épithélium qui les revêt. Peut-être existe-t-il entre les vaisseaux et l’épithélium une très mince lame amorphe de la nature des vitrées, cela est même infiniment probable, mais je n’ai pu m'en assurer. Dans les travées les plus fines, il existe un seul capillaire sanguin lequel peut avoir du reste des dimensions un peu différentes suivant qu’il est pris au milieu même du réseau capillaire, c’est-à-dire à égale distance de l’artère et de la veine, ou bien qu’il est plus rapproché de la veine et forme un de ses troncs d’origine. C’est à ces différences qu’il faut attribuer la légère inégalité des mailles du réseau et l’on conçoit que l’épaisseur des travées varie naturellement avec celle des vaisseaux qu’elles sont destinées à conduire. La question du revêtement épithélial ou mieux endothélial des travées, a été très discutée et cela se com- prend parce que la forme même de la surface qu’il recouvre se prête mal à sa recherche. La présence d’un endothélium dans le parenchyme pulmonaire, bien que rendue très vraisemblable par tout ce que l’on sait de la structure des autres poumons, ne pouvait pas cependant être regardée comme démontrée pour le poumon des Oiseaux, ainsi que laffirmait OPPEzL (p. 328, 1905). Les anciens auteurs, comme RAINEY (1849), l’avaient nié, ÊBERTH (1863) considérait que les travées du parenchyme étaient nues ou recouvertes seulement par places de cellules plates, et plus tard F. E. Scxurze (1871) parlait seulement d’un endothélium vraisemblablement présent. D’autres auteurs, il est vrai, WiLLrAMS (1859), SCHRÜDER 330 A. JUILLET VAN DER KoLrk (1860), avaient de bonne heure admis la présence d’un endothélium, mais sans en donner la démonstration. ELENz E. (1864) chercha par des injections au nitrate d'argent à mettre cet épithélium en évidence et put le démontrer sur les parois perforées des parabronches et sur embouchure des canaux qui en partent, mais il ne put pas le retrouver sur les voies aériennes les plus fines, et l’on comprend dès lors lopinion de Max BAER qui en 1896 admettait que les capillaires sanguins étaient nus dans le parenchyme pulmonaire. OPPEL (1905) admettait, comme il a été dit,la présence de cet épithélium sans que la démonstration en ait été faite et il n’a pas cherché lui-même à la donner. G. FIsCHER (1905) admet aussi cet épithélium sans en donner des figures ni indiquer qu’il ait fait des recherches spéciales pour le mettre en évidence. Je me suis efforcé de rechercher l’épithélium des fines voies aériennes à l’aide des injections de nitrate d’argent, poussées par la trachée suivant le procédé indiqué dans les méthodes générales, et aussi à l’aide d’injections de la même substance poussées par l’artère pulmonaire, espérant ainsi imprégner l’endothélium des plus fins capillaires aériens. Comme il fallait s’y attendre, les injections ne réussissent pas toujours avec le même succès ; mais en combinant les résultats obtenus après les deux procédés différents (injections par la trachée ou par l'artère pulmonaire), on arrive sans peine à démontrer partout la présence de l’endothélium. Les injections poussées par la trachée remplissent rarement tout le parenchyme d’une parabronche et s’arrêtent le plus souvent un peu au delà des vestibules, ce qui explique les résultats obtenus par ELENZ E. (1864). Ces injections décèlent la présence sur la paroi interne des vestibules d’une couche de cellules épithéliales plates en forme de losanges allongés, dont le grand axe est perpendiculaire à celui du vestibule (fig. x11). Ces cellules s’observent facilement jusque sur le commencement des capillaires aériens, mais faute de la pénétration plus profonde de l’injection, POUMON DES OISEAUX 331 elles ne peuvent être suivies à la surface de ces derniers. Les injections faites par l’artère pulmonaire viennent alors à appui des premières et montrent à la surface des capillaires aériens un revêtement continu de petites cellules endothéliales polyédriques irrégulières de forme et de dimensions, à cause de la différence de forme des surfaces qu’elles ont à recouvrir surfaces qui sont tantôt cylindriques sur les travées, tantôt à contours irréguliers à l’anastomose des travées. La figure xIIT représente ces cellules dans le parenchyme pulmonaire du Pigeon. Leur forme polygonale, leur diamètre peu considérable, leur position très superficielle sur les travées montrent indubitablement qu’elles appartiennent bien à l’épithélium de revêtement de ces dernières. Un autre fait est encore en faveur de cette opinion, c’est que le plus souvent è leurs . bords . perpendiculairement coupent 3 l'axe Fic. XIII. — Endothélium de revêtement des travées chez le Pigeon, Columba Liviu dom (L.). Imprégnation au nitrate de la d’argent. €. 4., Capillaires Gr. — 1 x 320. aériens ; end., — endo- thélium; tr., travée. travée qu’elles revêtent, par suite celui du vaisseau qui occupe cette travée, et qu’une semblable disposition ne s’observe jamais sur l’endothélium des capillaires sanguins dont les cellules sont allongées dans le sens du vaisseau et se terminent vers leurs extrémités par des pointes effilées dont les bords sont tout au plus obliques à l’axe du vaisseau lui-même. On ne voit pas sur ces préparations l’endothélium des capillaires sanguins et il ne faut point s’en étonner, car, étant donnée la brièveté des capillaires entre les anastomoses qui les réunissent, il est à peu près impossible de trouver une étendue suffisante de ces derniers pour que le dessin endothélial de leurs parois soit reconnaissable. Il se peut aussi que, dans ces capillaires, la paroi ne soit point divisée en cellules endothéliales, et à ce sujet on peut remarquer que les lames capillaires parallèles observées dans la paroi dépour- 392 A. JUILLET vue de parenchyme des hémiparabronches ne montrent point de divisions endothéliales. Il faut revenir encore sur la constitution du réseau formé par les capillaires aériens pour réfuter certaines opinions émises sur sa fine structure. RAINEY (1849), le premier, a bien compris la nature du parenchyme lorsqu'il le représente sous la forme d’un réseau continu à mailles sensiblement égales, mais il a eu le tort, me semble-t-il, de désigner ces mailles par le nom de « cellules aériennes ». Le mot « cellule » indique toujours une cavité et de plus, lorsqu'il s’agit du poumon, il a été pris souvent dans le sens d’« alvéole »; c’est-à-dire une petite niche creusée dans une paroi quelconque et ouverte d’un seul côté. Or jamais on ne rencontre rien de pareil chez les Oiseaux ; jamais il n’y a d’alvéole à proprement parler, parce qu’il n’y a jamais de surface pulmonaire, en entendant par surface une étendue de quelque importance formant une paroi plane ou légèrement courbe, maïs en somme appartenant à un plan déterminé. Tout le parenchyme pulmonaire des Oiseaux est disposé en travées, c’est-à-dire en minces cylindres anastomosés entre eux, formant un réseau à travers lequel l’air circule, enveloppant de toute part chaque travée qui se trouve totalement plongée dans le fluide respiratoire. C’est pourquoi la figure de RAINEY, toute imparfaite et schématique qu’elle soit, représente d’une façon plus exacte le parenchyme pulmonaire que celle de F. E. Scaurze (1871). Dans cette dernière, en effet, on voit encore, bien que d’une manière moins marquée que chez les Mammifères, les canalicules aériens partis des vestibules se terminer par des extrémités closes et présenter sur leur surface des bosselures rappelant un peu les alvéoles. Toute la différence entre le poumon des Oiseaux et celui des Mammifères semblerait alors résider en ce fait que les infundibula, au lieu d’être appendus à une grappe de bronchioles seraient tout simplement concentrés tout autour de la lumière de chaque parabronche. Rien dans cette figure ne met en lumière les communications si POUMON DES OISEAUX 333 fréquentes et si multiples des capillaires aériens, pas plus qu’il n’est question dans l’article de SCHUILZE des grandes communications si répétées entre les rameaux bronchiques plus grossiers. L’idée de retrouver des alvéoles dans le parenchyme pulmonaire des Oiseaux était tellement arrêtée qu’on la rencontre encore dans un travail récent de SUPINO (1899). IL faut absolument renoncer à cette manière de voir, comme le montrera encore avec plus d’évidence l’étude des capillaires de lhématose dont la disposition est tout à fait différente de celle des mêmes vaisseaux, là où il existe vraiment des alvéoles. Les figures de G.FIScHER (1905) permettent du reste de bien saisir la disposition fondamentale du parenchyme et elles ne représentent jamais d’alvéoles, mais simplement un réseau continu, à mailles égales, s’étendant dans tout le domaine d’une parabronche ou même passant dans celui d’une parabronche voisine. L’irrégularité des travées de ce réseau n’est troublée que çà et là par la présence de vaisseaux artériels ou veineux représentant les terminaisons des artères ou les origines des veines pulmonaires, et qui déterminent par leur présence l’apparition de travées plus volumineuses contrastant avec les fins réticulum voisins. CAPILLATRES SANGUINS. — L’injection du poumon par une masse de gélatine est difficile à réussir pour plusieurs raisons. D'abord la friabilité des capillaires est très grande et par suite la masse s’épanche souvent dans les capillaires aériens. Ensuite, le poumon des Oiseaux est toujours très gorgé de sang, même après la saignée préalable recommandée plus haut, et les globules qui occupent les vaisseaux s’opposent souvent au passage de l'injection en même temps qu'ils favorisent les ruptures. Il est donc difficile d'obtenir de bonnes préparations, d'autant plus que la rétraction de la masse de gélatine causée par le dureissement à l’alcool qui suit l’injection déforme les capillaires et rend plus difficile l'interprétation des figures. On peut cependant arriver à suivre la distribution des vaisseaux sanguins et voir qu’elle calque rigoureusement, 334 comme AZ JUILLET l’a déjà montré G. FIsCHER (1905), dans ses figures, la distribution des travées interposées aux capillaires aériens. Chaque travée est en effet parcourue par un vaisseau capillaire unique dans la plupart des cas, double peut-être dans les travée un peu plus grosses, et ce capillaire est entouré de tous côtés par l’air qui circule dans les capillaires aériens; réseau sanguin et réseau aérien sont étroitement entrelacés et enchevêtrés l’un dans l’autre. MAx BAER (1896) a déjà insisté sur cette différence entre le réseau de l’hématose chez les Oiseaux et les Mammifères. Il à fait remarquer que les capillaires sanguins, qu’il croyait à tort nus, sont entourés un à un et de tous côtés par le fluide aérien, tandis que dans la plus grande partie des alvéoles pulmonaires et sauf dans les endroits où deux alvéoles accolées n’ont qu’un seul réseau capillaire commun, l’air ne baigne qu’un côté des capillaires. Mais il faut insister davantage encore sur l’opposition qui existe entre les deux structures, car la forme réticulée du réseau capillaire des Oiseaux et sa disposition en un réticulum continu dans les trois directions de l’espace et plongé dans l’air qui entoure individuellement chacune de ses mailles, est absolument carac- téristique des Oiseaux et d’eux seuls. Partout ailleurs le réseau respiratoire est disposé en surface, sous la forme de mailles arrondies extrêmement régulières, étalées sur un seul plan et abordées par l’air d’un seul côté. Ici au contraire le réseau n’est plus disposé en surface mais en profondeur et se développe dans les trois directions de l’espace avec les mêmes caractères, toujours en contact de toutes parts avec l’air qui le pénètre de tous les côtés. Naturellement aussi ce réseau reproduit la forme des mailles dans lesquelles il circule ; il est donc plus régulier dans la partie externe des parabronches, plus irrégulier au contraire autour des vestibules qu’il entoure de mailles allongées transversalement à peu près de la même façon que les cellules épithéliales qui les revêtent. Ce réseau vasculaire a été plus exactement figuré par SCHULZE (1871) que le parenchyme lui-même et sa figure pourrait encore servir si elle n’était POUMON os. DES OISEAUX 335 accolée à celle du parenchyme à laquelle elle n’est certainement pas superposable ce qui la rend à peu près incompréhensible, ou plutôt ce qui ne permet point de voir sur elle les caractères distinctifs dont nous avons longuement parlé. On a signalé à différentes reprises chez les Mammifères des pores alvéolaires qui, criblant la paroi des alvéoles, la transforment en un treillis de capillaires anastomosés et les travaux récents de MARCHAND (1911) montrent que ces trous sont des formations normales chez les Mammifères. Il semble donc, dans ce cas, que le réseau vasculaire de l’hématose est disposé comme chez les Oiseaux en un véritable treillis dont les fils sont entourés par l’air de toute part ; mais cette disposition grillagée ne doit pas faire perdre de vue la différence capitale qui existe entre les poumons dans les deux classes. En effet le treillis vasculaire du poumon des Mammifères est toujours disposé dans un seul plan, celui de la paroi alvéolaire dans laquelle il à été secondairement produit par résorption de certaines parties de cette paroi, tandis que chez les Oiseaux, ce treillis s'étend dans les trois directions de l’espace en remplissant toute l’étendue comprise entre les vestibules et les limites extrêmes de chaque territoire parabronchique. La disposition particulière du réseau vasculaire chez les Oiseaux paraît en rapport avec la perméabilité à l’air de cet organe dans tous les sens, c’est-à-dire avec ce fait que l’air peut arriver au poumon aussi bien par son hile (trachée) que par sa périphérie (bronches récurrentes). La communication des cir- cuits intrapulmonaires de CAMPANA et la disposition que nous avons signalée des bronches récurrentes montrent la possibilité de cette double pénétration de l’air par les voies aériennes principales. Il n’est pas douteux qu’elle peut se faire aussi bien dans l’étendue des capillaires aériens, et que l’un d’entre eux peut recevoir de l’air tantôt par le vestibule auquel il est directement rattaché, tantôt par sa partie profonde en relation avec les autres capillaires d’une parabronche voisine dans le cas des Oiseaux bons voiliers. ARCH. DE ZOOL. EXP. ET GÉN. — 5€ SÉRIE. — T. IX. — (III). 12 Ÿ% 336 A. JUILLET CHAPITRE ANATOMIE VI COMPARÉE On manque presque totalement de données sur l’anatomie comparée du poumon. Les auteurs qui ont étudié l’appareil respiratoire ont signalé à diverses reprises les différences que présentent les sacs aériens dans les différentes espèces. On trouve déjà à cet égard des renseignements importants dans le travail de NATALIS GuizLor (1846) et il existe dans la thèse de ROCHÉ (1891) des données encore plus nombreuses que nous pourrons utiliser en partie, notamment pour ce qui regarde les rapports des sacs diaphragmatiques avec la face ventrale du poumon, rapports qui varient dans de grandes limites et dont les variations sont étroitement liées à la structure pulmonaire. Mais sur la structure proprement dite du poumon, on ne trouve pas dans la bibliographie des renseignements permettant d'établir une étude comparative. Parmi les auteurs qui ont étudié cette structure, deux des meilleurs, SAPPEY (1847) et CAMPANA (1875), se sont limités strictement à une seule espèce. G. Fiscer (1905) a examiné, il est vrai, un assez grand nombre d'espèces et a représenté les moulages des poumons de vingtneuf espèces d’Oiseaux, mais l’abondance de ses figures ne doit pas faire illusion sur la valeur des résultats qu’il a obtenus et qui est assez peu considérable. En effet, les moulages qu’il représente et qui ont été obtenus pour la plupart à l’aide de la photoxyline sont, quand on les examine d’un peu près, très défectueux ; beaucoup sont criblés de vacuoles qui défigurent le dessin des bronches superficielles, d’autres montrent une confluence de la masse à injection de plusieurs bronches voisines, enfin la plupart de ses figures se rapportent à la face costale du poumon qui au point de vue de la structure est la POUMON DES OISEAUX 337 moins importante parce qu’elle ne donne point naissance aux sacs aériens dont le mode d’insertion sur le poumon varie beaucoup et constitue l’un des traits les plus importants de la structure comparée de cet organe. D'autre part sur les trois figures de la face ventrale du poumon représentées par G. FISCHER, les orifices des sacs aériens ne sont point indiqués, si bien que toutes ses figures ne peuvent servir qu’à représenter la disposition des bronches dorsales et leurs principales ramifications superficielles. L’anatomie comparée des poumons des Oiseaux se heurte à un certain nombre de difficultés que les données antérieures sur la technique nécessitée par leur étude font suffisamment ressortir. Il faut absolument employer des moulages métalliques et des injections à l’alcool distendant l’appareil aérien. Or ces procédés sont coûteux et difficiles à appliquer, dans les espèces de très grande taille à cause des frais qu’elles entraînent, sur les espèces de très petite taille parce que les dissections sont très difficiles à cause de la délicatesse des parties ou que, après les injections métalliques, il est impossible de séparer les poumons des sacs aériens attachés à leur face ventrale. Aussi ne peut-on étudier cette face qui est pourtant, comme on le verra, celle dont la constitution varie le plus par le mode de distribution des sacs qui s’y attachent. Pour cette raison les résultats que je vais indiquer maintenant se rapportent seulement à un nombre limité d’espèces et n’ont aucunement la prétention de constituer une étude comparative du poumon des Oiseaux. Ils sont destinés simplement à montrer un certain nombre de dispositions particulières, faisant bien ressortir quelques-unes des différences susceptibles d’être présentées par le poumon et indiquant très nettement que la structure, du poumon du Poulet n’est point du tout le type unique de cet organe, mais que ce dernier peut présenter des formes assez différentes dans le détail tout en conservant les caractères fondamentaux qui font de ce poumon un organe si spécial dans la série pulmonaire. 338 A. JUILLET ForME pu PoUMON. — Nous avons déjà signalé (p. 231) les variations que pouvait présenter la pente craniale dans son développement et son inclinaison et nous faisions remarquer à ce propos comment cette pente, faiblement inclinée et bien développée chez le Poulet adulte, était par contre très inclinée et très courte chez le Poulet à l’éclosion +6 chez certaines espèces. Ces variations sont nécessairement en rapport avec la forme générale du poumon qui peut se rapprocher soit d’un triangle dont le sommet serait représenté par l'extrémité craniale, les côtés par le bord latéral et par la face médiale, la base par le bord caudal, soit d’un parallélipipède rectangle court. J’ai essayé d'indiquer ces variations de forme en établissant d’après mes moulages métalliques les rapports de la longueur à la largeur maxima du poumon. Il est à remarquer tout d’abord que ce rapport est toujours supérieur à 1, c’est-à-dire que le poumon est toujours ‘plus long que large, et ce rapport varie beaucoup soit dans le développement individuel à ses différentes périodes, soit à l’état adulte dans les différentes espèces. Chez le petit Poulet à l’éclosion (fig. 12 &), la pente craniale est très courte, le poumon est par ce fait coupé carrément à son sommet. Plus tard la pente craniale s’allonge et le poumon prend sa forme définitive (fig. 14 a). Aïnsi le rapport de la longueur à la largeur est de 1,11 à l’éclosion — 1,13 chez le Poulet de deux jours — 1,50 à quinze jours — 1,60 chez un jeune adulte, tandis que chez les individus âgés il atteint 1,83. Comme on le voit, l’augmentation est considérable puisqu'elle dépasse le 7/20 de la longueur totale. Si maintenant on examine l’adulte des différentes espèces étudiées, on trouve des poumons courts, de forme plus ou moins quadrilatérale comme celui du Poulet nouveau né, par exemple ceux de la Chevêche Noctua minor (Briss) 1,19, du Geai Garrulus glandarius (L.) 1,25, de la Mouette rieuse Larus ridibundus (L.), du Goéland Z. fuscus (L.) et L. argentatus (L.) 1,23 à 1,25, du Traquet-Motteux Saxicola ænanthe (L.) 1,27, du Martinet Cypselus apus (L.) 1,37, de la POUMON Perruche à collier Palaeornis DES OISEAUX torquata 339 1,45, du Bruant Æmbe- riza cirius (L.) 1,45. Viennent ensuite les poumons ayant à peu près les mêmes proportions que dans le Poulet jeune adulte ; ce sont le Verdier Ligurinus chloris (L.) 1,52, le Moineau Passer domesticus (L.) 1,61, le Pigeon Columba livia CORTE SL dom. (L.) 1,61, la Gallinule Gallinula chloropus (L.) 1,72. Le Canard se distingue par un allongement de son poumon tout à fait remarquable. Son indice dépasse de beaucoup les plus grands observés jusqu'ici : il est de 2,10 chez le Canard domestique. Le Canard sauvage Anas boschas (L) à, il est vrai, un poumon beaucoup plus court, 1,84. La Sarcelle Querguedula angustirostris (Menet), à un indice intermédiaire entre les deux précédents et qui est de 1,91. \| BRONCHES. — La disposition des bronches chez les différents Oiseaux qui nous avons observés, rappelle dans ses traits principaux celle qui a été décrite chez le Poulet. La face ventrale est toujours pourvue de gros troncs entobronchiques au nombre de quatre, la surface dorsale présente des ectobronches au nombre de six environ et qui se distinguent plus ou moins nettement suivant la différence qu’il y a entre leur calibre et celui de leurs rameaux immédiats. Plus ces derniers sont fins, plus les ectobronches contrastent avec eux par leur taille et sont facilement distinguées. Dans les poumons du type court, comme dans le Geaiï, la Chevêche, les ectobronches caudales l’emportent en diamètre sur les craniales. C’est le contraire pour les types allongés, comme le Canard où les ectobronches craniales sont très fortes. Il serait avantageux pour les descriptions comparatives de pouvoir donner un nom propre à chacune des grosses bronches pulmonaires, entobronches et ectobronches. G. FISCHER (1905) a fait dans ce sens une tentative qui aurait pu être heureuse, mais qui ne peut pas être suivie comme nous l’avons fait remar- quer p. 259. Il est en effet impossible d'admettre la nomencla- ture proposée par cet auteur puisqu'elle repose sur une détermination inexacte des rapports des sacs interelaviculaire et 340 A. JUILLET diaphragmatique antérieur avec l’arbre bronchique, et que le procédé choisi pour dénommer les bronches n’est pas applicable au point de vue comparatif parce qu’un même sac, le sac interclaviculaire en particulier, ne se rattache pas toujours à la même entobronche et peut naître chez le Canard, par exemple, sur la première; tandis qu’il se rattache plus habituellement à la troisième. C’est pourquoi nous avons cru devoir garder la nomenclature exposée au début (p. 254) et qui a l’avantage de ne pas donner le même nom à des formations différentes. RAPPORTS DE LA FACE VENTRALE AVEC LES SACS AÉRIENS. La face ventrale du poumon et les orifices des sacs aériens que l’on y observe sont un des points les plus variables de la structure du poumon, tant par le nombre et par la position des orifices des sacs, que par l’étendue de chacun de ces derniers en rapport avec cette face pulmonaire. J’ai représenté schématiquement dans les figures XIV à xXvIIT la face ventrale des poumons de différents oiseaux qui peuvent être pris comme types. L’étendue de chaque sac en rapport avec la face ventrale du poumon est limitée sur cette dernière par des contours pleins et l’on voit à la première inspection combien cette étendue varie dans les différents cas. Le sac le plus cranial est le sac cervical, les autres le suivent régulièrement et sont en allant d’avant en arrière, le sac interclaviculaire, les sacs diaphragmatiques antérieur et postérieur, enfin le sac abdominal. Dans le territoire réservé à chaque sac sur la face ventrale du poumon se voient ses orifices direct et récurrents tantôt bien séparés tantôt rapprochés les uns des autres. D’après mes recherches, portant sur vingt-quatre espèces appartenant à sept ordres différents, on peut distinguer tout d’abord deux types principaux, suivant que le sac interclaviculaire naît sur la troisième entobronche par un tronc qui lui est 2 POUMON DES OISEAUX 341 commun avec le sac diaphragmatique antérieur, ou bien qu’il prend son origine d’une manière indépendante, sur la première entobronche. TyPE I. — Ce type est caractérisé par la communication du sac diaphragmatique antérieur et du sac interclaviculaire, communication qui résulte de la naissance du sac interelaviculaire sur la troisième entobronche par un canal commun avec le sac diaphragmatique antérieur. Cette disposition du sac interclaviculaire, qui s’observe dans le plus grand nombre des cas (dix-neuf espèces sur vingt-deux) est celle qui a été décrite précédemment chez le Poulet. La structure présentée par cet animal constitue un type moyen auquel il est facile de ramener les autres par le défaut ou l'absence d’un orifice ou par le dédoublement d’un autre orifice. A Type moyen. — Ce type caractérisé par la présence de sept orifices a été observé dans des espèces appartenant à des groupes très différents : Gallus domesticus (L.), Perdix rubra (Brisson), Coturnix communis (Bonnaterre) Galliformes, Gallinula chloropus (L.) Gruiforme, Noctua minor (Briss.) Strix flammea (L.) Coractiformes. Chez le Poulet les orifices des sacs aériens sont au nombre de sept comme l’avait déjà montré CAMPANA (1875). Mais les données de cet auteur ont été, comme nous l’avons dit, absolu- ment oubliées, car on ne décrit en général que cinq orifices. ROCHÉ admet comme typique le nombre cinq « bien que ce nombre cinq, dit-il, ne soit pas absolument constant et puisse être dépassé, un réservoir ayant quelquefois deux orifices pulmonaires » (1891, p. 25). Pour ma part, j'ai toujours trouvé six orifices chez les Oiseaux qui en ont le moins, et j’en ai constamment rencontré sept chez le Poulet, où ils sont disposés de la manière suivante : (fig. xIv et fig. 14 à). 19 un orifice cervical, orifice direct du sac cervical O. c., inséré sur le rameau cranial de la première entobronche. Cet orifice est unique pour le territoire pulmonaire occupé par le sac cervical ; 20 deux orifices pour le sac interclaviculaire. L’orifice direct 342 A. JUILLET est placé en dedans de la bronche extra-pulmonaire et répond à l’ouverture du canal interclaviculaire né sur la troisième entobronche. L’orifice récurrent À. 1. est placé sur le bord latéral, immédiatement en avant de la cloison qui sépare le sac interclaviculaire du sac diaphragmatique antérieur. C’est un orifice polybronchique simple, comme l’indique CAMPANA : il a été bien figuré par la plupart des auteurs; 3° deux orifices pour le sac diaphragmatique antérieur. L’orifice direct ©. d. a. qui naît sur la troisième entobronche, est placé en dedans de la bronche externe au voisinage de la veine pulmonaire. Cet orifice est toujours signalé par les auteurs. L’orifice récurrent À. d. a. est placé sur le bord latéral, immédiatement en arrière de la cloison séparant le sac diaphragmatique antérieur du sac interclaviculaire et à peu de distance de l’orifice récurrent de ce dernier. Cet orifice est aussi polybronchique simple. Il n’a pas été aperçu par ROCHÉ (1891, p. 25) qui l’a sans doute confondu avec celui du sac interclaviculaire ; 49 l’orifice du sac diaphragmatique postérieur. IL est très développé et placé sur le bord latéral du poumon au voisinage de son extrémité caudale. Elliptique comme on l’a vu, il donne accès dans un grand nombre de bronches: l’une d’entre elles, plus volumineuse, est formée par le gros tronc latéral C. d. p. né sur la mésobronche et qui continue l’orifice direct de ce sac O. d. p. Les autres R. d. p. plus petits, l'entourent de toute part et appartiennent aux bronches récurrentes. Cet orifice, du type polybronchique, est polybronchique mixte, contrairement à ce que croyait CAMPANA, évident avec une puisqu'il est en rapport voie directe d’accès de l'air; 59 l’orifice du sac abdominal. IL est très développé et appartient au groupe des polybronchiques mixtes. Il reçoit la terminaison de la mésobronche comme canal direct O. ab. et émet autour de lui un certain nombre de bronches récurrentes R. ab. Les sacs aériens présentent d’autre part avec la face ventrale les rapports suivants (fig. XIV). ASE :LE POUMON DES OISEAUX 343 Toute la pente craniale n’est en rapport qu'avec deux sacs : le cervical et l’interclaviculaire. Le sac cervical répond à l’ex- trémité craniale du poumon et à la partie médiale de la pente Cranial + / Et /i Lateral Les À x À o FD X ER 0 ss ee DE E1 Métal #8 j R= Rd.a. C.d.p. Rdp. O.dp. Caudal Fi1G. XIV. — Schéma montrant les rapports des sacs aériens et de leurs orifices avec la face ventrale du poumon chez le Poulet. Gr. — 1 x 1,5. — B, bronche extra-pulmonaire ; O. d. p., Canal direct du sac diaphragmatique postérieur ; C’. à., canal du sac interclaviculaire ; Æ,, E,, E,, E,, première, deuxième, troisième et quatrième entobronches ; Mes, mésobronche ; O. «b., orifice direct du sac abdominal ; O. c., orifice direct du sac cervical ; O. d. «., orifice direct du sac diaphragmatique antérieur; C. d. p., orifice direct du sac diaphragmatique postérieur ; R. ab., orifices récurrents du sac abdominal; R. d. «., orifice récurrent du sac diaphragmatique antérieur; R. d. p., orifices récurrents du sac diphragmatique postérieur ; R. 2., orifice récurrent du sac interclaviculaire. craniale. Il s’étend aussi un peu en arrière sur la partie médiale de la pente caudale comme on l’a vu plus haut. Le sac interclaviculaire occupe la moitié latérale de la pente craniale en dehors du sac cervical. Il est limité en arrière par la lame résultant de l’accolement de sa paroi caudale avec la paroi craniale 344 A. JUILLET du sac diaphragmatique antérieur, lame qui s’insère sur la crête séparant les deux pentes du poumon. Le sac diaphragmatique antérieur est en rapport avec la plus grande partie de la pente caudale qu’il recouvre sur toute son étendue, sauf un petit espace situé du côté médial et qui est occupé par le prolongement caudal du sac cervical, et un petit espace triangulaire situé vers l’extrémité caudale et à l’extrémité latérale de cette pente : cet espace est occupé par le sac diaphragmatique postérieur. Cette grande extension du sac diaphragmatique antérieur est très particulière et ne se présente avec de telles proportions chez aucun des animaux que nous avons étudiés, sauf chez la Perdrix rouge Perdix rubra (Brisson) et la Caille Coturnix communis (Bonnaterre). RoCHÉ a signalé (1891, p. 69) le grand développement des sacs diaphragmatiques antérieurs chez certains Gallinacés (Faisan), mais, d’après le même auteur, il est d’autres Gallinacés où les deux sacs diaphragmatiques sont moins différents l’un de l’autre, et où le sac diaphragmatique postérieur couvre aussi une certaine partie de la pente caudale. J’ai observé le même fait sur certaines espèces appartenant au type moyen, par exemple chez la Gallinule Gallinula chloropus (L.), la Chevêche Noctua minor (Briss.), l’'Effraye Strix flammea (L.) où les deux sacs diaphragmatiques se partagent presque également le revêtement de la pente caudale (fig. 16). Le sac abdominal n’est, à proprement parler, pas en rapport avec la face ventrale dont il est séparé par une arcade saïllante suivant le bord caudal de cette face. Il n’est en rapport qu'avec le bord caudal et seulement dans la partie de ce dernier qui confine avec le territoire du sac diaphragmatique postérieur. B. Type réduit.— Ce type, que nous avons rencontré chez des Animaux appartenant à des ordres différents : Charadriiformes (Pigeon), Cuculiformes (Perruches) est caractérisé par l’absence de bronches récurrentes dans le sac interclaviculaire. A côté de ce fait essentiel, la description plus détaillée du Pigeon permet de bien caractériser ce type (fig. xv, et 17, 18, 20 a). POUMON DES OISEAUX 345 Les orifices des sacs aériens présentent la disposition suivante : 1° l’orifice du sac cervical, unique, n'offre rien de spécial ; 20 l’orifice du sac interclaviculaire est unique ; il est représenté par l’orifice du canal interclaviculaire C. à. analogue à celui du Poulet, et né comme chez ce dernier sur le conduit direct du sac diaphragmatique la troisième entobronche. Cet orifice est placé en dedans de la bronche extrapulmonaire. Il n’y a pas d’orifice antérieur près de son insertion sur récurrent ; 3° le sac diaphragmatique antérieur exami- Rd né sur le frais, paraît posséder Oa4p. un seul orifice, placé en dedans de la bronche extra-pulmo- Rdp . , naire dant et EDS l’orifice à figuré par FIG. XV. — Schéma montrant les rapports des sacs aériens et de leurs orifices avec la face ventrale du poumon chezle Pigeon Columba livia dom (L.) Gr.=1 X 2,5. — B., bronche extrapulmonaire ; C.d.p., canal direct du sac et VuUNG fig. 36, VocrT (1894, diaphragmatique postérieur ; C.i., canal du sac interelaviculaire ; £,, E,, E;, E,, première, deuxième, troisième et quatrième entobronches ; Mes.,mésobronche ;O.ab., orifice direct du sac abdominal; O.c., orifice direct du sac cervical; O.d.p., orifice di- P: 81 5) . : examiI- . Mais en nant d’un peu rect du sac difphragmatique postérieur; R.ab., orifices récurrents du sac abdominal; R.d.4., orifice récurrent du sac diaphragmatique antérieur ; R.d.p., orifices récurrents du sac diaphragmatique postérieur. près cet orifice on voit que le cerele qui l’entoure n’est en quelque sorte qu’un cadre commun pour deux trous parfaitement distincts dont l’un placé sur le prolongement du conduit direct est situé du côté médial O. d. a. tandis que l’autre, placé latéralement, conduit dans une bronche transversale se dirigeant 346 A. JUILLET vers le côté latéral, parallèlement au rameau transverse de la quatrième entobronche, et qui ne tarde pas à se ramifier en parabronches (2. d.a, fig. xv). Placée immédiatement sous le diaphragme ornithique cette bronche n’est point saillante sur les pièces simplement fixées à l'alcool, mais sur les moulages métalliques elle fait saillie à la face ventrale du diaphragme qu’elle repousse au-devant d’elle et son mode de distribution, aussi bien que sa diminution de calibre à partir de son origine, indiquent clairement que c’est une bronche récurrente (voir en particulier fig. 17). Nous trouverons du reste une bronche récurrente de même forme, mais plus étroitement dépendante du canal direct du sac diaphragmatique antérieur chez le Canard. Les moulages : figures 17, 18, 20 a représentent assez nettement cette disposition pour en permettre une étude plus complète. Dans la figure 17, cette bronche est encore réunie au moulage du sac diaphragmatique antérieur qui à été enlevé en partie à la scie. Dans la figure xv, les deux orifices direct et récurrent sont un peu trop écartés l’un de l’autre ; je n’ai pas pu les représenter autrement, pour la clarté du dessin, s’il m'avait été possible d'indiquer sur un semblable schéma les reliefs, je les aurais placés tous les deux aux extrémités du diamètre d’un cerele légèrement déprimé représentant l’orifice unique décrit par les auteurs ; 49 l’orifice du sac diaphragmatique postérieur est voisin du bord caudal du poumon dont il est cependant un peu moins rapproché que chez le Poulet, à cause de la plus grande étendue du sac diaphragmatique postérieur en rapport avec la face ventrale du poumon ; il appartient au type polybronchique mixte. Son canal direct C’, d. p., né sur la mésobronche, s’élargit distalement comme on peut s’en rendre compte sur certains moulages où ce canal étant très superficiel, il est aisé d’en suivre le trajet ; 5° l’orifice du sac abdominal est un orifice polybronchique mixte il ne présente rien de spécial. Les bronches récurrentes du sac abdominal À. ab, sont très faciles à suivre sur ies moulages. La figure 20 b en particulier le montre venant du POUMON DES OISEAUX 347 bord caudal du poumon pour s’étaler et s’anastomoser sur la face dorsale avec les circuits latéro-caudaux. Le territoire des sacs en rapport avec la face ventrale offre une disposition intermédiaire entre celle du Poulet et celle du Canard, en ce sens que le sac diaphragmatique antérieur occupe une plus grande étendue de la pente caudale que chez le Canard, mais recouvre moins complètement cette face que chez la Poule : le sac diaphragmatique postérieur s'étale sur le tiers caudal de la face ventrale dans toute son étendue transversale, comme le montrent encore certains moulages (fig. 18) où la crête transversale qui divise en deux la pente caudale répond à la cloison séparatrice des deux sacs diaphragmatiques. La Perruche à collier Palaeornis torquata et la Perruche ondulée Melopsittacus undulatus (Shaw.) présentent la même structure (fig. 21 a, 22 a). C. Type augmenté. — Il y a plus de sept orifices. Dans ce cas, observé chez onze espèces et par conséquent dans la moitié de celles que j'ai étudiées, l’augmentation du nombre des orifices est variable et nous distinguerons deux cas. a La structure ne diffère de celle observée chez le Poulet que parce que le sac diaphragmatique postérieur occupe une grande partie de la face ventrale du poumon en arrière de la traché (fig. XVI), et que l’orifice (polybronchique mixte) qu’il présente chez le Poulet est ici dédoublé en deux orifices distincts, l’un pour le canal direct placé plus médialement, l’autre pour les bronches récurrentes qui sont situées tout à fait latéralement, empiétant même sur la face dorsale du poumon. Cette disposition a été rencontrée chez le Geai Garrulus glandarius (L.), le Moineau, Passer domesticus (Brisson), le Bruant Æmberiza cirius (L.) le Verdier Ligurinus chloris (L.), le Pinson Fringilla caelebs (L.) le TraquetMotteux, Saxicola ænanthe (L.) (Passeriformes) le Martinet Cypselus apus (L.) (Coraciiforme), la Cresserelle Falco tinunculus (L.) (Falconiforme). Nous donnerons pour ce cas la description du Geai. Les orifices des sacs aériens présentent 348 A. JUILLET chez cet oiseau la disposition suivante : (fig. XVI et 23 a). 19 l’orifice cervical O.c. est unique et ne présente rien de spécial; 29 Je sac interclaviculaire possède deux orifices très nets. L’orifice direct est à l’extrémité du canal interclaviculaire C. 2. qui rattache ce sac à la troisième entobronche. Cet orifice est placé sur le bord internettde la bronche extrapulmonaire. L'autre, récur- rent, R.1., polybronchique simple,est placé sur le bord latéral du poumon, immédiatement en avant de la cloison qui sépare le sac diaphragmatique F1G. XVI. — Schéma montrant le rapport des sacs aériens et de leurs orifices avec la face ventrale du poumon chez le Geai, Garrulus glandarius (L.) Gr.=1 x 2,5. — B., bronche extrapulmonaire ; C.4.p., canal direct du sac diaphragmatique postérieur ; ©. à. canal du sac interclaviculaire; Æ,, E,, E., E,, première, deuxième, troisième et quatrième entobronches ; Mes., mésobronche ; O.ab., orifice direct du sac ROLE O.e., OHACe direct du sac cervical. O.d.a., orifice FE antérieur du . : Sac interclavilai x CUlAITE ; 30 Le sac dia- direct du sac diaphragmatique antérieur; O.d.p., orifice direct du sac diaphragmatique postérieur ; R.ab., orifices récurrents du sac abdominal; R.d.a., orifice récurrent du sac diaphragmatique phragmatique antérieur ; R.d.p., orifices récurrents du sac diaphragmatique térieur ; ÆR.12., orifice récurrent du sac interclaviculaire. antérieur à pos- sède lui pos- aussi à deux orifices, l’un direct, rattaché à la troisième entobronche et situé comme toujours en dedans de la bronche extra-pulmonaire, O. d. a. ; l’autre, récurrent À. d. a, situé en dehors de cette bronche, donne accès dans une grosse bronche transversale placée sous le diaphragme ornithique et qui émet, surtout du côté caudal, une série de parabronches entrant dans la constitution du réseau de la pente caudale. Cette bronche répond indubitablement à celle décrite chez le Canard et le Pi- POUMON DES OISEAUX 349 geon. Mais elle à pris d’une manière beaucoup plus frappante le type récurrent. Son orifice est du type monobronchique simple ; 4° le sac diaphragmatique postérieur contrairement à ce qui a été vu jusqu'ici possède deux orifices : un orifice direct O. d. p. monobronchique simple et un orifice récurrent polybronchique simple. Le premier est formé par le canal direct C. d. p.' né sur la mésobronche comme chez les autres Oiseaux. Mais ce canal au lieu de se prolonger sous la forme d’un long tube évasé à son sommet venant s’ouvrir au voisinage des bronches récurrentes, est court, également calibré sur tout son trajet, et s’ouvre à la surface ventrale, très peu en dehors d’une ligne verticale menée par le milieu du poumon, et à une grande distance de l’orifice des bronches récurrentes lequel est toujours placé sur le bord latéral même du poumon. Cet orifice des bronches récurrentes À. d. p. appartient au type poylbronchique simple. Sur les moulages métalliques il est remplacé par un gros conduit large et court, rattaché au sac diaphragmatique postérieur, et d’où l’on voit s’échapper un bouquet de bronches récurrentes passant en partie sur la face dorsale. Cette disposition qui n’est plus apparente sur la figure 23 b, le moulage ayant été brisé en ce point, est au contraire très nette sur les moulages de Verdier (fig. 26), de Traquet-Motteux (fig. 27), de Martinet (fig. 28) ; 59 l’orifice du sac abdominal est polybronchique mixte. Les bronches récurrentes À. ab. s’étalent sur la partie caudale du poumon et l’une d’elle plus volumineuse, très nettement indiquée sur la figure 23 &, suit le bord latéral jusqu’au point d'insertion des bronches récurrentes du sac diaphragmatique postérieur. Cette disposition se voit aussi très bien sur les figures 24, 26, 27, 28. Le poumon du Geai est un des moins allongés que nous avons rencontrés et présente de ce fait certaines particularités qui le distinguent aisément de ceux qui ont été étudiés jusqu'ici. Les territoires des sacs qui s’étalent sur la face ventrale sont en effet assez particuliers. 350 A. JUILLET Le sac cervical occupe toujours le sommet de la face ventrale. Par suite du racourcissement de la pente craniale les rapports du sac interclaviculaire avec cette dernière sont un peu différents de ce qu’ils étaient chez la Poule et chez le Pigeon. Il occupe seulement une bande transversale étroite de cette pente, entre le bord latéral et le bord médial. Le sac diaphragmatique antérieur est également très étroit et ne s’étend que sur le quart antérieur de la pente caudale du poumon. ROCHÉ (1891, p. 75) a déjà signalé chez les Passereaux cette inégalité des sacs diaphragmatiques antérieurs et postérieurs et la prépondérance de ces derniers. Le sac diaphragmatique postérieur occupe en effet la presque totalité de la pente caudale. Cette disposition s’observe aisément sur les moulages des poumons et des sacs aériens du Moineau, du Verdier, du Traquet-Motteux et du Martinet . b. Dans un autre type observé seulement dans le genre Larus (Charadriiformes) (fig. xvir et 29 & et b), le nombre des orifices monte à neuf par l’apparition d’un sac aérien nouveau, qui n’a point été signalé jusqu'ici, le sac cervical latéral qui naît un peu en dehors du sac cervical typique, par un orifice direct rattaché au rameau latéral de la première entobronche. Mais l’apparition de ce sac n’est pas l’unique différence qui sépare ces Oiseaux de ceux du groupe précédemment décrit. La distribution des orifices des sacs diaphragmatiques présente aussi des différences importantes. Le sac diaphragmatique antérieur, plus développé que dans les Passereaux et qui couvre à peu près la moitié de la pente caudale, présente ici trois orifices : un orifice direct ©. d. a. rattaché au canal du sac interclaviculaire et deux orifices récurrents distincts R. d. a : l’un est placé à peu près au milieu de la face ventrale un peu en dehors de la bronche extra-pulmonaire, caudalement à l’orifice récurrent du sac interclaviculaire, l’autre est placé tout à fait latéralement, empiétant en partie sur la face dorsale. D'autre part le sac diaphragmatique postérieur moins développé que chez les Passereaux, et qui recouvre seulement à POUMON DES OISEAUX 301 peine la moitié de la pente caudale, n’a plus ses orifices direct et récurrent séparés comme dans le type précédent. Ces orifices sont au contraire réunis en un seul orifice polybronchique mixte comme c’est le cas pour la Poule, le Pigeon et le Canard. Cette réunion de deux sortes d’orifices du sac diaphragmatique postérieur compense le dédoublement de la \|LR, FIG. XVII. — Schéma montrant les rapports des sacs aériens etde leursorifices avec la face ventrale du poumon chez la Mouette rieuse. Larus ridibundus (L.) Gr. = 1 x 2. B., bronche extra-pulmonaire ; C‘.d.p., canal direct du sac diaphragmatique postérieur ; C.2., canal du sac interclaviculaire ; £,, E,, E,, E,, première, deuxième, troisième et quatrième entobronches ; Mes., mésobronche ; O.ab., orifice direct du sac abdominal ; O.c., orifice direct du sac cervical; 0°.c’., orifice direct du sac cervical latéral; O.d.a., orifice direct du sac diaphragmatique antérieur ; O.d.p., orifice direct du sac dia- phragmatique postérieur ; R.ab., orifices récurrents du sac abdominal; R.d.4., orifices récurrents du sac diaphragimatique antérieur ; ÆR.d.a., orifices récurrents du sac diaphragmatique postérieur : Z2.2., orifice récurrent du sac interclaviculaire. bronche récurrente du sac diaphragmatique antérieur observé dans le type précédent & et fait qu’en définitive il n’y a que neuf orifices, le nouvel orifice étant dû au sac cervical supplémentaire. Ces dispositions ont été observées chez la Mouette rieuse Larus ridibundus (L.), chez le Goéland argenté Larus argentatus (Brünn) et chez L. fuscus (L.). ARCH. DE Z0OL. EXP. ET GÉN. — SEE 5° SÉRIE. — T. IX. — (Il). 23 302 A. JUILLET TyPe 11. — Le second type diffère du premier en ce que le sac interclaviculaire naît par un conduit propre sur la première entobronche et ne communique jamais avec le diaphragmatique antérieur. Ce type est réalisé avec de légères modifications spécifiques, chez le Canard Anas boschas (L.), lOie Anser F1G. XVIII. — Schéma montrant les rapports des sacs aériens et deleursorifices avec la face ventrale du poumon chez le Canard Anas boschas (L.) = 1 x 0,8. — B., bronche extrapulmonaire ; C!d.p., canal direct du sac diaphragmatique postérieur ; C.i., orifice du sac interclaviculaire ; E,, E,, E,, E,, première, deuxième, troisième et quatrième entobronches ; Mes., mésobronche ; O.ab., orifice direct du sac abdominal; O.c., orifice direct du sac cervical; O.d.a., orifice direct du sac diaphragmatique antérieur ; O.d.p., orifice direct du sac diaphragmatique postérieur ; R.ab., orifices récurrents du sac abdominal; R.d.a., orifices récurrents et bronche récurrentes du sac diaphragmatique antérieur ; R.d.p., orifices récurrents du postérieur ; R.i., orifices récurrents du sac interclaviculaire. sac diaphragmatique domesticus (L.) et la Sarcelle Querquedula angustirostris (Menet). Il y a chez ces animaux sept orifices qui sont répartis de la manière suivante : 19 un orifice direct pour le sac cervical; 2 un orifice direct et un orifice récurrent pour le sac interclaviculaire ; 3° un orifice direct et un orifice récurrent parfois dédoublé en deux orifices voisins l’un et l’autre, pour le POUMON DES OISEAUX 303 diaphragmatique antérieur ; 4° un orifice unique, polybrone chique mixte pour le diaphragm tique postérieur et de mêmpour le sac abdominal. Nous ajouterons quelques détails pour le Canard domestique. Les orifices des sacs aériens très faciles à observer se présentent de la façon suivante (fig. XVII). 1° L’orifice du sac cervical est placé comme chez le Poulet, sur le gros tronc cranial de la première entobronche, mais à cause de l’allongement général de la pente craniale du poumon, il est situé très en avant, de plus le sac cervical se prolonge fortement en arrière le long du bord médial de la face ventrale du poumon. 20 les orifices du sac interclaviculaire sont au nombre de deux. L’orifice direct C. 1. naît comme je le disais plus haut, sur la première entobronche au voisinage de son insertion sur le vestibule et très près de la bronche extra-pulmonaire. Cet orifice n’a pas été vu par SAPPEY. L’orifice récurrent R.1., polybronchique simple, est situé comme chez la Poule, sur le bord latéral de la face ventrale du poumon immédiatement en avant de la cloison séparant le sac interclaviculaire du sac diaphragmatique antérieur; 30 le sac diaphragmatique antérieur offre des orifices assez mal décrits jusqu’iei et qui méritent d'attirer l’attention. L’orifice direct O.d. a est formé par un tube naissant sur la troisième entobronche à son origine, et qui se dirige ventralement pour venir s’épanouir à la surface ventrale du poumon en formant le sac lui-même. Avant d'atteindre cette surface il émet vers le bord latéral une grosse bronche, placée immédiatement sous le diaphragme ornithique, et qui se ramifie abondamment dans le parenchyme pulmonaire. Comment doit-on envisager cette bronche ? Faut-il la considérer comme une bronche ordinaire ou comme une bronche récurrente ? Bien que je n’aie pas suivi son développement, il me semble qu’il faut la rattacher à ce groupe de bronches, parce qu’elle naît du canal direct du sac, c’est-à-dire du sac lui-même et rentre par suite dans la 304 A. JUILLET définition que j'ai donnée des bronches récurrentes. D’autre part, il semble bien qu’au point de vue physiologique cette bronche soit plutôt disposée pour recevoir l’air revenant du sac que l'air y allant. Sa position à angle droit sur le canal direct du sac fait que l’air arrivant de la trachée se dirige plus facilement dans le sac que dans cette bronche, tandis que, lors du retour de l’air, elle se trouve plus directement placée sur son trajet. Enfin, une troisième raison tendà nous faire considérer cette bronche comme une récurrente, c’est que chez le Pigeon, le Geai, les Passereaux, la bronche récurrente du même sac a exactement la même disposition que cette bronche transversale du Canard, bien qu’elle naïsse un peu plus latéralement qu’elle. L’orifice direct du sac diaphragmatique antérieur est placé sur la face ventrale du poumon immédiatement en arrière de cette bronche transversale. C’est le seul qui ait été représenté par SAPPEY ; mais il existe un ou deux orifices de bronches récurrentes dans le territoire du sac diaphragmatique antérieur. Ces deux orifices À. d. a. placés un peu en arrière de la cloison qui sépare ce sac de l’interclaviculaire, appartiennent au type polybronchique simple et donnent naissance à des bronches récurrentes du sac diaphragmatique antérieur; 40 l’orifice du sac diaphragmatique postérieur, placé sur le bord latéral de la pente caudale du poumon, est vaste, moins rapproché de l’orifice abdominal que dans le Poulet. Cette position est due à la plus grande extension du sac diaphragmatique postérieur qui occupe la moitié de la pente caudale. L’orifice du sac, placé derrière le bord cranial du sac diaphragmatique postérieur est, à cause de cela, situé plus près du milieu de la pente caudale. C’est un orifice polybronchique mixte renfermant un grand nombre de bronches, dont la directe se reconnaît d'emblée à son aspect infundibuliforme, son plus grand diamètre étant tourné vers son extrémité distale. L’extrémité élargie de cette bronche directe est placée immédiatement au-dessous du diaphragme ornithique à tra- POUMON DES OISEAUX 355 vers lequel on la distingue aisément ainsi que les nombreuses parabronches qui criblent sa paroi. Elle est entourée de bronches récurrentes nombreuses, mais il est bien évident que vu sa forme conique, les nombreuses parabronches qui débouchent au voisinage de son ouverture doivent aussi remphr le rôle de récurrentes, l’air de retour du sac tendant plutôt à gagner le poumon par ces parabronches qu’à revenir dans la mésobronche et dans la trachée par l'extrémité proximale rétrécie du conduit direct. 5° l’orifice du sac abdominal est un orifice ovale, bien déve- loppé, du type polybronchique mixte. Sa bronche directe est formée par l’extrémité de la mésobronche qui s’effile graduellement en arrivant jusqu'à lui et qui est entourée de bronches récurrentes nombreuses À. ub., dont l’une, plus volumineuse que les autres, forme un gros tronc dirigé d’arrière en avant sur la face dorsale ‘du poumon. On peut la suivre aisément pendant un certain temps en lui voyant émettre des ramifications qui se perdent dans le réseau bronchique de cette région. Cette bronche que l’on peut reconnaître sur le moulage figure 31, a été également représentée par G. FISCHER dans la figure 5, Taf. IV (1905) du moulage d’Anas crecca (L.) mais l’auteur n’a pas su en saisir exactement la nature. La distribution des sacs aériens par rapport à la face ventrale du poumon est à peu près la même que chez le Poulet, sauf pour ce qui regarde les sacs diaphragmatiques qui se partagent à peu près également l’étendue de la pente caudale, comme je l’ai indiqué précédemment et pour l’allongement dans le sens caudal du sac cervical. Si maintenant on jette un regard d'ensemble sur les dispositions présentées par les différentes espèces, on voit d’abord pour ce qui regarde le type I, que les différences anatomiques ne coïncident point du tout exactement avec les ordres, puisque des animaux du même ordre offrent des dispositions tout à fait différentes ; ainsi les genres Columba (L.) et 356 A. JUILLET Larus (L.) qui appartiennent tous les deux aux Charadrii- formes présentent l’un le type I réduit (Columba) avec six orifices, l’autre au contraire (Larus) le type IT augmenté avec neuf orifices. De même parmi les Coraciiformes deux genres appartenant à l’ancien groupe des Rapaces nocturnes Strix et Noctua nous présentent le type Î moyen (sept orifices) tandis que Cypselus offre le type I augmenté (huit orifices). Enfin, on trouve avec la même disposition des espèces de deux ordres différents. Le type [ moyen comprend des Galliformes, un Gruiforme et des Coractiformes. — Le type I réduit comprend à la fois un Charadriiforme (Colomba) et des Cuculiformes (Palaeornis). Au contraire tous les Passériformes que nous avons observés et qui appartiennent à trois familles, celle des Corvidés (Garrulus), des Turdidés (Saxicola), des Fringillidés (Emberiza, Passer, etc.), présentent tous le même type à huit orifices, auquel appartient aussi un Falconiforme, Falco tinunculus (L.). \| Pour le type II, nous ne l’avons rencontré que dans le groupe des Ansériformes qui présente comme on le sait des caractères très primitifs. Il se peut donc que ce type représente la forme initiale dans laquelle les sacs aériens naîtraient tous indépendamment les uns des autres, chacun sur une entobronche propre et nous regrettons de n’avoir pu nous procurer d’autres types primitifs pour voir s’il en est bien ainsi. COMPARAISON AVEC LA DE LA STRUCTURE STRUCTURE DE CELUI DU POUMON DES DES AUTRES AMNIOTES. OISEAUX Une comparaison détaillée du poumon des Oiseaux et des autres Amniotes demanderait des études personnelles sur le poumon des Reptiles qui me font défaut. On ne peut en effet utiliser dans ce but les schémas de poumons de Reptiles qui ont été donnés par divers auteurs entre autres par MrILAntr et par MILLER ; mais si je manque de données personnelles pour la solution complète du problème, je puis cependant POUMON faire ressortir certains DES points OISEAUX d'anatomie 357 microscopique qui indiquent une différence profonde entre le poumon des Oiseaux et celui des autres Animaux. La comparaison du poumon des Oiseaux avec celui des Mammifères montre qu’il y a entre eux des divergences considérables. Sans doute, comme on l’a dit à propos de l’embryologie, on retrouve dans l’insertion des premières bronches sur la bronche souche, la disposition linéaire que l’on rencontre aussi chez les Mammifères, seulement au lieu des quatre lignes placées sur les parois externe, interne, antérieure et postérieure de la bronche souche que l’on observe chez les Mammifères, il n’existe ici que deux rangées, l’une à trajet spiroïde, pour les entobronches et les ectobronches, l’autre plus rectiligne pour le groupe des bronches secondaires latérales de CAMPANA. Il est en outre assez facile tout en tenant compte des différences de situation chez les Oiseaux, superficielle, profonde chez les Mammifères, de retrouver dans les ectobronches un groupe épartériel, dorsal à l’artère pulmonaire, et dans les entobronches le pendant du groupe hypartériel prédominant des Mammifères. Cette comparaison à été faite: elle est rapportée par G. FiIscHeR (1905, p. 20 et 21) qui fait remarquer que les bronches dorsales sont plus irrégulières et plus variables dans les différentes espèces que les ventrales, comme l’a déjà indiqué NAaRATH (1892) pour celles des Mammifères. Mais toutes ces comparaisons n’ont pas une portée bien grande. En réalité le poumon des Oiseaux est construit sur un tout autre modèle que celui des Mammifères et on peut ajouter que celui de tous les pulmonés. Il dérive évidemment d’une ébauche strictement homologue à celle du poumon des autres Amniotes. Il se développe également pendant les premiers jours suivant un modèle qui ne diffère point fondamentalement de celui de ces derniers, mais à partir du moment où les bronches récurrentes des sacs aériens apparaissent et où les parabronches s’anastomosent entre elles, il prend un type tout à fait spécial 308 A. JUILLET qui s'éloigne de plus en plus de la forme originelle commune et qui ne peut plus être comparé aux autres types dérivés de cette dernière sans forcer extrêmement les rapports indiqués. Les sacs aériens correspondent véritablement à une partie de l’arbre trachéo-bronchique, et la découverte des bronches récurrentes confirme absolument cette comparaison, en montrant que les sacs aériens se comportent absolument comme les bronches, puisqu'ils rentrent à nouveau dans le poumon, et comme les bronches vont s’anastomoser avec les bronches opposées. Il ne s’agit point là d’anastomoses latérales comme l’imaginait MILLER (1893) pour les parabronches des Oiseaux, anastomoses latérales qu’il comparait aux perforations latérales qui peuvent s'établir entre les différentes loges du poumon de certains Reptiles par résorption d’une partie de leur paroi commune. Seules les anastomoses des capillaires aériens du parenchyme pulmonaire des Oiseaux seraient à la rigueur comparables à ces anastomoses latérales des Reptiles. Mais l’anastomose de conduits aériens, tels que les parabronches est tout autre chose, et constitue un caractère que l’on ne retrouve point ailleurs. Il ne faut pas oublier, en effet, que ces anastomoses entre les parabronches ne sont point des fusions latérales s’effectuant entre des culs-de-sac à leur terminaison et sur les points où il existerait un contact intime entre eux. Ce sont des anastomoses terminales qui se font entre les deux extrémités de deux parabronches, parties des faces opposées du poumon, et qui s'effectuent partout avec une régularité admirable, sans qu'il y ait le moindre changement de calibre entre les deux parabronches qui s’unissent entre elles et sans que l’on puisse trouver jamais une seule parabronche terminée en cul-de-sac, dans tout le parenchyme pulmonaire. Cette disposition est évidemment en rapport avec la marche du courant aérien dans le poumon. Dans l’immense majorité des Amniotes autres que les Oiseaux, le courant qui apporte l’oxygène s'effectue toujours dans une seule direction, c’està-dire de la trachée vers la surface repsiratoire. Sans doute POUMON CPE DES OISEAUX 309 il existe chez de nombreux Reptiles des diverticules du poumon, soustraits à la fonction de lhématose et qui, constituant à cause de cela des réservoirs où l’air conserve son oxygène intact, peuvent distribuer ensuite cet air au parenchyme pulmonaire : mais il ne s’agit point ici encore d’une fonction bien régularisée et comportant une différenciation de l’appareil chargé d’assurer son mécanisme. Dans les Oiseaux la question est bien différente. Les réservoirs aériens ne constituent pas simplement des diverticules capables d’accumuler l’oxygène qu’ils diffuseront ensuite d’une manière plus ou moins parfaite dans un milieu aérien appauvri, mais ces réservoirs aériens prennent des connexions secondaires avec le parenchyme pulmonaire ; ‘ils se complètent par des voies de retour qui peuvent amener dans le poumon un courant d'air neuf, en l’y répartissant par des voies préformées admirablement disposées pour ce service, et qui conduisent dans la partie périphérique, et en quelque sorte terminale de l’arbre bronchique et non pas dans sa partie centrale, comme peuvent le faire les sacs des Reptiles. Le poumon se trouve donc parcouru par deux courants d'air pur, l’un qui vient de la trachée, l’autre qui vient des sacs : ces deux courants alternatifs étant toujours constitués par de l'air pur, il s’en suit que même dans le temps de l’expiration, le poumon fonctionne chez les Oiseaux comme dans l’inspiration la plus active et la plus favorable. Enfin, ce mouvement alternatif de l’air dans les circuits bronchiques, toujours continus, ne s’effectue point seulement pendant le temps où les mouvements respiratoires peuvent être régulièrement assurés par le jeu des cavités thoraco-abdominales, mais aussi pendant le vol, où il est produit par les mouvements alaires. Comme on l’a fait remarquer plus haut, c’est à partir d’un certain moment du développement, et en réalité du dixième au treizième jour que ces propriétés spéciales du poumon des Oiseaux s’accusent et à partir de là, elles ne font que s’accentuer davantage, car elles portent plutôt sur la disposition du 360 A. JUILLET parenthyme que sur les grandes voies d’accès de l’air. En effet, le parenchyme occupe une situation tout à fait spéciale. Au lieu d’être formé par les extrémités des culs-de-sac terminaux des ramifications bronchiques, elles-mêmes fort riches, il naît, avec une régularité admirable, de petits conduits radiés, les vestibules, qui criblent sans la moinäre interruption la paroi des parabronches, en formant tout autour de chacune d’elles un revêtement continu et admirablement régulier, comparable à des poils grossiers insérés en rayonnant sur toute la surface d’un cylindre. Là, par conséquent, point de bronchioles de longueur variable, portant, comme les ramifications d’une grappe, les infundibula irréguliers et lobulés accommodant leur forme aux espaces laissés par leurs voisins, mais au contraire, un manchon de parenchyme formé par les capillaires aériens issus des vestibules et qui donne à tout l’ensemble une disposition qui avait frappé depuis longtemps les premiers observateurs. Enfin, en dernière analyse et comme conséquence histologique de cette structure si particulière, il faut mentionner un fait unique dans la disposition de la surface respiratoire et qui contraste avec la structure de celle-ci, chez tous les autres Vertébrés quels qu’ils soient, aussi bien branchiés que pulmonés. Chez les Oiseaux, il n’y a plus une surface respiratoire, e’est-àdire des étendues planes parcourues par un réseau vasculaire également plan, à mailles arrondies et parfaitement régulières, comme on l’observe aussi bien dans les lamelles branchiales que dans les alvéoles pulmonaires des Mammifères, mais au contraire un treillis formé par la substance propre du poumon, c’est-à-dire par des travées minces, recouvertes d’endothélium et contenant les capillaires sanguins entrelacés avec un treillis complémentaire de cavités aériennes. Aussi CAMPANA exprimait-il très bien les caractères essentiels de leur poumon lorsqu'il disait (1875, p. 222): « chez les Oiseaux, les capillaires sanguins sont absolument environnés d’air et les capillaires pneumatiques sont de même partout environnés de sang ». La structure macroscopique aussi bien que microscopique du ne — POUMON DES OISEAUX 361 poumon des Oiseaux, est tout entière en corrélation avec le reste de l’organisation de ces animaux comme l’a déjà fait remarquer VIALLETON (1910, p. 431). Les détails du fonctionnement des sacs ont pu prêter à des interprétations divergentes pour ce qui à trait au mécanisme du fonctionnement de ces sacs, et nous ne ferons que rappeler la théorie de l’antagonisme de certains groupes de sacs soutenue par SaAPPEy et niée par SOUM. Il n'entre pas dans le cadre de nos recherches de prendre parti pour l’une ou l’autre de ces opinions, mais nous pouvons affirmer que la structure pulmonaire ne laisse absolument aucun doute sur le rôle des sacs et sur leur valeur, comme organes destinés à faire passer dans le poumon, l’air oxygéné qu’ils ont mis en réserve, Cet air a été en même temps échauffé et chargé de vapeur d’eau, comme l’a fait remarquer SOUM qui insiste sur l’espèce de suppléance que les sacs peuvent jouer, à ce dernier point de vue, vis-à-vis des glandes sudoripares. Conclusions La face ventrale du poumon des Oiseaux ne se comporte point, comme on l’admet implicitement, de la même manière dans toute son étendue; elle peut être divisée par une crête transversale, passant à peu près par l’insertion de la bronche entra-pulmonaire en deux parties : une tournée vers la tête, la pente craniale, l’autre tournée vers la queue, la pente caudale. Cette dernière seule est en rapport avec le diaphragme ornithique uni aux faisceaux musculaires costaux qui ont été décrits depuis longtemps. Elle peut donc seule être considérée comme répondant à la portion du poumon soumise à l’action de ce diaphragme, et toutes les considérations physiologiques émises sur le rôle de ce dernier ne peuvent s'appliquer qu'à elle. La bronche souche (mésobronche), qui traverse le poumon 362 A. JUILLET dans toute sa longueur et s’ouvre en arrière dans le sac abdominal, émet des bronches secondaires qui peuvent être divisées en deux groupes : Les ectobronches (HuxLEY) destinées à la face ventrale du poumon, les entobronches (HUXLEY) réservées à sa face dorsale. Ces bronches traversent perpendiculairement le poumon et, arrivées à la face qu’elles doivent occuper, se ramifient d’une manière pennée en bronches de troisième ordre, les parabronches (HUXLEY) qui conservent un même diamètre sur toute leur longueur. La première entobronche fournit les ramifications de toute la pente craniale, tant du côté latéral que du côté médial. La deuxième et la troisième entobronches donnent des rameaux principalement du côté médial. La quatrième entobronche est semi-pennée et s'étale seulement du côté latéral en fournissant la majeure partie des rameaux de la pente caudale. Les ectobronches donnent aussi des rameaux dirigés vers le bord médial et des rameaux latéraux. De la face profonde des ectobronches et des entobronches, partent des parabronches qui se comportent comme les superficielles. Les parabronches venues des entobronches vont s’anastomoser à plein canal avec celles des ectobronches, aussi bien à la surface que dans la profondeur du poumon et forment ainsi des circuits bronchiques, bien vus par CAMPANA. A cause du mode de ramification pennée des bronches, il y à deux sortes de circuits bronchiques: 10 Les circuits médiaux, formés par l’anastomose des parabronches venues des faces dorsale et ventrale du poumon et qui se dirigent du côté médial ; la ligne d’anastomose des parabronches qui forment ces circuits est très visible, elle est située sur la face médiale du poumon (face vertébrale) tout près du bord dorsal de cette face. 20 Les circuits latéraux. Les circuits latéraux sont formés par les parabronches qui se dirigent sur le bord latéral du poumon, mais ils présentent, au moins en partie, une plus grande POUMON DES OISEAUX 363 complexité que ceux du côté médial ce qui force à les subdiviser en deux groupes: a. Les circuits latéraux craniaux qui sont formés par l’anastomose des parabronches venues des rameaux latéraux de la première entobronche et de la première ectobronche. Ces rameaux s’anastomosent suivant une ligne très nette, située sur la face dorsale du poumon parallèlement à son bord latéral et tout près de ce dernier. Ces circuits comme les circuits médiaux sont simples, parce que les parabronches qui les constituent ne présentant que de rares anastomoses transversales qui les réunissent entre elles gardent leur trajet parallèle et ne forment point un réseau compliqué, comme cela s’observe dans les circuits caudaux, où ces anastomoses sont très multipliées, et où la direction parallèle des parabronches situées dans leurs intervalles, fait place à une véritable disposition en mailles. b. Circuits latéraux caudaux. Formés par les anastomoses des branches latérales des ectobronches et des entobronches, ces circuits sont énormément compliqués par l'intervention des bronches récurrentes qui se mêlent à eux et qui les transforment en un véritable labyrinthe de voies aériennes extrêmement embrouillé. Les bronches récurrentes sont des bronches issues des sacs interclaviculaire, diaphragmatiques antérieur et postérieur et abdominal, qui rentrent dans le poumon où elles se divisent en donnant des parabronches qui s’anastomosent à plein canal avec celles des circuits latéraux caudaux. La distribution des bronches récurrentes a été donnée en détail pour chaque sac. Nous ferons remarquer ici seulement, ce caractère général et constant, qu’elles siègent toujours très près du bord latéral du poumon sinon sur ce bord lui-même, et que certaines ont une tendance à passer sur sa face dorsale, comme on le voit toujours pour les bronches récurrentes du sac abdominal, et dans certains cas (Passeriformes) pour les bronches récurrentes du sac diaphragmatique postérieur. Il faut remarquer aussi que, 24 364 A. JUILLET sauf les bronches récurrentes du sac interclaviculaire, qui du reste peuvent manquer, toutes ces bronches appartiennent à la pente caudale et se trouvent en rapport étroit avec les muscles du diaphragme ornithique qui, quels que soient leurs autres effets, ont toujours celui de faciliter leur béance. Le développement montre de bonne heure la tendance générale des grosses bronches à se diriger vers la périphérie, et dans les ébauches pulmonaires du sixième au huitième jour, toutes les ramifications bronchiques sont situées à la surface du poumon, dont le centre est occupé exclusivement par un mésenchyme abondant et la bronche souche. Au huitième jour, les parabronches qui vont former les circuits médiaux, sont déjà rapprochées sur la face médiale et vont manifestement au-devant les unes des autres. En même temps les entobronches et les ectobronches qui leur ont donné naissance produisent par leur face profonde des bourgeons parabronchiques destinés à l’intérieur du poumon. Le développement des bronches récurrentes des sacs aériens, s'effectue entre le huitième et le dixième jour, les détails en sont donnés dans le texte. L’anastomose des parabronches qui va fermer les circuits pulmonaires, s’observe dès le treizième jour. Elle est précédée par une bifurcation en Y de l’extrémité de chaque parabronche, si bien que les parabronches d’un mêma circuit ne sont pas exactement dans le prolongement l’une de l’autre, mais alternent et sont réunies par un court segment oblique, de même diamètre et de même structure qu’elies-mêmes. A partir du neuvième jour, autour de cartaines parabronches, le mésenchyme se distribue en prismes limités par des bourgeons vasculaires pleins, formant un cadre polygonal ayant le même centre que la parabronche qu’il entoure. Cette disposition s'étend peu à peu à toutes les parabronches. Un peu plus tard, le dixième jour, l’épithélium de chaque parabronche est entouré par une couche continue, très mince POUMON DES OISEAUX 365 de fibres musculaires lisses, puis à partir du treizième jour, cet épithélium pousse des culs-de-sac radiés qui végètent tout autour de la parabronche dans le prisme mésenchymateux qui l’entoure. Vers le seizième jour, l'extrémité de ces culs-de-sac se bifurque et se prolonge par des culs-de-sac plus étroits, qui s’allongent jusque vers le cadre vasculaire limitant le prisme parabronchique, mais ne le dépassent pas. Cette bifurcation des culs-de-sac permet de leur distinguer deux parties : une partie proximale plus large, qui donne directement dans la lumière de la parabronche, c’est le vestibule : une partie périphérique formée de conduits plus fins, terminés en culs-de-sac et qui s’ouvrent dans le vestibule, ce sont les capillaires aériens. Vers la fin de l’incubation ces capillaires aériens forment un manchon serré tout autour de la lumière de la parabronche. Ils ne tardent pas à s’anastomoser entre eux, de manière à former un véri- table labyrinthe aérien dans chaque parabronche. Je n'ai pu observer la formation de ces anastomoses qui doit être extrêmement rapide, elle n’existe pas encore à la fin du dixneuvième jour et au vingt et unième elle est déjà aussi compliquée qu’elle l’est chez l’adulte. Le développement du poumon des Oiseaux explique les différences essentielles qu’il offre par rapport à celui des Mammifères. Les bronches ne se terminent jamais chez les Oiseaux en culs-de-sac, elles communiquent toutes entre elles, en formant des circuits qui peuvent être abordés par l'air pur, par l’une ou par l’autre de leurs extrémités, suivant que cet air viendra de la trachée ou des sacs aériens. Le parenchyme pulmonaire ne forme point de culs-de-sac compliqués à paroi plus ou moins bosselée et revêtue d’alvéoles. IL constitue un réseau de travées minces, parcourues par des capillaires sanguins et revêtues d’un endothélium que le nitrate d’argent met en évidence. Il en résulte que le réseau sanguin de l’hématose ne forme pas une surface plane, mise en contact avec l’air d’un seul côté ou de 366 A. JUILLET deux côtés à la fois lorsque une même lame vasculaire est commune à deux alvéoles adossées, mais que chaque capillaire sanguin est entouré par l’air de toutes parts. Il n’y a donc point dans le poumon des Oiseaux de surfaces respiratoires, formées par des réseaux, à peu près plans de capillaires décrivant des mailles extrêmement régulières, comme c’est le cas pour l’appareil respiratoire des autres Vertébrés, même pour les branchiés, mais un véritable labyrinthe sanguin développé dans les trois directions de l’espace et pénétré par l’air de tous les côtés. Cette disposition déjà entrevue par RAINEY et bien décrite par CAMPANA a été éclairée par la démonstration que j'ai pu donner du développement embryologique des capillaires aériens et de la présence d’un endothélium à leur surface, contrairement à ce que pensait Max BAER (1896), qui niaït la présence de cet endothélium. INDEX BIBLIOGRAPHIQUE (1 1896. Barr (Max). Beiträge zur Kenntnis. Atemwerkzeuge bei den Vôgeln. der Anat. und Physiol. d. (Zettschr. f. wissensch. Zool. Bd. 61, p. 420-498). 1905. BertTezrt (D.). Ricerche di Embriologia e di Anatomia comparata sul diaframma e sull’apparechio respiratorio dei vertebrati. (Archivio di Anatomia e di Embriologia. Vol. IV, p. 593-8%#4, Tav. LXXIX-LXX XIII). 1875. CAMPANA. Physiologie de la respiration chez les Oiseaux. Anat. de Pappareil pneumatique pulmonaire, des faux diaphragmes, des séreuses et de l'intestin chez le Poulet (p. 1-385, pl I-XVI, 1863. 1864. Paris, Masson). EBerTu. Ueber den feineren Bau der Lunge. (Zeuschr. f. wissensch. 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Mémoire sur lappareil de la respiration dans les Oiseaux (Ann. Sc. Nat. T. 5. (3), p. 25-87, PL IIT-IV). 1897. HARDIVILLER (A. D’). Développement et homologation des bronches principales, chez les Mammifères (Lapin). (Thèse de doctorat en Méd. 1882. Huxzey. Lille). On the respiratory organs of Apteryx (Proced. Zool. Soc. p. 960). Cité d’après le Zoologischer Jahresbericht de Naples. 1911. JUILLET (A.). a. Rapports des sacs aériens et des bronches chez leSOseaux (CR Acad Sc Paris. E_M152)1p. 1024). b. Observations comparatives sur les rapports du poumon et des sacs aériens chez les Oiseaux. (C. R. Acad. Se. Paris. 12152; p. 1930): c. Phases avancées du développement du poumon Poulet. (C. R. Soc. Biologie, T. Lxx, p. 985). d. Face ventrale du poumon (C. R. Soc. Biologie, 1835. Laurx. Nouveau p27167284pl.MI Manuel T. LxxI, des Oiseaux chez le et diaphragme. p. 230). de lAnatomiste. 2e Edit. (Paris 1835, VIT): 1911. MarcHanD (R.). Les pores alvéolaires du poumon chez les animaux. (CR Soc Biologie, T. xx, p. 912). 1893. Mrccer (W. S.). The Structure Vol. 8, p. 165-188). of the lung. (Journ. of Morphol. 1857. MiLxe Enwarps (H.). Leçons sur la Physiologie et Anatomie comparée de l’homme et des animaux. (Vol. IT, p. 399. Paris, Masson). 1902. Moser (Fanny). Beiträge zur vergleichenden chichte der Wirbeltierlunge. (Amphibien, ARCH. DE ZOOL. EXP. ET GÉN. — 5% SÉRIE. — T, IX. — (III). EntwicklungsgesReptilien, Vôgel, 24 A. JUILLET 368 Säuger). (Archio. fur mikrosk. Anatomie. Bd 60, p. 587-667, Taf. XXX-XXXIII). \| 1892. NaARATH (A.). Vergleichende Anatomie des Bronchialbaumes. (Ver. d. Anat. Ges. auf. d. VI. Vers. in Wien 1892, p. 168-174). 1905. OPPEL (A.). Lehrbuch der vergleichenden mikroskopischen Anatomie der Wirbeltiere. (VIe Teil. Atmungsapparat, p. 824, Taf. I-IV). 1835. OwEx (Ricu). Aves. (The Cyclopædia of Anat. and Physiol. Vol. I, 1835-1836). 1866. Owex (Ricx). Comparative Anatomy and Physiolog of Vertebrates. (Vol. II 1866. Longmans, Green and C9, London.) 1849. RaINEY (G.). On the minute structure of the Lung of the Bird. (Medico. Chirungic. Trans. XXXII, p. 47. Cité d’après Williams, voyez ci-après). 1891. RocHé (G.). Contribution à l'étude de l'anatomie comparée “Ex réservoirs aériens d’origine pulmonaire chez les Oiseaux. (Ann. d. sc. nat. Zool. Vol. XI (7), p. 1-120, PL I-IV). 1847. SAPPEY (PH. C.). Recherches sur l'appareil respiratoire des Oiseaux, (Paris, Germer-Baillière et Thèses sciences, Paris p.1-92, PL IV. 1847). SCHIMKEWITSCH (W.) Vergleich. Anat. d. Wirbeltiere, traduction allemande par Maier et Sukatschoff (Stuttgart p. 652). 1871. ScHULZE (F. E.) Manual of human and comparative histology by S. Stricker. (Vol. IT, p. 68-72. London Sydenham Society). 1908. ScHuLZE (F.E.). Die Lungen d. african. Strausses (Sitzung. d. Preus. Akad. d. Wissenchaft). Cité d’après le Jahresbericht de G. Schwalbe. 1866. SELENKA (Emir). Beiträge zur Entwickl. der Luftsäcke des Huhns. (Zeitschr. f. wiss. Zool. Bd. 16, p. 178-182, PI. VIII). 1896. SIEFERT (ERNST ) Uber die Atmung der Reptilien und Vôügel. (Arch. {. d. ges. Physiol. Bd. 64. p.321-506, Taf. III. IV-V). 1896. SouM. (J. M.). Recherches physiologiques sur l'appareil respiratoire des Oiseaux. (Ann. de l’Université de Lyon X XVIII, p. 1-126). 1899. SuPINO (FELICE). Ricerche sulla struttura del po'mone negli ucelli. (Atti della Societa Veneto-Trentina di Scienze naturali resid. in Padova. Ser. 2. Vol. 3. Fasc. 2, p. 306-316). Cité d’après Oppel. 1910. 1909. VIALLETON (L.) Précis de technique histologique et embryologique. 1911. VIALLETON (L.) Eléments de (O. Doin et fils, Paris 790 p.). 1911. VIALLETON (2° édit. O. Doin et fils, Paris. P. 467, PI. I-XIT). (L.) et JUILLET Morphologie des (A.). Sur la technique Vertébrés, des injections POUMON DES OISEAUX d’alliages fusibles en anatomie Biologie, T. LxxI, p. 249). 369 microscopique. (C. R. 1894 Vocr et E. Yunc. Traité d’Anatomie comparée pratique, C. Reinwald, Vol. II, p. 989). 1906. WiepersHEIM (R.). Vergleichende Anat. d. Wirbeltière Jena G. Fischer). 1859. Wiczrams. Respiration. (The Vol. V. suppl. London). Cyclop. of Anat. Soc. (Paris, (6° Auflage. and Physiol. 1900. ZumsTeiN (J.). Ueber den Bronchialbaum der Säuger und Vôgel. (Srtzungsber. Ges. z. Beford. d. ges. Naturviss. p. 39-48). 1891-92. Zumsrein (J.). Ueber Korrosions präparate. Sitzungsber Ges. z. Beford.d.ges. Naturwiss. Marburg. 1891-92). EXPLICATION DES PLANCHES A TOUTES LES FIGURES. . LETTRES COMMUNES B., bronche extra-pulmonaire. C. a., capillaire aérien. C. d. p., canal direct du sac diaphragmatique postérieur, E,, première entobronche. E,, deuxième entobronche. E,, troisième entobronche. E,, quatrième entobronche. dorsal de la deuxième entobronche. E, tr., rameau transversal de la quatrième entobronche. Ect,, première ectobronche. Ect,, deuxième ectobronche. Ect,, troisième ectobronche, ectobronche. Ect,, cinquième ectobronche. Ect,, sixième ectobronche, Mes., postérieur. \| Pb., parabronche, R. ab., orifice récurrent du sac abdominal. R. d. «., orifice récurrent du sac diaphragma- E,, d., rameau Ect,, quatrième O. d. p., orifice direct du sac diaphragmatique mésobronche. O. ab., orifice direct du sac abdominal, O. c., orifice direct du sac cervical, 0. ce. à. d., orifice commun des sacs interclaviculaire et diaphragmatique antérieur, tique antérieur. R. d. p., orifice récurrent du sac diaphragmatique postérieur. R. i., orifice récurrent du sac interclaviculaire, S. ab., sac abdominal. S. ce. sac cervical. $. d. a., sac diaphragmatique antérieur. an., anastomose. art., artère. b. s. L., bronche secondaire externe ep., épithélium. end., endothélium, hen., hematies. L. pb., lame parabronchique. m., muscles, tr.; travée. v., Vaisseau, vest., vestibule, PLANCHE XV FiG. 1. Schéma pour montrer la distribution des entobronches, des bronches récurrentes et des circuits broncniques sur la face ventrale du poumon du Poulet Gallus domesticus (L.). Gr. = 1 x 2. — Le rameau dorsal de la deuxième entobronche et le canal interclaviculaire n’ont pas été représentés. FIG. 2. Schéma pour montrer la distribution des ectobronches, des bronches récurrentes et des 2 circuits bronchiques sur la face dorsale du poumon du Poulet Gr. = 1 x LA A. JUILLET 370 FIG. FIG. Fi. 3. Schéma pour montrer la distribution des parabronches et des circuits médiaux sur la face médiale du poumon de Poulet. Gr. = 1 x 2. 4, Reconstruction du poumon droit d’un embryon de Poulet à la fin du sixième jour : la reconstruction est vue par le bord latéral. D. Reconstruction du poumon droit d’un embryon de Poulet au huitième jour ; la reconstruction est vue par la face médiale. Cette figure est surtout destinée à montrer la marche des parabronches dorsales et ventrales les unes au devant des autres sur la face médiale. PLANCHE XVI FIG. . Embryon de Poulet au dixième jour. Coupe transversale d’une parabronche de son territoire mésenchymateux propre. Gr. = 1 x 400. F1G. . Anastomoses F1G. let (treizième jour). Gr. — 1 x 400. . Début de la formation des capillaires aériens. des parabronches coupe transversale. Gr. = Fi1G. . Stade plus avancé du pour entourée former les circuits bronchiques. Embryon de PouEmbryon de Poulet (treizième jour), 1 x 400. développement des capillaires aériens (quinzième jour). Gr. = 1 x 400. PLANCHE Fi&. FIG. 10, XVII Différenciation des vestibules et des capillaires aériens (seizième jour). Gr. — . Coupe transversale chyme d’une respiratoire. parabronche de Poulet Safranine Picro-indigo-carmin. {PLANCHE 1 x 400. (deux jours après l’éclosion). Gr. = 1 x Paren- 400. XVIII Fi. . Poulet âgé de 2 jours. Injection métallique (Darcet). Gr. = 1 x 2. — «., face ventrale (poumon droit) ; b., face médiale (poumon gauche) ; e, face dorsale (poumon droit). Fi1G. . Poulet de 15 jours. Poumon droit, face dorsale. Injection métallique (Wood). Gr. = 1 x 1,5. F1, . Poulet adulte. Injection métallique (Wood), Gr. = Nat. —a, FIG. sale (poumon droit). . Noctua minor (Briss). Injection Fic. Fi. face ventrale ; b., face dor- métallique (Wood). Gr. nat. — «a., face ventrale ; b., face dorsale (poumon gauche). Une portion du sac abdominal a été laissée appendue à ses bronches récurrentes. 16. Gallinula chloropus (L.). Poumon gauche face ventrale. Injection métallique (Wood). Gr. = 1 x 1,8. — La pente caudale est divisée en deux moitiés par une crête transversale séparant les deux sacs diaphragmatiques. 17e Columba livit dom. (L.). Poumon droit face ventrale. Injection métallique (Wood). Gr. = 1 x 1,5.— Au dessous de la bronche extra-pulmonaire se voit une portion du sac diaphragmatique antérieur sur laquelle s’insère nettement du côté latéral la bronche récurrente de ce sac .On voit également partir du côté droit de cette masse métallique le canal interclaviculaire. Fic. 18. Columba livia dbm. (L.). Poumon gauche face ventrale. Injection métallique (Wood). Gr. = 1 x 1,5. — Cette figure montre très bien le rameau transverse de la quatrième entobronche et la crête transversale divisant la pente caudale en deux étages superposés pour les deux sacs diaphragmatiques. FiG. 19. Columba livia dom. (L.). Poumon droit, facem édiale. Injection métallique (Wood). Gr. = 1 x 1,5. — Pour montrer les parabronches internes, les parabronches de la couche externe ont été détruites. Columba livia dom. (L.). Injection métallique (Wood). Gr. = 1 x 1,5. — a face venFIG. : trale, b face dorsale (poumon droit). Pendant le refroidissement du métal la face dorsale s’est légèrement creusée sur son milieu par suite d’une mauvaise position du sujet. Fi1G. Palaeornis torquata. Injection métallique (Wood). Gr. = 1 x 1,2. — «a, face ventrale b, face dorsale (poumon droit) Sur le bord latéral de la pente caudale (fig. a) on voit l'insertion du sac diaphragmatique postérieur sous la forme d’une masse métallique faisant légèrement saillie vers le milieu de la pente caudale : plus bas apparait également l'insertion du sac abdominal, POUMON DES OISEAUX PLANCHE LUTER . Passer domesticus (Brisson). Poumons ventrale ; b, face dorsale poumon 371 XIX et sacs l'1G. aériens. Injection métallique (Wood) Gr. = 1 X 1,2. — Par suite de l’obliquité donnée à la pièce pour la photographier, le poumon droit semble se continuer directement dans le gauche. En réalité, ils sont séparés suivant la ligne peu marquée qui passe à 3 millimètres du bord gauche. 2, Melopsitacus undulatus (Shaw). Injection métallique (Wood). Gr. = 1 x 1,2. — «, face FiG. . Garrulus glandarius (L.). Injection métallique (Wood). Gr. = 1 x 1,8. — «, face ventrale : droit). b, face dorsale (poumon gauche). Sur l’angle inférieur et latéral de la figure &, on voit très facilement les bronches Ji. . Passer domesticus — 116, Gr. = (Brisson). récurrentes Poumon du sac abdominale, droit, face ventrale. Injection métallique (Wood). 1 x 2. 26. Ligurinus chloris (L.). Poumons et sacs aériens vus par la face gauche, Injection métalli- que (Darcet), Gr. = 1 x 2. 110, 27. Saxicola ænanthe (L.). Poumons et sacs aériens vus par la face droite. Injection métallique (Wood). Gr.= 1 x 1,5.— Les impressions costales sont bien accentuées sur le sac diaphragmatique postérieur et sur l’abdominal. De ce dernier partent des bronches récurrentes qui s’insèrent au niveau de l’avant-dernière empreinte costale ; les bronches récurrentes du sac diaphragmatique postérieur sont placées beaucoup plus en avant, sur le sillon costal le plus antérieur. F1G. . Cypselus apus (L.). Poumons et sacs aériens vus par la face droite. Injection métallique (Wood). Gr.= 1 X 1,5. — On voit nettement ici les bronches du sac abdominal, malgré une cassure transversale sur leur tronc etles bronches récurrentes du sac diaphragmatique postérieur peu éloignées des précédentes. PLANCHE FiG. XX 29. Larus ridibundus (L.). Injection métallique (Wood). Gr. = 1 x 1,2. — «, face ventrale, b, face dorsale (poumon gauche). Au niveau de la lettre b, se voient bien les bronches récurrentes du sac diaphragmatique postérieur. FIG. 30. Anas boschas (L.). Poumon droit, face ventrale. Injection métallique (Wood). Gr. nat. Du côté latéral, sur la pente caudale, la partie superficielle du moulage a été détruite et on aperçoit les parabronches profondes. 31. Anas boschas (L.). Poumon droit, face dorsale. Injection métallique (Wood). Gr. nat. Sur l'angle inférieur droit on voit très bien les bronches récurrentes du sac abdominal. FIG. 32. Métis de Canard domestique. Poumon gauche face ventrale, Injection métallique (Darcet). FiG. Gr. nat. SECONDE THESE PROPOSITIONS Botanique. — DONNÉES PAR LA FACULTÉ Respiration chez les végétaux. Minéralogie. — Gites cuprifères. Vu et approuvé. Paris, le 19 Juin 1911. LE DOYEN DE LA FACULTÉ DES SCIENCES, PaAuz APPELL Vu et permis d'imprimer : LE VICE-RECTEUR DE L'ACADÉMIE L. LIARD DE PARIS. ET LP Arch. de Zool. Exp° et Gén° Cranial 7 —= Lateral Caudal ÆEcté l re \| : \| A Dorsal Ect 3 'cty \| Juillet et Aimé del. \| POUMON \| If \|\| \| 3? Série, Tome IX P1 XV Cranial à Lateral Lith.AnstvE.A.Furiee, Leipzig. > OISEAUX. ( e ) Soie RABAT À DE RE E : @- © AS. Juillet et Aimé del. b- 1 ® : \ 5° Série, Tome ARE EL] A2 6 ï \| IX Re Et i Æ en SET op Se “aD PS). & Ç © LS (PErh Sr >= Lith.AnstvE£EAFanrke, (OISEAUX. PI XVI. Leipzig S ea << a Z == = À à 5 - : E nF CR ri :: E 4 un Lo] +. E o [ob] : : = ‘= 4 ë Si 8 À. " WU,/, \| Ï \| \| à ÿ 5 \| CU \| \| N È às F : J & Photographies. POUMON 10 œ Photographies. Arch. de Zool. 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https://openalex.org/W2597870570	https://www.mdpi.com/1424-8220/17/4/733/pdf?version=1490952252	English	null	Patients’ Data Management System Protected by Identity-Based Authentication and Key Exchange	Sensors	2,017	cc-by	9,065	Alexandra Rivero-García 1, Iván Santos-González 1, Candelaria Hernández-Goya 1, Pino Caballero-Gil 1,* and Moti Yung 2 1 Department of Computer Engineering and Systems, University of La Laguna, 38206 Tenerife, Spain; ariverog@ull.edu.es (A.R.-G.); jsantosg@ull.edu.es (I.S.-G.); mchgoya@ull.edu.es (C.H.-G.) 2 Computer Science Department, Snapchat and Columbia University, New York, NY 10027, USA; 1 Department of Computer Engineering and Systems, University of La Laguna, 38206 Tenerife, Spain; ariverog@ull.edu.es (A.R.-G.); jsantosg@ull.edu.es (I.S.-G.); mchgoya@ull.edu.es (C.H.-G.) 2 Computer Science Department, Snapchat and Columbia University, New York, NY 10027, USA; moti@cs.columbia.edu 1 Department of Computer Engineering and Systems, University of La Laguna, 38206 Tenerife, Spain ariverog@ull.edu.es (A.R.-G.); jsantosg@ull.edu.es (I.S.-G.); mchgoya@ull.edu.es (C.H.-G.) 1 Department of Computer Engineering and Systems, University of La Laguna, 38206 Tenerife, Spain; ariverog@ull.edu.es (A.R.-G.); jsantosg@ull.edu.es (I.S.-G.); mchgoya@ull.edu.es (C.H.-G.) 2 Computer Science Department, Snapchat and Columbia University, New York, NY 10027, USA; moti@cs.columbia.edu g ( ); j g ( ); g y ( ) 2 Computer Science Department, Snapchat and Columbia University, New York, NY 10027, USA; moti@cs.columbia.edu * Correspondence: pcaballe@ull.edu.es * Correspondence: pcaballe@ull.edu.es Academic Editor: Vittorio M. N. Passaro Received: 31 January 2017; Accepted: 28 March 2017; Published: 31 March 2017 Academic Editor: Vittorio M. N. Passaro Received: 31 January 2017; Accepted: 28 March 2017; Published: 31 March 2017 Abstract: A secure and distributed framework for the management of patients’ information in emergency and hospitalization services is proposed here in order to seek improvements in efﬁciency and security in this important area. In particular, conﬁdentiality protection, mutual authentication, and automatic identiﬁcation of patients are provided. The proposed system is based on two types of devices: Near Field Communication (NFC) wristbands assigned to patients, and mobile devices assigned to medical staff. Two other main elements of the system are an intermediate server to manage the involved data, and a second server with a private key generator to deﬁne the information required to protect communications. An identity-based authentication and key exchange scheme is essential to provide conﬁdential communication and mutual authentication between the medical staff and the private key generator through an intermediate server. The identiﬁcation of patients is carried out through a keyed-hash message authentication code. Thanks to the combination of the aforementioned tools, a secure alternative mobile health (mHealth) scheme for managing patients’ data is deﬁned for emergency and hospitalization services. Different parts of the proposed system have been implemented, including mobile application, intermediate server, private key generator and communication channels. Alexandra Rivero-García 1, Iván Santos-González 1, Candelaria Hernández-Goya 1, Pino Caballero-Gil 1,* and Moti Yung 2 Apart from that, several simulations have been performed, and, compared with the current system, signiﬁcant improvements in efﬁciency have been observed. Keywords: identity-based cryptosystem; identity-based authentication and key exchange; mHealth; keyed-hash message authentication code; Android; NFC sensors sensors sensors Sensors 2017, 17, 733; doi:10.3390/s17040733 Keywords: identity-based cryptosystem; identity-based authentication and key exchange; mHealth; keyed-hash message authentication code; Android; NFC 1. Introduction One of the most innovative paradigms of the last years in the healthcare sector is the integration of mobile devices in the practice of medicine and public health, known as mHealth. Its signiﬁcance stems from the ﬂexibility provided by the use of mobile devices. However, the potential security problems that arise from the use of mobile devices and their wireless interface must be carefully addressed due to the strict privacy requirements of medical data. In the work [1], a few recommendations to solve some of these problems are explained in detail. This paper presents a secure and distributed system for the management of patients’ data in emergency and hospitalization services with the primary goal of improving efﬁciency and security. In particular, several cryptographic protocols are used to protect the conﬁdentiality of the communications and the access control to patient records. The risk of patient misidentiﬁcation is an issue to which health authorities pay a lot of attention, in order to try to avoid dangerous consequences such as medication errors, incorrect surgical procedures, Sensors 2017, 17, 733; doi:10.3390/s17040733 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors Sensors 2017, 17, 733 2 of 16 etc. [2]. In spite of that, statistical data on this subject are worrying. For example, in the UK, the National Health Service received more than 24,000 statements on misidentiﬁcations of patients in 2006–2007 [3]. One of the bases of the proposal is the use of Near Field Communication (NFC) [4], speciﬁcally NFC wristbands, for automatic patient identiﬁcation. Unlike other technologies such as Radio Frequency IDentiﬁcation (RFID) [5], Bluetooth or Wi-Fi [6], NFC is not oriented to continuous data transmission because it requires a temporal contact between the devices that interact in order to allow the exchange of information in a quick and timely manner. Although at ﬁrst glance the distance factor for transmitting information may seem a limitation, it is actually a key point of this technology. The need for proximity between devices limits the types of attacks that can be launched. In addition, not requiring pairing between devices facilitates its use by medical staff. 1. Introduction Apart from the NFC wristbands , the other main components of the system are: a mobile device associated to each member of the medical staff, the intermediate server that hosts a web service, an NFC reader and writer for allocating wristbands once patients have been identiﬁed, and a second server in charge of producing information to protect the exchange of information. This work is organized as follows. Section 2 provides some related works while Section 3 gives a general description of the proposed system. The topic of patient identiﬁcation through NFC tags and keyed-Hash Message Authentication Code (HMAC) schemes in emergency and hospitalization services is dealt with in Section 4. The protection of communications between the medical staff and the intermediate server through Identity-Based Authentication and Key Exchange is proposed in Section 5. A brief security analysis is provided in Section 6. The implementation of parts of the proposal and simulations of the system are explained in Section 7. Finally, a few conclusions and future works close the paper. 3. System Overview In most current health systems, when a patient arrives at a hospital, the ﬁst step that the staff must do is to identify her/him. Patient identiﬁcation is usually performed through the veriﬁcation of a health identiﬁcation card. Then, the patient is evaluated by a healthcare staff member who analyses the information collected during admission and adds the results of new assessments if required. Afterwards, the patient may be seen by a specialist. Before each one of these actions, the process of patient identiﬁcation must be repeated. This current system has several drawbacks. Doctors must check the patient record before assisting her/him. In order to do it, depending on the particular case, they can make such a consultation through printed documentation or by using a computer. If paper documentation is used, it is usually generated as a batch for a set of patients. For example, three medical records may be printed at a time so that a doctor can check and attend those three patients one after the other. Once they are attended, the doctor should leave the records and repeat the process with a new group of patients. This arrangement produces heterogeneous information because some data may be updated on computers while other data are kept in printed format. In addition, updates made by doctors are not changed in the central system in real time. In this approach, health workers have to deal with a lot of documentation, which leads to consuming considerable time and resources. On the other hand, each member of the medical staff has to visit several patients at each turn, which may generate wrong patient identiﬁcations, with serious consequences in some cases. A solution to these issues is described in this work, which consists in the implementation of a secure system based on NFC wristbands and mobile devices that allow for eliminating patient misidentiﬁcation and rationalizing the use of time in patient care. The proposal involves substantial changes with respect to the traditional system. When patient identiﬁcation is performed for the ﬁrst time, an NFC wristband is assigned to him/her. Speciﬁcally, the NTAG21x ICs [17] wristband, which follows the pattern set by the NFC Forum (association that regulates the NFC standards) [18] is recommended here. This wristband will not be used to store any sensitive patient data. The only data stored on the wristband is an identiﬁer assigned by the server. 2. Related Works Recently, different solutions to solve speciﬁc problems in the management of the patients’ data have been proposed. The security in healthcare applications is one of the most important points, even more when wireless sensor networks are used [7]. The speciﬁc case of wireless body area networks is very useful in healthcare, as shown in [8], where sensors are wearable devices that allow for obtaining, computing and distributing information about patients. The data shared by these networks are usually stored as electronic health records so that the protection of the privacy of these records is very important. In the work [9], a system using an attribute-based infrastructure is proposed to preserve the privacy of the data. In that system, the registration of patients and doctors is performed with a username and a password, and the identiﬁcation of the patient is a private user-index. The so-called Personal Health Record (PHR) facilitates the management of medical records in a centralized way. One of the improvements obtained with the use of PHR is that patients can analyse their own information. In the work [10], a proposal of this kind of system is proposed based on the use of PHR in cloud computing, where an attribute-based encryption protocol is used to obtain the information. However, in that paper, the identiﬁcation of patients and authentication of doctors are not explained. Sharing medical care information in a secure way is proposed in the work [11], thanks to the use of role-based secure messaging services. The main problem of this approach is that a speciﬁc e-mail provider is used, and that the authentication is based on external tools. In the paper [12], the authors introduce a mutual authentication scheme to improve the security of automatic systems for medication through RFID bracelets and cryptography based on elliptic curves. However, the used protocol does not provide a mechanism for session key generation to protect conﬁdentiality. There are proposals for medical systems that base their operation on the use of NFC bracelets. In the work [13], an example is shown where it is assumed that every day each patient uses a bracelet. If there is an emergency that requires checking of medical data, these are read using a mobile Sensors 2017, 17, 733 3 of 16 application. 2. Related Works A disadvantage of that application is that any user who has the application could perform an information query because there is no security mechanism implemented. application. A disadvantage of that application is that any user who has the application could perform an information query because there is no security mechanism implemented. In the work [14], a system for the management of medical staff rounds is described that uses NFC wristbands and mobile devices. However, it does not specify whether security services are deployed or not. The authors of the publication [15] developed an attack on a mutual authentication system based on RFID tags deﬁned in [16]. Speciﬁcally, they show that it is possible to trace the tags so the protection of patient privacy is not guaranteed. At the same time, a new protocol is deﬁned that solves the privacy problem and improves the efﬁciency of the system. An authentication scheme for the RFID tag is introduced, but it does not take into account the security of RFID readers. Apart from this, the server used to manage the information is also in charge of generating and storing the keys. The proposal presented here includes automatic patient identiﬁcation, mutual authentication between server and medical staff, and protection of conﬁdentiality in the communications between the mobile device and the server. Conﬁdentiality between the mobile device and wristband is warranted by the need for proximity to establish the NFC connection. Thus, this integration of security tools provides a robust solution that improves patient management and daily routine of medical staff. Sensors 2017, 17, 733 Sensors 2017, 17, 733 4 of 16 This wristband can be deployed both in the inpatient and emergency areas. It can be even assigned before the patient arrives to hospital, in the ambulance, where the identiﬁcation and the writing procedures could be done through a mobile phone. The data stored in the wristband allow any member of the medical staff with the right permissions access to the patient record identifying the patient with the simple gesture of bringing a mobile device close to the wristband. Thanks to the use of wristbands, the system prevents confusion when identifying patients and increases efﬁciency in the development of medical tasks. In addition, wristbands are fully recyclable, so when a patient leaves the hospital, its wristband is reset to be used by another patient. The system is designed to work with two separated servers, here referred to as the intermediate server and second server. On the one hand, the intermediate server manages access permissions to patients’ data on the basis of medical staff shifts. On the other hand, the second server uses a Private Key Generator (PKG) to manage the information related to keys. The use of two different physical servers is proposed to add a new security layer in the management of the keys. With this separation, different ﬁrewalls can be added to each server independently and different secure rules can be applied in the communications between them. Speciﬁcally, the limitation of the communication of the private key server to intra-communication (intranet communications) is advisable. In other words, the communication of the PKG with the extranet can be denied and just some interactions with the intermediate server can be allowed through, for example, an intranet. In this way, if the intermediate server is corrupted by an attacker, both the private key generator and the server keys should not be involved. Although having two servers is more expensive than having just one, since nowadays the value of a dedicated server in the cloud is about $50 a year, we consider that this is a very low value when compared with the security that it brings to the proposed system. The protection of patients’ data is a paramount objective in the healthcare environment. This is why security is one of the pillars of the described solution. Sensors 2017, 17, 733 A keyed-Hash Message Authentication Code (HMAC) is applied for automatic patient identiﬁcation, and IDentity-based (ID-based) cryptography is used to protect conﬁdentiality of patient records. The security of the communication between doctors and the intermediate server is based on an ID-based Authentication and a Key Exchange (AKE) scheme that provides mutual authentication between doctors and the server through a PKG. Next, the details on how these security tools are used in the proposed framework are included. 3. System Overview This identiﬁer is generated through a process that will be discussed below. Such a generation takes into account the physical identiﬁer of the wristband (similar to the Media Access Control (MAC) address number of computers) together with the patient record number. Note that this information will be written on the wristband in a completely secure way. 4. Automatic Patient Identiﬁcation As aforementioned, when a patient arrives at a hospital, the ﬁrst step is the identiﬁcation through his/her credentials. After that, in the proposal, an NFC wristband is assigned to him/her so that each patient is identiﬁed through an HMAC generated by the intermediate server by using the physical identiﬁer of the wristband and the patient record number. If a patient does not have a medical record in the system, it is automatically created with some basic ﬁelds, such as name, age, country, etc. The generation of the HMAC can be seen in Figure 1. The system sends the physical identiﬁer of the wristband to the intermediate server, and two 64-byte arrays denoted as ipad and opad are generated as in the work [19], where some default values are assigned to them during the initialization of the HMAC generation. New arrays denoted by ipadmsk and opadmsk are generated through an bit level exclusive OR operation on ipad and opad respectively, and the master secret key (msk). Then, with the physical identiﬁer of the wristband Tag (idTag) and the Patient Record Number (PatRecN), the system uses a SHA3-512 hash function [20] to generate the HMAC value. Firstly, the hash function is applied to the concatenation of ipadmsk, idTag and PatRecN. Secondly, the output of this hash function concatenated with the opadmsk is the input to another hash function so that the HMAC is the ﬁnal result. This output is stored in the NFC wristband to be used as patient identiﬁer. Sensors 2017, 17, 733 5 of 16 When trying to access to a patient data, his/her NFC wristband must be read through a doctor’s device, which sends the data obtained from the wristband, corresponding to the physical identiﬁer of the wristband and the HMAC, to the server. The server veriﬁes the authenticity of the bracelet and the doctor’s access permissions. If the veriﬁcation is positive, the authentication protocol described later is used each time a member of the medical staff needs to access to patients’ data. Figure 1. Keyed-hash message authentication code Generation. Figure 1. Keyed-hash message authentication code Generation. The protection of communications is achieved through an ID-based scheme. In this type of public key cryptography schemes, any text can act as a valid public key with a PKG. 4. Automatic Patient Identiﬁcation The intermediate server sends to the second server idTag, HMAC and PatRecN. 10 The PKG analyses and veriﬁes the association and the result of this veriﬁcation is sent to the 9. The intermediate server sends to the second server idTag, HMAC and PatRecN. 9. The intermediate server sends to the second server idTag, HMAC and PatRecN. 10 The PKG analyses and veriﬁes the association and the result of this veriﬁcation is sent to the 0. The PKG analyses and veriﬁes the association, and the result of this veriﬁcation is sent to intermediate server. 11. If the HMAC veriﬁcation is right, the server sends the medical record values to the mobile device where the doctor can read, edit or add data. The values corresponding to a patient can be modiﬁed until a new patient’s wristband is read. 11. If the HMAC veriﬁcation is right, the server sends the medical record values to the mobile device where the doctor can read, edit or add data. The values corresponding to a patient can be modiﬁed until a new patient’s wristband is read. Figure 2. Medical record consultation. Figure 2. Medical record consultation. 4. Automatic Patient Identiﬁcation The main reason to choose this approach for the proposal is the simpliﬁcation of management because in this way it is not necessary to deﬁne a public key infrastructure. Furthermore, an ID-based scheme was chosen because of its low computational complexity and its efﬁciency in terms of memory and usability. The description of all the steps of the communication ﬂow during a medical record consultation between the participants in the system is included below (see Figure 2): 1. A member of the hospital admission staff receives the basic patient’s data. 2. This patient’s information is sent to the intermediate server. The identiﬁcation of the patient is analysed at the server. If the patient is registered in the system, the server stores any new information and the system sends the veriﬁcation to the web application. Otherwise, the server generates a new user identiﬁcation and stores the corresponding data. g p g 3. The assignation of a wristband to a patient starts with the reading of the physical identiﬁcation of the tag idTag through an NFC reader. g p g 3. The assignation of a wristband to a patient starts with the reading of the physical identiﬁcation of the tag idTag through an NFC reader. 4. The idTag is sent from the web application to the intermediate server, which links the idTag with the patient’s medical record of number PatRecN and sends these values to the PKG in the second server. 5. The PKG generates the HMAC value with idTag, PatRecN and the pre-calculated values ipadmsk and opadmsk. The HMAC value is sent to the intermediate server, which sends it to the web application. pp 6. The HMAC value is stored in the NFC tag of the patient’s wristband. 7. When a doctor wants to identify a patient, he/she has to touch the NFC wristband with his/her mobile device to read both idTag and HMAC. Sensors 2017, 17, 733 6 of 16 8. The stored values are sent from the mobile device to the intermediate server where the wristband is identiﬁed. The medical record is then loaded. 8. The stored values are sent from the mobile device to the intermediate server where the wristband is identiﬁed. The medical record is then loaded. 8. The stored values are sent from the mobile device to the intermediate server where the wristband is identiﬁed. The medical record is then loaded. 9. 5. Secure Communication Communications between the mobile device of each member of the medical staff and the intermediate server are encrypted with an ID-based scheme. Communications between the mobile device of each member of the medical staff and the intermediate server are encrypted with an ID-based scheme. A crucial element of the proposal is the Private Key Generator in the second server because it is in charge of generating private keys for medical staff. The identiﬁer used in the system for each member of the medical staff is the corresponding number of registered medical practitioner. Speciﬁcally, the system is based on the proposal described in [21], but adapted to a more secure infrastructure where the PKG and the intermediate server are separated. As seen in Figure 3, on the one hand, there are different devices assigned to doctors, which are smartphones or tablets with NFC reader and Wi-Fi. On the other hand, each patient has an NFC wristband. The intermediate server is the controller of the communication between the medical staff and the PKG. In particular, the intermediate server has a public Application Programming Interface (API) for doctors’ communication and other hospital computers and a private API for communication with the PKG. Finally, the PKG is in charge of the authentication and veriﬁcation of each communication. This is why server keys are stored in the PKG. The protection of communications is achieved through an ID-based scheme. The ﬁrst approach of this type of scheme was proposed by Shamir [22] in 1985 to avoid certiﬁcate management in asymmetric key systems. In this public key cryptography schemes, any text can be used as a valid public key. Speciﬁcally, the public key is usually extracted from some user’s identity information, such as name, email or health identiﬁcation. Following Shamir’s idea, Boneh and Franklin proposed a practical 7 of 16 Sensors 2017, 17, 733 ID-based encryption system [23] based on the Weil pairing. Starting from this proposal, multiple ID-based schemes have been introduced. Some basic ID-based schemes may be identiﬁed depending on the type of security service to be implemented: ID-based encryption schemes [24–26], ID-based signature schemes [27,28], ID-based signcryption schemes [29,30], ID-based group key exchange protocols [31,32] and ID-based AKE [33,34]. ID-based encryption system [23] based on the Weil pairing. Starting from this proposal, multiple ID-based schemes have been introduced. 5. Secure Communication Some basic ID-based schemes may be identiﬁed depending on the type of security service to be implemented: ID-based encryption schemes [24–26], ID-based signature schemes [27,28], ID-based signcryption schemes [29,30], ID-based group key exchange protocols [31,32] and ID-based AKE [33,34]. Figure 3. System’s communication ﬂow. Figure 3. System’s communication ﬂow. In the proposed system, mobile devices are used to manage patients’ information. These devices have energy and computing capability limitations, so they should not depend on heavy cryptographic computations. Taking this into account, some protocols based on a client-server paradigm have been applied. One of the most used techniques to reduce the online cost is ofﬂine pre-computation. In the ofﬂine pre-computation used in this proposal, a few random values called ephemeral secrets are required to perform some operations in advance. These values are stored in the memory of the mobile device until the system requires them in the online step. The attack called Ephemeral-Secret-Leakage (ESL) [35,36] might be launched in the online step. In order to be resistant to ESL attacks, the present proposal uses an ID-based AKE protocol that does not use bilinear pairings. Speciﬁcally, the scheme is based on the ESL-secure ID-based AKE protocol [21] that uses an ESL-secure signature scheme [37] to manage the client-to-server authentication and the Tate pairing [38], which is faster than the basic Weil pairing [39]. Computable: There exists an efﬁcient algorithm to compute ˆe(P, Q), ∀P, Q ∈G. • Computable: There exists an efﬁcient algorithm to compute ˆe(P, Q), ∀P, Q ∈G. Let a cyclic group (G, +) have prime order q and P′ be a generator of G; then, the following mathematical assumption based on the so-called Elliptic Curve Difﬁe-Hellman (ECDH) [40] problem can be considered. Given P′, aP′, bP′ ∈G for unknown a, b ∈Zq, the ECDH problem consists of computing abP′. No probabilistic polynomial time exists to allow an adversary to compute abP′ with a non-negligible probability. Two different types of hash functions are used. On the one hand, two map-to-point hash functions: H1 : {0, 1}∗→G∗, H2 : {0, 1}∗→G∗. On the other hand, a one-way hash function: On the other hand, a one-way hash function: f : {0, 1}∗→{0, 1}n, where the size of the message is deﬁned by n. where the size of the message is deﬁned by n. We assume that to concatenate a point P to a number N, the coordinates (Px, Py) of the point P are concatenated separately so that P\|\|N is equivalent to Px\|\|Py\|\|N. The four steps needed for the ID-based AKE scheme are: Setup, Extract, Mutual Authentication and Session Key Generation. • Setup: The initial parameters are established and the PKG in the second server generates the master public key mpk and the master secret key msk. For that, a prime q based on some private data k ∈Z, two groups G and GT of order q and a symmetric bilinear pairing map ˆe : G × G →GT are selected. P ∈G is randomly chosen and the hash functions H1, H2 and f are used (see Figure 4). • Setup: The initial parameters are established and the PKG in the second server generates the master public key mpk and the master secret key msk. For that, a prime q based on some private data k ∈Z, two groups G and GT of order q and a symmetric bilinear pairing map ˆe : G × G →GT are selected. P ∈G is randomly chosen and the hash functions H1, H2 and f are used (see Figure 4). Figure 4. Setup phase. Figure 4. Setup phase. • Extract: The private group key for each member of the medical staff based on its ID is generated. The intermediate server generates a random number l, and sends it together with ID to the second server. 5. Secure Communication The notations used within this paper are introduced below: • G, GT : cyclic groups; • P, P′ : generators of the group G; • ˆe : a bilinear map from G × G to GT; • msk : randomly selected master secret key; • mpk : master public key; • ID : IDentiﬁcation of a registered medical practitioner; • pgkID : private group key of the medical practitioners with ID; • H1, H2, f : hash functions; • {0, 1}n : space of all n-length binary vectors; • {0, 1}∗: space of all binary strings of any length; • G∗: multiplicative group G\{0}; • G, GT : cyclic groups; ′ • P, P′ : generators of the group G; msk : randomly selected master secret key; • ID : IDentiﬁcation of a registered medical practitioner; • pgkID : private group key of the medical practitioners with ID; • {0, 1}n : space of all n-length binary vectors; • {0, 1}∗: space of all binary strings of any length; • G∗: multiplicative group G\{0}; 8 of 16 Sensors 2017, 17, 733 • x r←S : an element x is randomly selected from a set S; • \|\| : concatenation; • \|\| : concatenation; • == : comparison. • == : comparison. Next, the mathematical basis used in the system is described. Considering two cyclic groups (G, +) and (GT, ·) of the same prime order q, there is a symmetric bilinear map pairing ˆe : G × G →GT with the following properties: • Bilinear: ∀P, Q ∈G and ∀a, b ∈Z, ˆe(aP, bQ) = ˆe(P, Q)ab; • Bilinear: ∀P, Q ∈G and ∀a, b ∈Z, ˆe(aP, bQ) = ˆe(P, Q)ab; • Non-degenerate: ∃P1, P2 ∈G that ˆe(P1, P2) ̸= 1. This means that if P is generator of G, then ˆe(P, P) is a generator of Q; Computable: There exists an efﬁcient algorithm to compute ˆe(P, Q), ∀P, Q ∈G. Then, the PKG computes different values to obtain a private group key pgkID, which is calculated taking into account the master private key mpk. Finally, in the intermediate server, a secure channel is created based on an ECDH to share the private information with the doctor. A session key is generated to encrypt the information, obtaining an encrypted message C with a Snow 3G stream cipher algorithm [41] (see Figure 5). • Extract: The private group key for each member of the medical staff based on its ID is generated. The intermediate server generates a random number l, and sends it together with ID to the second server. Then, the PKG computes different values to obtain a private group key pgkID, which is calculated taking into account the master private key mpk. Finally, in the intermediate server, a secure channel is created based on an ECDH to share the private information with the doctor. A session key is generated to encrypt the information, obtaining an encrypted message C with a Snow 3G stream cipher algorithm [41] (see Figure 5). 9 of 16 Sensors 2017, 17, 733 Figure 5. Extract phase. Figure 5. Extract phase. Figure 5. Extract phase. • Mutual Authentication: Some ofﬂine computations are performed by the client to be able to perform mutual authentication. After the extract phase, the client generates and sends to the server a 3-tuple. Then, the server parses the tuple to obtain the values, and generates some new parameters. Afterwards, the server sends all the information to the PKG, who is in charge of the veriﬁcation of the bilinear map pairing. If the veriﬁcation is OK, the PKG sends a parameter to the server, otherwise a call “close” is sent back to the server to ﬁnish the communication. If the veriﬁcation was OK, the server generates a tuple, which is sent to the client, which authenticates the server. If this authentication is OK, the client generates a parameter for its own authentication against the server and sends it back to the intermediate server, which veriﬁes the user authentication (see Figure 6). If everything is OK, both client and server continue to the next step, which is the session key generation. Note that in the veriﬁcation step carried out in the second server, the PKG checks whether the condition ˆe(P, V) == ˆe(W1, W)ˆe(mpk, W2) is fulﬁlled in order to accept the communication. Computable: There exists an efﬁcient algorithm to compute ˆe(P, Q), ∀P, Q ∈G. The justiﬁcation of that condition is explained below: ˆe(P, V) = ˆe(P, T + DID2) = ˆe(P, (r + DID1) · W + DID2) = ˆe(P, (r + l + h · msk) · W + msk · QID2) = ˆe(P, (r + l) · W + msk · (h · W + QID2) = ˆe(P, (r + l) · W) · ˆe(P, msk · (h · W + QID2)) = ˆe(P · (r + l), W) · ˆe(P · msk, h · W + QID2) = ˆe(P · r + P · l, W) · ˆe(mpk, h · W + QID2) = ˆe(U1 + QID1, W) · ˆe(mpk, W2) = ˆe(W1, W) · ˆe(mpk, W2). ˆe(P, V) = ˆe(P, T + DID2) = ˆe(P, (r + DID1) · W + DID2) = ˆe(P, (r + l + h · msk) · W + msk · QID2) = ˆe(P, (r + l) · W + msk · (h · W + QID2) = ˆe(P, (r + l) · W) · ˆe(P, msk · (h · W + QID2)) = ˆe(P · (r + l), W) · ˆe(P · msk, h · W + QID2) = ˆe(P · r + P · l, W) · ˆe(mpk, h · W + QID2) = ˆe(U1 + QID1, W) · ˆe(mpk, W2) = ˆe(W1, W) · ˆe(mpk, W2). = ˆe(W1, W) · ˆe(mpk, W2). • Session Key Generation: When both server and client have been mutually authenticated, the session key (see Figure 7) is generated, at the same time, on the client side and on the server side. Afterwards, the exchanged messages are encrypted using the produced session key. In particular, the communication exchange between server and doctor is performed using the stream cipher SNOW 3G [42] using the obtained session key. 10 of 16 Sensors 2017, 17, 733 Figure 6. Mutual authentication phase. Figure 6. Mutual authentication phase. Figure 6. Mutual authentication phase. Figure 7. Session key generation. 6 S it A l i Figure 6. Mutual authentication phase. Figure 6. Mutual authentication phase. Figure 7. Session key generation. Figure 7. Session key generation. 6. Security Analysis This section includes a brief review on the protection provided by the system against different types of attacks. A spooﬁng attack and/or cloning of the card would be hardly successful in the proposal, since these types of attacks involve the generation of the HMAC, but this generation requires the server master private key, the ID of a registered medical practitioner and the patient record number. Even if an external attacker obtains this information, the combination between the physical identiﬁer of the NFC wristband and the patient record number is unique. If the attacker corrupts the data of the wristband, the system can easily detect it because the server can know that the information used by the attacker does not coincide with the stored data. Thus, one of the strong points of the proposal is that the data saved on the NFC wristbands are not sensitive data. Sensors 2017, 17, 733 11 of 16 If an attacker wants to emulate the wristband with an Android device, the attack is detected because the application is restricted only to read passive NFC tags. Denial of Service attacks based on overloading the server with a large number of false requests are restricted because only those requests associated to the ID of a registered medical practitioner will take effect. Once the corresponding private key is assigned, additional requests from the same number will not be attended. Regarding Man in the Middle attacks, they would be easily detectable because the number of members who are allowed to make requests to the server is limited to those who are working at the time of the request. Regarding ESL attacks, the corresponding security level is based on the primitive of the ESL-secure ID-AKE protocol for mobile client-server environments included in the scheme proposed. In addition, there is a client-to-server authentication that prevents an adversary can impersonate a legitimate medical staff person to share information with the server through the use of the ECDH problem. In the case of the server-to-client authentication, the same reasoning is applicable. The protection is provided through a mutual authentication scheme and a SNOW 3G stream cipher with shared secret key obtained by an ECDH scheme. Apart from this, a key agreement procedure is deﬁned to obtain the key required to encrypt all the communications between the server and the clients through the same stream cipher. This key agreement provides protection under known-session-key attacks. 6. Security Analysis Finally, an implicit implicit key conﬁrmation is used based on a random oracle model, in order to offer partial forward secrecy in this model. The system design includes two servers, the intermediate server and the second server with the private key generator. The use of two different physical servers is proposed to have an additional security layer for key management. In this way, ﬁrewalls can be added independently to each server, and different secure rules can be applied in the communications between them. In other words, the communication of the private key generator with the Extranet might be denied, and just some interactions with the intermediate server might be allowed through, for example, an Intranet. Furthermore, if the intermediate server is corrupted by an attacker, the key generator will not be affected. 7. Performance Analysis Some basic prototypes of the proposal have been implemented on the client side, and the implementation is mainly formed by a web and a mobile application. On the server side, the prototype contains a data server and the private key generator. The web application is in charge of the management of patients’ and doctors’ information, and of the assignment of doctors to patients (see Figure 8). Figure 8. Web application. Figure 8. Web application. Thanks to the mobile application, doctors can easily identify and analyse the patient record. The integration of the application with the NFC sensor of the smartphones has been implemented for the Android platform in order to read and write NFC tags in a secure way (see Figure 9). 12 of 16 Sensors 2017, 17, 733 Figure 9. Mobile application. (left) View of registration; (right) View of patient data. Figure 9. Mobile application. (left) View of registration; (right) View of patient data. The data model for patient information is based on the clinic history containing is information related with afﬁliation data and health care data. Among the information related to the health, medical staff can analyse: reason for consultation (or hospitalization), personal history (allergies, habits, medical history, surgical history, family history, social history or current treatment), family background, current illness, anamnesis by organs, physical exploration results, differential diagnosis, supplementary tests, diagnostic trial and even the current therapeutic plan. In the back-end side, the server was implemented based on a Model-View-Controller design pattern and with a non-relational database. On the one hand, the public access to the server was limited to restrict the access to the PKG, generated by a REpresentational State Transfer (REST) API. On the other hand, the internal communication between the server and the PKG is generated by a local REST API that has external restrictions, which means that a query can be generated only in the local site of the server. The prototypes have undergone some tests. Since the HMAC generation depends on the server computer capabilities, different traces of the communication system were collected and evaluated. During these tests, the intermediate server was a computer with a quad core processor (Intel(R) Core(TM) i7-3537U CPU @ 2000 GHz) (Intel Corporation, Santa Clara, CA, USA), 4 GB of RAM memory, 1 TB of storage memory and the Windows 10 Pro version (×64 bits). 7. Performance Analysis This computer was used to access to the web application (see Figure 8) and patients’ data. The private key generator was a similar computer, with an Intel i7-4702MQ processor (Intel(R) Core(TM) CPU @ 2000 GHz) (Intel Corporation, Santa Clara, CA, USA), 8 GB of RAM memory, 1 TB of storage memory and Windows 10 operating system version (×64 bits). As the client, a Samsung Galaxy S6 (Samsung Electronics, Suwon, Korea) with Exynos 7420 octa core processor (Samsung Electronics, Suwon, Korea) (4 × 2.1 GHz Cortex-A57 4 × 1.5 GHz Cortex-A53), 3 GB of RAM memory, 32 GB of storage memory and the 6.0 Android version was used. In these experiments, an amount of 100 data packets were collected to be analysed in order to obtain results related with the overall response time. The network used to perform all the tests was made up of a Tp-link TL-WR841N Wi-Fi router that theoretically offers a maximum transfer rate of 300 Mbps and an Internet connection of 100 Mbps. All communications were tested in the laboratory with the prototypes, obtaining a transmission time of less than 1 s. Taking these results into account, it may be stated that less than a minute is necessary both to assign a NFC wristband to a patient and to read patients’ data. Sensors 2017, 17, 733 13 of 16 Apart from implementing different parts of the proposal to show its feasibility, several simulations of its behaviour in a real environment have been done to measure some parameters that indicate improvements with respect to the current system. One of the most important points of this proposal is to save time on the tasks of the medical staff. Currently, before assisting a patient, the doctor must review the patient record, which, in many cases, is printed and located in speciﬁc areas. The mobility feature of the devices used in the proposed system allows for reducing the time spent by medical staff on roaming because they will not need to go to the documentation area and so they can attend patients without limitations. Some simulations on the distribution of a ﬂoor in a real hospital (see Figure 10) have been performed. In this example, the orange zones are the areas where the hospital has the documentation areas for the doctors. Figure 10. Hospital map. Figure 10. Hospital map. 7. Performance Analysis For the simulations, the map of the ﬂoor was divided into four parts so that each one was run in a quarter of the map. A grid map of each part was generated to evaluate the route that doctors follow to visit each patient (see Figure 11). The best route in the current system consists of visiting patients located in adjoining rooms. In the simulation, it is assumed that there are two patients in each room. Figure 11. Grid map of the simulated area. The time required for the doctor’s route depends on the number of patients that a doctor can visit at once, that is to say, the size of the batches of patients that he/she has to visit before going back to the documentation area. In the proposed system, the time required for the doctor’s route is always constant because it is assumed that he/she does not have to go to the documentation area. In Figure 12, a representation of the grid units covered by the physician can be observed. Blue bars reﬂect the grids covered in the current system while red ones show those required by the proposed system. Figure 11. Grid map of the simulated area. Figure 11. Grid map of the simulated area. Figure 11. Grid map of the simulated area. The time required for the doctor’s route depends on the number of patients that a doctor can visit at once, that is to say, the size of the batches of patients that he/she has to visit before going back to the documentation area. In the proposed system, the time required for the doctor’s route is always constant because it is assumed that he/she does not have to go to the documentation area. In Figure 12, a representation of the grid units covered by the physician can be observed. Blue bars reﬂect the grids covered in the current system while red ones show those required by the proposed system. 14 of 16 Sensors 2017, 17, 733 Figure 12. Time required for a doctor’s route. Figure 12. Time required for a doctor’s route. The improvement observed in the time consumed by each doctor in the route to attend patients in batches is increased by the saving on the time that the identiﬁcation of patients requires since, with the proposed system, this identiﬁcation is automatic thanks to the mobile devices of doctors and NFC wristbands of patients. Acknowledgments: Research was supported by TESIS2015010102, TESIS2015010106, RTC-2014-1648-8, TEC2014-54110-R, MTM2015-69138-REDT, DIG02-INSITU and IDI-20160465. References 1. Oakes, R.; Allen, C. mHealth Security: Best Practices and Industry Trends. Available online: http://www.oracle.com/us/corporate/proﬁt/archives/opinion/011813-oakes-allen-1899091.html (accessed on 29 March 2016). 1. 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These two issues are addressed in this work through the proposal of a distributed framework for the secure management of patients’ information. The proposed system is based, on the one hand, on NFC wristbands assigned to patients and mobile devices assigned to medical staff, and, on the other hand, on two servers to manage patients’ data and to generate private keys separately. A modiﬁcation of an ID-based Authentication and Key Exchange Protocol resistant to Ephemeral-Secret-Leakage attacks for mobile devices is presented in this paper to provide mutual authentication between server and health staff. Speciﬁcally, the proposed protocols include client-to-server authentication, server-to-client authentication, key agreement, implicit key conﬁrmation and secure channel to share keys. This is part of a work in progress. All the curves used in the performed beta implementation were chosen according to the National Institute of Standards and Technology suggestions [40] over Fp, but in the next implementations, new curves over F2 p, and in particular the new curve called FourQ, will be used to try to improve efﬁciency [43] in the implementation of the ECDH protocol on the most used processors in current smartphones. Furthermore, a future version of the system will include a robust and secure anonymity scheme based on pseudonyms. The issue of non-traceability of patients is also an open issue. Finally, although some simulations were generated and the system was tested in the laboratory with some medical staff, in the immediate future, the system will be deployed in a real hospital to analyse the real improvements contributed by this proposal compared with the traditional method. 15 of 16 15 of 16 Sensors 2017, 17, 733 Author Contributions: All the authors conceived the system, developed the algorithms and wrote the paper. Alexandra Rivero-García and Iván Santos-Gonález performed the experiments. Candelaria Hernández-Goya, Pino Caballero-Gil and Moti Yung revised the work. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References A ﬂexible role-based secure messaging service: Exploiting IBE technology for privacy in health care. technology for privacy in health care. 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https://openalex.org/W4213387137	https://www.researchsquare.com/article/rs-101772/v1.pdf?c=1604625680000	English	null	Hemoglobin A1c Levels Affect Visit-to-visit Variability of Lipid Profiles in Patients Undergoing Elective Percutaneous Coronary Intervention: A Retrospective Study	Research Square (Research Square)	2,020	cc-by	5,725	Hemoglobin A1c Levels Affect Visit-to-visit Variability of Lipid Profiles in Patients Undergoing Elective Percutaneous Coronary Intervention: A Retrospective Study p y Duanbin Li Zhejiang University School of Medicine Sir Run Run Shaw Hospital Cao Wang Zhejiang University School of Medicine Sir Run Run Shaw Hospital Liding Zhao Zhejiang University School of Medicine Sir Run Run Shaw Hospital Yi Luan Zhejiang University School of Medicine Sir Run Run Shaw Hospital Ya Li Zhejiang University School of Medicine Sir Run Run Shaw Hospital Guosheng Fu Zhejiang University School of Medicine Sir Run Run Shaw Hospital Zhaoyang Chen Xiehe Affiliated Hospital of Fujian Medical University Wenbin Zhang (  3313011@zju.edu.cn ) Department of Cardiology, Sir Run Run Shaw Hospital, School of Medicine Zhejiang University, 3 East Qingchun Road, Hangzhou, Zhejiang Province, People’s Republic of China. https://orcid.org/0000-0003- 4746-9570 Research Keywords: Hemoglobin A1c, HbA1c, Variability, Lipid profiles, Percutaneous coronary intervention Posted Date: November 5th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-101772/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Conclusion HbA1c is a potential risk factor for the variability of LDL-C, non-HDL-C, TC and TG in patients undergoing elective PCI. Research Page 1/17 Results A total of 4,445 patients were enrolled in the study. The median age and value of HbA1c were 64yrs old and 5.9%, respectively, 64% had hypertension and 25.5% diabetes. The variability of low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), total cholesterol (TC), and triglyceride (TG) was each significantly higher in patients with HbA1c≥6.5% than those with HbA1c＜6.5% group. Multivariate linear regression indicated that HbA1c level was a potential risk factor for the variability of LDL-C, non-HDL-C, TC and TG, which was independent of the mean values of lipids. Subgroup analyses demonstrated that the relationship between HbA1c and the variability of LDL-C, non- HDL-C, TC, and TG did not importantly vary across several subgroups. These results remained consistent when lipid variability was represented by the standard deviation (SD), coefficient of variation (CV) and variability independent of the mean (VIM), respectively. Background Both hemoglobin A1c (HbA1c) levels and visit-to-visit variability of lipid profiles are risk factors for cardiovascular disease (CVD). We conducted a retrospective cohort study to explore the relationship between HbA1c and lipid variability. Methods We retrospectively collected baseline and follow-up data on patients who underwent elective percutaneous coronary intervention (PCI) from January 2009 to April 2019. Univariate and multivariate linear regression analyses were performed to assess the association between HbA1c and lipid variability. Subgroup analyses employed multivariate linear regression analyses. Background Cardiovascular disease (CVD) remains the leading cause of death globally and is one of the most common complications of diabetes mellitus (DM)[1, 2]. Hemoglobin A1c (HbA1c) is usually regarded as an indicator representing the average blood glucose levels over the past 2-3 months and is often employed to assess glycemic control[3]. Several epidemiological studies[4, 5] have demonstrated associations between elevated HbA1c levels and adverse CVD outcomes in patients with type 2 DM. Moreover, even within the normal reference range, increased blood glucose levels are associated with an increased risk of CVD[6]. Study subjects The study population comprised all consecutive patients who attended the Sir Run Run Shaw Hospital, Zhejiang University in China, from January 2009 to April 2019. Eligibility criteria for inclusion into the study were: (1) patients who underwent elective PCI, (2) sufficient clinical information such as HbA1c an lipid profiles were available at baseline and follow-ups, (3) patients were followed up at 1, 3, 6, 9, and 12 months following PCI according to the prescribed follow-up procedures. Patients with acute myocardial infarction (MI), active cardiopulmonary diseases, heart failure, severe liver and/or renal insufficiency, cancer, acute or chronic infection and other serious diseases were excluded from the study. A total of 4,445 patients were finally enrolled in the study. All PCI procedures were performed by experienced interventional cardiologists using the recommended guidelines[15]. Baseline and follow-up measurements of exposures were ascertained from fasting venous blood samples (at least 8 hrs overnight). Levels of HbA1c, LDL-C, HDL-C, non-HDL-C, TC and TG were determined at each follow-up. Standard follow-up procedures and examinations were employed for all patients who had undergone elective PCI. The Ethics Committee of Sir Run Run Shaw Hospital of Zhejiang University approved the study (20200803-34). Page 2/17 Page 2/17 Page 2/17 Traditionally, mean values of indicators or exposures are often employed to estimate the prognostic risk for CVD. For example, higher low-density lipoprotein cholesterol (LDL-C) levels and lower high-density lipoprotein cholesterol (HDL-C) levels are associated with an increased risk of CVD[7, 8]. However, if there is wide variability in the exposure, the mean value will no longer be reliable. In recent years, more attention is being focused on the variability of these exposures. With particular emphasis on lipid profiles, the visit- to-visit variability of LDL-C, HDL-C, non-high-density lipoprotein cholesterol (non-HDL-C), total cholesterol (TC) and triglyceride (TG) has each been demonstrated to be a potential risk factor for CVD[9-13]; these associations are independent of the mean levels of the exposures and traditional cardiovascular risk factors. Based on intravascular ultrasound examination, elevated variability of LDL-C has been shown to independently promote the progression of atherosclerosis, which is the crucial underlying pathology of CVD[14]. In general, lipid variability, an independent risk factor for CVD, is attracting considerable attention. Traditionally, mean values of indicators or exposures are often employed to estimate the prognostic risk for CVD. For example, higher low-density lipoprotein cholesterol (LDL-C) levels and lower high-density lipoprotein cholesterol (HDL-C) levels are associated with an increased risk of CVD[7, 8]. However, if there is wide variability in the exposure, the mean value will no longer be reliable. In recent years, more attention is being focused on the variability of these exposures. With particular emphasis on lipid profiles, the visit- to-visit variability of LDL-C, HDL-C, non-high-density lipoprotein cholesterol (non-HDL-C), total cholesterol (TC) and triglyceride (TG) has each been demonstrated to be a potential risk factor for CVD[9-13]; these associations are independent of the mean levels of the exposures and traditional cardiovascular risk factors. Based on intravascular ultrasound examination, elevated variability of LDL-C has been shown to independently promote the progression of atherosclerosis, which is the crucial underlying pathology of CVD[14]. In general, lipid variability, an independent risk factor for CVD, is attracting considerable attention. Both HbA1c and the variability of blood lipids are risk factors for CVD. However, whether there is any relationship between HbA1c and the variability of lipids is still unclear. Therefore, the purpose of this study is to investigate the relationship between HbA1c and lipid variability in patients undergoing elective percutaneous coronary intervention (PCI). Definitions The level of HbA1c was defined as the mean value during a 1-year follow-up period and was calculated using values of HbA1c measured at follow-up. Diabetes mellitus was defined as a fasting serum glucose≥126 mg/dL, a history of diabetes or the current use of antidiabetic medications. Hypertension was defined as blood pressure≥140/90 mmHg, a documented history of hypertension or on anti- hypertensive medications. Regular statin therapy was defined as atorvastatin≤20mg or rosuvastatin≤10mg per day. Intensive statin therapy was defined as atorvastatin≥40mg or rosuvastatin≥20mg per day. Particular attention was paid to patients’ compliance with statin use. Heart failure was defined by ejection fraction (EF) < 40% or N-terminal pro B-type natriuretic peptide (NT-pro BNP) > 2000 pg/ml without renal failure. Body mass index (BMI) was calculated as weight in kilograms, divided by height in meters squared. Glomerular filtration rate (GFR) was estimated using the Japanese Society of Nephrology equation as follows: estimated GFR (eGFR) (mL/min/1.73 m2) = 194×serum creatinine-1.094×age-0.287 (×0.739 for women)[17]. Assessment of variability in lipid profiles Baseline and follow-up values of lipids were measured by a blood chemistry analyzer (Hitachi 747; Hitachi, Tokyo, Japan). The variability of lipids reflected the degree of fluctuation of individual blood lipid levels during the 1-year follow-up period. To assess the lipid variability more comprehensively, the Page 3/17 Page 3/17 following 3 indicators were used:(1) standard deviation (SD); (2) coefficient of variation (CV): The CV of blood lipids was defined as follows, CV=(SD/mean)×100 (%); and (3) variability independent of the mean (VIM): VIM was calculated as (SD/meanβ)×100 (%), in which β was derived from the fitting of the curve and was the regression coefficient based on the natural logarithm of SD and the natural logarithm of the mean[16]. following 3 indicators were used:(1) standard deviation (SD); (2) coefficient of variation (CV): The CV of blood lipids was defined as follows, CV=(SD/mean)×100 (%); and (3) variability independent of the mean (VIM): VIM was calculated as (SD/meanβ)×100 (%), in which β was derived from the fitting of the curve and was the regression coefficient based on the natural logarithm of SD and the natural logarithm of the mean[16]. Statistical analysis Continuous variables were presented as median (interquartile range) or mean (standard deviation). Categorical variables were represented as frequency (%). Baseline characteristics were compared between patients with HbA1c＜6.5% and HbA1c≥6.5% using chi-square tests for categorical variables and nonparametric tests for continuous variables. Univariate and multivariate linear regression analyses were used to evaluate the relationship between HbA1c and lipid variability. Subgroup analyses employed multivariate linear regression analyses. A value of P<0.05 (2 sided) was considered statistically significant. Statistical analysis was performed using SPSS software version 22.0 (SPSS Inc., Chicago, IL, USA). appeared to have higher BMI (24.73[23.38-26.67] vs. 24.62[22.48-26.00]); p<0.001), more likely to have hypertension (70.0% vs. 62.5%; p < 0.001) than those with HbA1c＜6.5%. Table 1 summarizes the baseline characteristics of the enrolled patients categorized by HbA1c levels. Patient characteristics Table 1 Baseline characteristics of patients Table 1 Baseline characteristics of patients Page 5/17 Characteristics Total (n=4445) HbA1c＜6.5% (n=3538) HbA1c≥6.5% (n=907) P- value Demographic information Age (Years) 64.00(58.00- 70.00) 64.00(58.00- 70.00) 64.00(58.00- 71.00) 0.158 Male, N (%) 3187(71.7) 2549(72.0) 638(70.3) 0.391 BMI, (kg/m2) 24.73(22.77- 26.18) 24.62(22.48- 26.00) 24.73(23.38- 26.67) ＜ 0.001 Current smoking, N (%) 1102(24.8) 890(25.2) 212(23.4) 0.388 Hypertension, N (%) 2845(64.0) 2210(62.5) 635(70.0) ＜ 0.001 Type 2 diabetes, N (%) 1133(25.5) 327(9.2) 806(88.9) ＜ 0.001 Previous MI, N (%) 164(3.7) 129(3.6) 35(3.9) 0.786 Previous PCI, N (%) 342(7.7) 278(7.9) 64(7.1) 0.581 LDL-C<1.8 mmol/L, N (%) 3729(83.9) 2984(84.3) 745(82.1) 0.127 Laboratory examination HbA1c (%) 5.9(5.50-6.70) 5.7(5.40-6.00) 7.6(6.90-8.70) ＜ 0.001 LDL-C (mean, mmol/L) 1.78(1.49-2.13) 1.78(1.50-2.12) 1.77(1.47-2.18) 0.834 HDL-C (mean, mmol/L) 1.02(0.88-1.20) 1.05(0.90-1.22) 0.97(0.83-1.14) ＜ 0.001 non-HDL-C (mean, mmol/L) 2.46(2.10-2.90) 2.47(2.10-2.87) 2.44(2.08-2.95) 0.806 TC (mean, mmol/L) 3.66(3.21-4.16) 3.67(3.25-4.12) 3.62(3.15-4.23) 0.219 TG (mean, mmol/L) 1.36(1.04-1.82) 1.35(1.04-1.78) 1.38(1.05-1.95) 0.051 LDL-C (SD) 0.47(0.27-0.75) 0.45(0.25-0.74) 0.50(0.29-0.76) 0.014 HDL-C (SD) 0.13(0.08-0.18) 0.13(0.08-0.18) 0.13(0.08-0.18) 0.577 non-HDL-C (SD) 0.43(0.25-0.70) 0.42(0.25-0.70) 0.47(0.27-0.73) 0.004 TC (SD) 0.59(0.35-0.93) 0.56(0.33-0.90) 0.66(0.39-1.00) ＜ 0.001 TG (SD) 0.33(0.19-0.55) 0.32(0.19-0.52) 0.37(0.20-0.65) ＜ 0.001 ( ( ( 97.70) 99.01) 98.11) eGFR (mL/min/1.73 m2) Baseline medication ACEI, N (%) 1102(24.8) 876(24.8) 226(24.9) 0.897 ARB, N (%) 1565(35.2) 1180(33.4) 385(42.4) ＜ 0.001 Beta blocker, N (%) 2614(58.8) 2092(59.1) 522(57.6) 0.429 CCB, N (%) 1325(29.8) 1020(28.8) 305(33.6) 0.007 Aspirin, N (%) 4294(96.6) 3424(96.8) 870(95.9) 0.244 Clopidogrel, N (%) 3592(80.8) 2889(81.7) 703(77.5) 0.007 Ticagrelor, N (%) 805(18.1) 613(17.3) 192(21.2) 0.008 Statin, N (%) 4401(99.0) 3507(99.1) 894(98.6) 0.151 Intensive statin, N (%) 845(19.0) 660(18.7) 185(20.3) 0.064 Statin combined with ezetimibe, N (%) 956(21.5) 764(21.6) 192(21.2) 0.818 Baseline medication Data are expressed as median (25%-75%) or n (%). Total indicates total group; SD, standard deviation; BMI, body mass index; MI, myocardial infarction; LDL-C < 1.8 mmol/L , LDL-C < 1.8 mmol/L at the first- month follow-up after percutaneous coronary intervention (PCI); LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride; eGFR, estimated glomerular filtration rate; ACEI, angiotensin-converting enzyme inhibitors; ARB, angiotensin receptor blocker; CCB, calcium channel blocker Patient characteristics Page 4/17 The 4,445 patients who had undergone elective PCI at the Sir Run Run Shaw Hospital between January 2009 and April 2019 comprised 3,538 males and 907 females. Overall, the median age of subjects was 64 (58-70) years old, with a median BMI of 24.73 (22.77-26.18) kg·m-2, 64% had hypertension and 25.5% had type 2 diabetes mellitus. The median value of HbA1c was 5.9%, with the interquartile range from 5.5% to 6.7% for all subjects. The variability of LDL-C, non-HDL-C, TC and TG was each significantly higher in patients with HbA1c≥6.5% than in those with HbA1c＜6.5%. Patients with HbA1c≥6.5% Page 4/17 appeared to have higher BMI (24.73[23.38-26.67] vs. 24.62[22.48-26.00]); p<0.001), more likely to have hypertension (70.0% vs. 62.5%; p < 0.001) than those with HbA1c＜6.5%. Table 1 summarizes the baseline characteristics of the enrolled patients categorized by HbA1c levels. appeared to have higher BMI (24.73[23.38-26.67] vs. 24.62[22.48-26.00]); p<0.001), more likely to have hypertension (70.0% vs. 62.5%; p < 0.001) than those with HbA1c＜6.5%. Table 1 summarizes the baseline characteristics of the enrolled patients categorized by HbA1c levels. appeared to have higher BMI (24.73[23.38-26.67] vs. 24.62[22.48-26.00]); p<0.001), more likely to have hypertension (70.0% vs. 62.5%; p < 0.001) than those with HbA1c＜6.5%. Table 1 summarizes the baseline characteristics of the enrolled patients categorized by HbA1c levels. Results of univariate and multivariate linear regression for the variability o Based on clinical considerations and differences in baseline characteristics according to HbA1c categories, the following factors were included in the univariate analysis: HbA1c, age, gender, BMI, current smoking, hypertension, eGFR, types of statin, intensive statin treatment, statin combined with ezetimibe and the corresponding mean values of lipids. Factors that were significant in the univariate analyses (p<0.05) were included in multivariate linear regression analysis. Multivariate analysis for the effect of HbA1c on lipid variability was conducted in all patients and demonstrated that HbA1c was a potential risk factor for the variability of lipids, which included LDL-C (regression coefficients of HbA1c on SD [β] 0.014, standard error [SE] 0.004, P-value＜0.001), non-HDL-C ([β] 0.023, [SE] 0.004, P-value＜0.001), TC ([β] 0.041, [SE] 0.005, P-value＜0.001), and TG ([β] 0.033, [SE] 0.005, P-value＜0.001). Besides, HbA1c remained a potential risk factor for the variability of LDL-C, non- Page 7/17 Page 7/17 HDL-C, TC and TG when CV and VIM were employed to represent lipid variability, respectively (see Table 2 to 5). Page 8/17 Results of the multivariate linear regression in subgroups In multivariate subgroup analyses, the relationship between HbA1c and the variability of LDL-C, non-HDL- C, TC, and TG did not significantly vary across several subgroups such as non-diabetes, diabetes, atorvastatin, rosuvastatin, regular statin, intensive statin and statin-ezetimibe combined therapy. Figures 1-3 present the results of subgroup analyses when lipid variability was represented by SD. Similarly, when CV and VIM were employed to represent lipid variability, respectively, subgroup analysis results were consistent with results when using SD. (see Table 6). Page 9/17 Page 9/17 Discussion The main findings of this study are summarized as follows: (1) HbA1c level was a potential risk factor for the variability of LDL-C, non-HDL-C, TC and TG. The results were consistent when SD, CV, and VIM were used to represent lipid variability, respectively; (2) Subgroup analysis demonstrated that the effect HbA1c on the variability of LDL-C, non-HDL-C, TC, and TG remained similar in several relevant subgroups. In patients without diabetes, the significant positive correlations between HbA1c and lipids variability remained, including LDL-C, non-HDL-C, TC and TG. Elevated visit-to-visit variability of blood lipids is associated with different adverse outcomes or organ dysfunction, including CVD, obstructive sleep apnea, renal function decline and cognitive decline[18-20]. Based on the Treating to New Targets (TNT) trial, the visit-to-visit variability of LDL-C, HDL-C and TG was demonstrated to be an independent predictor of cardiovascular events[21, 22]. Moreover, Lee et al.[12] confirmed that the visit-to-visit variability of non-HDL-C was associated with major adverse cardiovascular and cerebrovascular events (MACCE) in patients who had undergone PCI. Furthermore, a study based on 3.6 million people in the general population showed that the variability of TC was associated with the risk of all-cause mortality, MI and stroke[23]. A wealth of data suggests that the visit- to-visit variability of lipid profiles is independently associated with the risk of CVD. However, the underlying mechanisms for the association are still unclear. Similarly, HbA1c has been identified as a risk factor for CVD and confirmed by many epidemiological studies[4, 5, 24, 25]. The current study has demonstrated the potential role of HbA1c as a risk factor for the variability of LDL-C, non-HDL-C, TC, and TG. To further explore the relationship between HbA1c and blood lipid variability, we postulate the following underlying mechanisms based on findings from previous studies. Insulin resistance (IR) is considered one of the underlying mechanisms, which play an essential role in glucose and lipid metabolism. In this study, IR may be ubiquitous among subjects. A quarter of the patients had a history of type 2 DM, which is very likely to be accompanied by IR[26]. Besides, within the normal range of glucose tolerance, patients with CVD have more IR compared to those without CVD, which suggests that patients without type 2 DM in this study may also have IR[27]. Conclusion HbA1c is a potential risk factor for the variability of LDL-C, non-HDLC, TC, and TG in patients undergoing elective PCI. Discussion IR not only affects HbA1c levels by reducing glucose transport into cells and glycogen synthesis, but is also closely related Page 10/17 Page 10/17 to dyslipidemia[28]. Although the existing evidence is not sufficient to ultimately demonstrate the effect of IR on lipid variability, the evidence for the role of IR in lipid metabolism appears to suggest this possibility. IR leads to dyslipidemia via the following ways: (1) increased TG, (2) decreased HDL-C, and (3) the appearance of small dense LDL particles[29]. Moreover, hyperinsulinemia, which is the response of insulin resistance, stimulates the synthesis and secretion of very LDL and promotes LDL-C transport into arterial smooth muscle cells[30, 31]. In general, given the high prevalence of IR in our patients and its effects on glucose and lipid metabolism, IR could be the potential mechanism behind our findings[32]. Based on analyses of baseline characteristics, the variability of LDL-C, non-HDL-C, TC and TG showed significant differences between categories of HbA1c. However, this finding was not observed for the variability of HDL-C. Unlike other blood lipids, HDL-C is often called “good cholesterol,” which exerts multiple beneficial functions within the cardiovascular system[33]. There is a negative correlation between HbA1c and HDL-C levels. Reduced HDL-C levels often accompany elevated HbA1c[34]. To some extent, it is possible that this negative correlation weakened the influence of HbA1c on the variability of HDL-C, hence the null findings. Both HbA1c and blood lipid variability are risk factors for adverse CVD outcomes. Based on the data of patients undergoing elective PCI, this study clarified the relationship between HbA1c and blood lipid variability. First, insights on the relationships promote the understanding of the role of HbA1c and lipid variability in CVD development. Second, these findings provide new perspectives for further understanding the relationship between glucose metabolism and lipid metabolism, which are closely related in vivo. Limitations There are a number of limitations to consider. First, being a single-center retrospective study, it is limited by inherent biases. Second, we had no data on antidiabetic medications, which may affect lipid metabolism or improve insulin sensitivity. Third, this study examined the effect of long-term blood glucose levels on lipid variability. Indicators of short-term blood glucose levels or glucose tolerance such as random glucose, fasting plasma glucose and 2h post-OGTT glucose, were not included in this study, Acknowledgements The authors would like to express their gratitude to EditSprings (https://www.editsprings.com/) for the expert linguistic services provided. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding This work was supported by grants from the National Natural Science Foundation of China (82070408) , Zhejiang Natural Science Fund (LY18H020007) and Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (2021RC014). Competing interests The authors declare that they have no competing interests. Abbreviations HbA1c: hemoglobin A1c; CVD: cardiovascular disease; PCI: percutaneous coronary intervention; LDL-C: low-density lipoprotein cholesterol; non-HDL-C: non-high-density lipoprotein cholesterol; TC: total cholesterol; TG: and triglyceride; SD: standard deviation; CV: coefficient of variation; VIM: variability Page 11/17 Page 11/17 independent of the mean; DM: diabetes mellitus; MI: myocardial infarction; EF: ejection fraction; BMI: Body mass index; eGFR: estimated Glomerular filtration rate; ACEI: angiotensin-converting enzyme inhibitors; ARB: angiotensin receptor blocker; CCB: calcium channel blocker. independent of the mean; DM: diabetes mellitus; MI: myocardial infarction; EF: ejection fraction; BMI: Body mass index; eGFR: estimated Glomerular filtration rate; ACEI: angiotensin-converting enzyme inhibitors; ARB: angiotensin receptor blocker; CCB: calcium channel blocker. Ethics approval and consent to participate The study was given approval by the Ethics Committee of Sir Run Run Shaw Hospital of Zhejiang University. Consent for publication Not applicable. Authors’ contributions WB Z and ZY C conceived and designed the study. DB L organized these data and drafted the manuscript with the help of C Y, LD Z and Yi L. DB L analysed the data. Ya L drawed the pictures. WB Z, ZY C and GS F detected any errors in the whole process. All authors have read and approved the manuscript for submission. 7. Katzmann JL, Laufs U: New Insights in the Control of Low-Density Lipoprotein Cholesterol to Prevent Cardiovascular Disease.Curr Cardiol Rep 2019, 21:69. 7. Katzmann JL, Laufs U: New Insights in the Control of Low-Density Lipoprotein Cholesterol to Prevent Cardiovascular Disease.Curr Cardiol Rep 2019, 21:69. 8. Gordon DJ, Probstfield JL, Garrison RJ, Neaton JD, Castelli WP, Knoke JD, Jacobs DR, Jr., Bangdiwala S, Tyroler HA: High-density lipoprotein cholesterol and cardiovascular disease. Four prospective American studies.Circulation 1989, 79:8-15. 9. Boey E, Gay GM, Poh KK, Yeo TC, Tan HC, Lee CH: Visit-to-visit variability in LDL- and HDL-cholesterol is associated with adverse events after ST-segment elevation myocardial infarction: A 5-year follow- up study.Atherosclerosis 2016, 244:86-92. 10. Han BH, Han K, Yoon KH, Kim MK, Lee SH: Impact of Mean and Variability of High-Density Lipoprotein-Cholesterol on the Risk of Myocardial Infarction, Stroke, and Mortality in the General Population.J Am Heart Assoc 2020, 9:e015493. 10. Han BH, Han K, Yoon KH, Kim MK, Lee SH: Impact of Mean and Variability of High-Density Lipoprotein-Cholesterol on the Risk of Myocardial Infarction, Stroke, and Mortality in the General Population.J Am Heart Assoc 2020, 9:e015493. 11. Wan EYF, Yu EYT, Chin WY, Barrett JK, Mok AHY, Lau CST, Wang Y, Wong ICK, Chan EWY, Lam CLK: Greater variability in lipid measurements associated with cardiovascular disease and mortality: A 10- year diabetes cohort study.Diabetes Obes Metab 2020, 22:1777-1788. 11. Wan EYF, Yu EYT, Chin WY, Barrett JK, Mok AHY, Lau CST, Wang Y, Wong ICK, Chan EWY, Lam CLK: Greater variability in lipid measurements associated with cardiovascular disease and mortality: A 10- year diabetes cohort study.Diabetes Obes Metab 2020, 22:1777-1788. 12. Lee EY, Yang Y, Kim HS, Cho JH, Yoon KH, Chung WS, Lee SH, Chang K: Effect of visit-to-visit LDL-, HDL-, and non-HDL-cholesterol variability on mortality and cardiovascular outcomes after percutaneous coronary intervention.Atherosclerosis 2018, 279:1-9. 13. Zhao L, Xu T, Li Y, Luan Y, Lv Q, Fu G, Zhang W: Variability in blood lipids affects the neutrophil to lymphocyte ratio in patients undergoing elective percutaneous coronary intervention: a retrospective study.Lipids Health Dis 2020, 19:124. 13. Zhao L, Xu T, Li Y, Luan Y, Lv Q, Fu G, Zhang W: Variability in blood lipids affects the neutrophil to lymphocyte ratio in patients undergoing elective percutaneous coronary intervention: a retrospective study.Lipids Health Dis 2020, 19:124. 14. Clark D, 3rd, Nicholls SJ, St John J, Elshazly MB, Kapadia SR, Tuzcu EM, Nissen SE, Puri R: Visit-to- visit cholesterol variability correlates with coronary atheroma progression and clinical outcomes.Eur Heart J 2018, 39:2551-2558. 14. References Page 12/17 Page 12/17 1. Joseph P, Leong D, McKee M, Anand SS, Schwalm J-D, Teo K, Mente A, Yusuf S: Reducing the Global Burden of Cardiovascular Disease, Part 1: The Epidemiology and Risk Factors.Circulation research 2017, 121:677-694. 2. 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Levine GN, Bates ER, Blankenship JC, Bailey SR, Bittl JA, Cercek B, Chambers CE, Ellis SG, Guyton RA, Hollenberg SM, et al: 2011 ACCF/AHA/SCAI Guideline for Percutaneous Coronary Intervention: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines and the Society for Cardiovascular Angiography and Interventions.Catheter Cardiovasc Interv 2012, 79:453-495. 16. Rothwell PM, Howard SC, Dolan E, O'Brien E, Dobson JE, Dahlöf B, Sever PS, Poulter NR: Prognostic significance of visit-to-visit variability, maximum systolic blood pressure, and episodic hypertension.The Lancet 2010, 375:895-905. 17. Matsuo S, Imai E, Horio M, Yasuda Y, Tomita K, Nitta K, Yamagata K, Tomino Y, Yokoyama H, Hishida A: Revised equations for estimated GFR from serum creatinine in Japan.American journal of kidney diseases : the official journal of the National Kidney Foundation 2009, 53:982-992. 18. Smit RA, Trompet S, Sabayan B, le Cessie S, van der Grond J, van Buchem MA, de Craen AJ, Jukema JW: Higher Visit-to-Visit Low-Density Lipoprotein Cholesterol Variability Is Associated With Lower Cognitive Performance, Lower Cerebral Blood Flow, and Greater White Matter Hyperintensity Load in Older Subjects.Circulation 2016, 134:212-221. 19. Ng G, Boey E, Frampton C, Richards AM, Yeo TC, Lee CH: Obstructive sleep apnea is associated with visit-to-visit variability in low-density lipoprotein-cholesterol in patients with coronary artery disease.Sleep Breath 2017, 21:271-278. 20. Yan Y, Huang Y, Zhou D, Tang S, Feng Y-q: Visit-to-Visit Variability in Total Cholesterol Correlates with the Progression of Renal Function Decline in a Chinese Community-Based Hypertensive Population.Kidney and Blood Pressure Research 2019, 44:727-742. 20. Yan Y, Huang Y, Zhou D, Tang S, Feng Y-q: Visit-to-Visit Variability in Total Cholesterol Correlates with the Progression of Renal Function Decline in a Chinese Community-Based Hypertensive Population.Kidney and Blood Pressure Research 2019, 44:727-742. 21. Bangalore S, Breazna A, DeMicco DA, Wun CC, Messerli FH, Committee TNTS, Investigators: Visit-to- visit low-density lipoprotein cholesterol variability and risk of cardiovascular outcomes: insights from the TNT trial.J Am Coll Cardiol 2015, 65:1539-1548. 21. Bangalore S, Breazna A, DeMicco DA, Wun CC, Messerli FH, Committee TNTS, Investigators: Visit-to- visit low-density lipoprotein cholesterol variability and risk of cardiovascular outcomes: insights from the TNT trial.J Am Coll Cardiol 2015, 65:1539-1548. Figures Figures Figure 1 Multivariate analysis for the effect of HbA1c on lipid variability in patients with and without diabetes. Multivariate linear regression results for the effect of HbA1c on lipid variability, which was represented by 7. Katzmann JL, Laufs U: New Insights in the Control of Low-Density Lipoprotein Cholesterol to Prevent Cardiovascular Disease.Curr Cardiol Rep 2019, 21:69. 22. Waters DD, Bangalore S, Fayyad R, DeMicco DA, Laskey R, Melamed S, Barter PJ: Visit-to-visit variability of lipid measurements as predictors of cardiovascular events.J Clin Lipidol 2018, 12:356- 366. 22. Waters DD, Bangalore S, Fayyad R, DeMicco DA, Laskey R, Melamed S, Barter PJ: Visit-to-visit variability of lipid measurements as predictors of cardiovascular events.J Clin Lipidol 2018, 12:356- 366. 23. Kim MK, Han K, Kim HS, Park YM, Kwon HS, Yoon KH, Lee SH: Cholesterol variability and the risk of mortality, myocardial infarction, and stroke: a nationwide population-based study.Eur Heart J 2017, 38:3560-3566. 23. Kim MK, Han K, Kim HS, Park YM, Kwon HS, Yoon KH, Lee SH: Cholesterol variability and the risk of mortality, myocardial infarction, and stroke: a nationwide population-based study.Eur Heart J 2017, 38:3560-3566. 24. Saydah S, Tao M, Imperatore G, Gregg E: GHb level and subsequent mortality among adults in the U.S.Diabetes Care 2009, 32:1440-1446. 24. Saydah S, Tao M, Imperatore G, Gregg E: GHb level and subsequent mortality among adults in the U.S.Diabetes Care 2009, 32:1440-1446. 25. Palta P, Huang ES, Kalyani RR, Golden SH, Yeh HC: Hemoglobin A1c and Mortality in Older Adults With and Without Diabetes: Results From the National Health and Nutrition Examination Surveys (1988-2011).Diabetes Care 2017, 40:453-460. 25. Palta P, Huang ES, Kalyani RR, Golden SH, Yeh HC: Hemoglobin A1c and Mortality in Older Adults With and Without Diabetes: Results From the National Health and Nutrition Examination Surveys (1988-2011).Diabetes Care 2017, 40:453-460. 26. Shulman GI: Cellular mechanisms of insulin resistance.The Journal of Clinical Investigation 2000, 106:171-176. 26. Shulman GI: Cellular mechanisms of insulin resistance.The Journal of Clinical Investigation 2000, 106:171-176. Page 14/17 Reaven G: Insulin resistance and coronary heart disease in nondiabetic individuals.Arterioscler Thromb Vasc Biol 2012, 32:1754-1759. Yaribeygi H, Farrokhi FR, Butler AE, Sahebkar A: Insulin resistance: Review of the underlying molecular mechanisms.Journal of Cellular Physiology 2018, 234:8152-8161. Ormazabal V, Nair S, Elfeky O, Aguayo C, Salomon C, Zuniga FA: Association between insulin esistance and the development of cardiovascular disease.Cardiovasc Diabetol 2018, 17:122. Porter Karen E, Riches K: The vascular smooth muscle cell: a therapeutic target in Type 2 diabetes? Clinical Science 2013, 125:167-182. Scherer T, Lindtner C, O'Hare J, Hackl M, Zielinski E, Freudenthaler A, Baumgartner-Parzer S, Tödter K, Heeren J, Krššák M, et al: Insulin Regulates Hepatic Triglyceride Secretion and Lipid Content via Signaling in the Brain.Diabetes 2016, 65:1511-1520. 7. Katzmann JL, Laufs U: New Insights in the Control of Low-Density Lipoprotein Cholesterol to Prevent Cardiovascular Disease.Curr Cardiol Rep 2019, 21:69. P R Shepherd BBK: Glucose transporters and insulin action-implications for insulin resistance and diabetes mellitus.The New England Journal of Medicine 1999, 341:248-257. Toth PP: Cardiology patient page. The "good cholesterol": high-density lipoprotein.Circulation 2005, 11:e89-e91. Pacilli A, De Cosmo S, Trischitta V, Bacci S: Role of relationship between HbA1c, fibrinogen and HDL- cholesterol on cardiovascular disease in patients with type 2 diabetes mellitus.Atherosclerosis 2013, 228:247-248. 27. Reaven G: Insulin resistance and coronary heart disease in nondiabetic individuals.Arterioscler Thromb Vasc Biol 2012, 32:1754-1759. 28. Yaribeygi H, Farrokhi FR, Butler AE, Sahebkar A: Insulin resistance: Review of the underlying molecular mechanisms.Journal of Cellular Physiology 2018, 234:8152-8161. 29. Ormazabal V, Nair S, Elfeky O, Aguayo C, Salomon C, Zuniga FA: Association between insulin resistance and the development of cardiovascular disease.Cardiovasc Diabetol 2018, 17:122. 30. Porter Karen E, Riches K: The vascular smooth muscle cell: a therapeutic target in Type 2 diabetes? Clinical Science 2013, 125:167-182. 31. Scherer T, Lindtner C, O'Hare J, Hackl M, Zielinski E, Freudenthaler A, Baumgartner-Parzer S, Tödter K, Heeren J, Krššák M, et al: Insulin Regulates Hepatic Triglyceride Secretion and Lipid Content via Signaling in the Brain.Diabetes 2016, 65:1511-1520. 31. Scherer T, Lindtner C, O'Hare J, Hackl M, Zielinski E, Freudenthaler A, Baumgartner-Parzer S, Tödter K, Heeren J, Krššák M, et al: Insulin Regulates Hepatic Triglyceride Secretion and Lipid Content via Signaling in the Brain.Diabetes 2016, 65:1511-1520. 32. P R Shepherd BBK: Glucose transporters and insulin action-implications for insulin resistance and diabetes mellitus.The New England Journal of Medicine 1999, 341:248-257. 32. P R Shepherd BBK: Glucose transporters and insulin action-implications for insulin resistance and diabetes mellitus.The New England Journal of Medicine 1999, 341:248-257. 33. Toth PP: Cardiology patient page. The "good cholesterol": high-density lipoprotein.Circulation 2005, 111:e89-e91. 33. Toth PP: Cardiology patient page. The "good cholesterol": high-density lipoprotein.Circulation 2005, 111:e89-e91. 34. Pacilli A, De Cosmo S, Trischitta V, Bacci S: Role of relationship between HbA1c, fibrinogen and HDL- cholesterol on cardiovascular disease in patients with type 2 diabetes mellitus.Atherosclerosis 2013, 228:247-248. 34. Pacilli A, De Cosmo S, Trischitta V, Bacci S: Role of relationship between HbA1c, fibrinogen and HDL- cholesterol on cardiovascular disease in patients with type 2 diabetes mellitus.Atherosclerosis 2013, 228:247-248. Figure 1 Multivariate analysis for the effect of HbA1c on lipid variability in patients with and without diabetes. Multivariate linear regression results for the effect of HbA1c on lipid variability, which was represented by Page 15/17 Page 15/17 SD. non-DM indicates group without diabetes; DM, patients with diabetes; β-SD, regression coefficients of HbA1c on SD; CI, confidence intervals. Figure 2 Multivariate analysis for the effect of HbA1c on lipid variability in Atorvastatin and Rosuvastatin therap groups. Multivariate linear regression results for the effect of HbA1c on lipid variability, which was represented by SD. Ator indicates atorvastatin therapy group; Rosu, rosuvastatin therapy group; β-SD, regression coefficients of HbA1c on SD; CI, confidence intervals. Figure 2 Multivariate analysis for the effect of HbA1c on lipid variability in Atorvastatin and Rosuvastatin therapy groups. Multivariate linear regression results for the effect of HbA1c on lipid variability, which was represented by SD. Ator indicates atorvastatin therapy group; Rosu, rosuvastatin therapy group; β-SD, regression coefficients of HbA1c on SD; CI, confidence intervals. Page 16/17 Page 16/17 Figure 3 Multivariate analysis for the effect of HbA1c on lipid variability in the regular statin, intensive statin, and statin-ezetimibe combined therapy groups. Multivariate linear regression results for the effect of HbA1c on lipid variability, which was represented by SD. RS indicates regular statin therapy without ezetimibe; IS, intensive statin therapy without ezetimibe; RS+E, regular statin therapy combined with ezetimibe; β-SD, regression coefficients of HbA1c on SD; CI, confidence intervals. Figure 3 Multivariate analysis for the effect of HbA1c on lipid variability in the regular statin, intensive statin, and statin-ezetimibe combined therapy groups. Multivariate linear regression results for the effect of HbA1c on lipid variability, which was represented by SD. RS indicates regular statin therapy without ezetimibe; IS, intensive statin therapy without ezetimibe; RS+E, regular statin therapy combined with ezetimibe; β-SD, regression coefficients of HbA1c on SD; CI, confidence intervals. Multivariate analysis for the effect of HbA1c on lipid variability in the regular statin, intensive statin, and statin-ezetimibe combined therapy groups. Multivariate linear regression results for the effect of HbA1c on lipid variability, which was represented by SD. RS indicates regular statin therapy without ezetimibe; IS, intensive statin therapy without ezetimibe; RS+E, regular statin therapy combined with ezetimibe; β-SD, regression coefficients of HbA1c on SD; CI, confidence intervals. Page 17/17
https://openalex.org/W2960324301	https://europepmc.org/articles/pmc6640189?pdf=render	English	null	The Statistical Evaluation of Treatment and Outcomes in Head and Neck Squamous Cell Carcinoma Clinical Trials	Frontiers in oncology	2,019	cc-by	13,637	Edited by: Edited by: Dirk Van Gestel, Free University of Brussels, Belgium Edited by: Dirk Van Gestel, Free University of Brussels, Belgium Reviewed by: Marianne Paesmans, Institut Jules Bordet, Belgium Markus Brunner, Medical University of Vienna, Austria Correspondence: Catherine Fortpied catherine.fortpied@eortc.org Keywords: statistical design, statistical analysis, clinical trial, head and neck cancer, treatment, comorbidities, patient population, endpoint Reviewed by: Marianne Paesmans, Institut Jules Bordet, Belgium Markus Brunner, Medical University of Vienna Austria Reviewed by: Marianne Paesmans, Institut Jules Bordet, Belgium Markus Brunner, Medical University of Vienna, Austria Correspondence: Catherine Fortpied catherine.fortpied@eortc.org The Statistical Evaluation of Treatment and Outcomes in Head and Neck Squamous Cell Carcinoma Clinical Trials The purpose of this article is two-fold: to help statisticians confronted with the design, implementation and analysis of clinical trials and new to the ﬁeld of head and neck cancer; but also to sensitize research physicians with the role, the tasks and the challenges faced by the medical statisticians. These two purposes altogether will hopefully encourage and enable ﬂuid communication between the research physician and the medical statistician and the understanding of each other’s ﬁeld and concerns. In particular, the methodological challenges resulting from the heterogeneity of the head and neck cancer, the complexity of the treatments and the associated comorbidities are presented with examples borrowed from medical literature and from the practical experience of the authors in this ﬁeld. REVIEW published: 12 July 2019 doi: 10.3389/fonc.2019.00634 published: 12 July 2019 doi: 10.3389/fonc.2019.00634 INTRODUCTION Medical research and conduct of clinical trials is inconceivable today without statistical expertise. This is oﬃcially acknowledged in Europe since 1990 when the Committee for Proprietary Medicinal Medical research and conduct of clinical trials is inconceivable today without statistical expertise. This is oﬃcially acknowledged in Europe since 1990 when the Committee for Proprietary Medicinal Products (CPMP) (1) adopted a Note for Guidance covering the subject of Good Clinical Practice (GCP). This note stated that “access to biostatistical expertise is necessary before and throughout the entire procedure, commencing with designing of the protocol and ending with completion of the ﬁnal report.” Specialty section: This article was submitted to Head and Neck Cancer, a section of the journal Frontiers in Oncology The purpose of this paper is two-fold: to help statisticians confronted with the design, implementation and analysis of clinical trials and new to the ﬁeld of head and neck cancer; but also to sensitize research physicians with the role, the tasks and the challenges faced by the medical statisticians. These two purposes altogether will hopefully encourage and enable ﬂuid communication between the research physician and the medical statistician and the understanding of each other’s ﬁeld and concerns. Received: 06 March 2019 Accepted: 26 June 2019 Published: 12 July 2019 According to the title of the paper, the ﬁrst section explains what are the requirements to conduct a rigorous statistical analysis of a clinical trial: in particular the predeﬁned analysis plan, the data, and the software. Then moving to the ﬁeld of head and neck cancer, we will explain how the heterogeneity of the disease, the multimodality nature of treatments and the associated comorbidities inﬂuence the methods used to design and analyze clinical trials in this ﬁeld. Key methodological and statistical concepts are explained and illustrated with examples borrowed from medical literature and from the practical experience of the authors. Keywords: statistical design, statistical analysis, clinical trial, head and neck cancer, treatment, comorbidities, patient population, endpoint Computer Software Validity Diﬀerent statistical approaches can lead to diﬀerent numerical results and hence inﬂuence the interpretation of the trial. The statistician will need to choose among the diﬀerent possible statistical methods. The choice will depend on the nature of the data (e.g., continuous, binary, categorical, or time-to-event), the underlying assumptions about the statistical distribution (e.g., non-parametric, semi-parametric, or fully parametric), and the amount of data (e.g., asymptotic methods for large samples or exact methods for small samples). A demonstration of this, although based on observational data outside the medical ﬁeld, is provided in the confronting article by Silberzahn et al. (2), which reports how diﬀerent analytical methods can lead to diﬀerent results. Lack of prespeciﬁcation may aﬀect the trial’s validity by allowing the researchers to consciously or unconsciously select the analysis approach that provides the most favorable results. It is therefore important that these decisions are prespeciﬁed before seeing the trial data. This is why, and as stated in International Conference on Harmonization (ICH) E9 guideline (3), the study protocol must include the main features of the data analysis: deﬁnition of the analysis populations, timing of interim and ﬁnal analyses, precise deﬁnition of the study endpoints, methods used for estimation, conﬁdence intervals, and hypothesis testing; adjustment of signiﬁcance and conﬁdence levels; subgroup analyses. In addition, with the pre-speciﬁcation of the analyses and more speciﬁcally of the hypotheses tests, the extent of multiplicity is clearly stated (multiple endpoints, multiple comparisons of treatments, repeated evaluations over time, interim analyses) and measures to control the risk of overall Type I error, i.e., of false positive ﬁndings, can be taken. Unplanned analyses are sometimes conducted. For example, when new questions based on the observed data emerge or when heterogeneity of the treatment eﬀect across subgroups of patients needs to be assessed. When reporting and publishing the results of a clinical study, as stated in Consolidated Standards of Reporting Trials (CONSORT) guidelines (4), results from these post-hoc analyses have to be clearly distinguished from the results of the preplanned analyses. As the former cannot lead to ﬁrm conclusions, they are solely considered as exploratory and hypothesis generating. To ensure the completeness and the appropriateness of the written statistical analysis plan, a second statistician should ideally validate it. Based on the statistical analysis plan, the statistician will process the data statistically. Citation: Fortpied C and Vinches M (2019) The Statistical Evaluation of Treatment and Outcomes in Head and Neck Squamous Cell Carcinoma Clinical Trials. Front. Oncol. 9:634. doi: 10.3389/fonc.2019.00634 July 2019 \| Volume 9 \| Article 634 Frontiers in Oncology \| www.frontiersin.org Statistical Analysis of HNSCC Treatment/Outcomes Fortpied and Vinches Computer Software Validity He/she will produce a descriptive analysis in the form of tables and graphical displays and an inferential analysis consisting of estimated eﬀect sizes, their precision (such as 95% conﬁdence interval) and signiﬁcance (p-values). The credibility of the numerical results of the analysis depends on the quality and validity of the softwares used, either externally or internally written (3). A validated software and programming language, such as used in the Statistical Analysis System (SAS R⃝, Cary NC, USA) should be used to produce the statistical analysis outputs. The study statistician should develop the statistical analysis programs speciﬁc to his/her study using built- in SAS procedures and in-house programmed SAS macros that automate repetitive data processes. To ensure the correctness of the results, a second, independent statistician should validate the analysis program, by independent programming, at least for the analysis of the primary and key secondary endpoints of the study. WHAT MAKES A STATISTICAL ANALYSIS A GOOD STATISTICAL ANALYSIS? standard operating procedures should constitute a safeguard against poor data management. From the Statistical Analysis to the Publication The role of statistics is to translate information into knowledge, which is, according to the renowned statistician Stephen Senn, the challenge that faces statisticians (6). When browsing the Royal Statistical Society website (7), one can read that the medical statistician will see his/her work “inﬂuence clinical practice, help guide public health education and policies, or add to current knowledge, sometimes leading to further research studies.” It is not enough to produce statistical outputs, statistical judgment must also be exercised for the interpretation and presentation of the results. The statistician will make sure that the conclusions are presented or disclosed in a manner that fairly reﬂects the evidence supported by the results. In this regard, we would like to caution about the use or overuse or even misuse of p-values: not only because the concept of the p-value is often misunderstood but also because it is not a substitute for medical judgement. P-values should not be provided alone but should be accompanied with eﬀect sizes and their precision in order to be able to assess the clinical relevance of the results (8, 9). All research results should be published, irrespective of the ﬁndings (both positive and negative, statistically signiﬁcant, or not). As stated by Tam et al. (10), “Non publication of clinical trials breaks an implicit contract with trial participants, institutional review boards, and study sponsors and society in general.” As a measure to prevent publication bias, the International Committee of Medical Journal Editors (ICJME) (11) recommends editorial decisions not to be driven by the clinical trial results but by the originality, the quality and the contribution to scientiﬁc knowledge. Furthermore, regulatory bodies, Food and Drug Administration (FDA) and European Medicines Agency (EMA), have initiated moves toward greater transparency by requesting that the aggregated results of (drug) clinical trials are disclosed in public domain to US ClinicalTrials.gov (since 2007 and then extended to non-licensed The Medical Statistician: From the Start, in Close Interaction With the Research Physician, the Data Manager and the Clinical Operations Team • Assesses the quality of the clinical database for the intended analysis, in terms of completeness and consistency • Develops the analysis programs to produce the required tables, graphical displays and inferential analyses A statistical analysis can only be translated into knowledge if the study has been adequately designed to answer the key research questions of the study. The medical statistician develops an appropriate design that will ensure the trial to provide the answer to its objectives within the limits of existing statistical methodology, starting from the rationale, the objectives and the clinical background deﬁned by the research physician. The designs consists of the statistical and methodological setup of the trial, including elements such as randomization, stratiﬁcation, planning of the statistical testing of primary and secondary endpoints, adjustment of those comparisons for covariates, sample size calculation taking into account type I and type II errors. It is at this point that the statistician and the research physician need to interact closely. Discussions should identify the practical constraints of the study, particularly in terms of potential accrual and overall study duration. With these elements in hand, the statistician will propose a statistical design for the study. Several options are typically discussed before a ﬁnal design is agreed upon. • Writes the statistical analysis report • Contributes to the presentation and publication of the study results • Submits the ﬁnal study results to EMA European Clinical Trials Database (EudraCT) and to US • Submits the ﬁnal study results to EMA European • Submits the ﬁnal study results to EMA Europea Clinical Trials Database (EudraCT) and to US Submits the ﬁnal study results to EMA Europea Clinical Trials Database (EudraCT) and to US ClinicalTrials.gov public websites The medical statistician is thus involved from the start up to the end in a study life cycle. Table 1 provides a synthetic list of his/her responsibilities. The Clinical Data • Contributes to the deﬁnition of the study objectives and to the selection of the primary and secondary endpoints • Proposes one or several statistical designs until ﬁnal selection • Proposes one or several statistical designs until ﬁnal selection • Computes the sample size and the study duration • For randomized studies, speciﬁes the method and parameters of the randomization procedure • Deﬁnes accurately the trial endpoints and the methods of assessment • Designs the early stopping rules and interim monitoring plan • Writes the statistical analysis plan • Monitors the assumptions underlying the statistical design • Interacts with the Independent Data Monitoring Committee, when pre-speciﬁed in the study protocol or in case of unanticipated issues • Contributes to the amendment of the study protocol in case of major changes to the statistical considerations of the trial During the study conduct At each interim, ﬁnal or long-term data analysis Frontiers in Oncology \| www.frontiersin.org The Clinical Data Accuracy, consistency, completeness and reliability of the clinical data is obviously critical for the analysis and interpretation of the study. Data management processing involves several steps and usually a high number of actors. The design of the case report forms and the clinical database are developed by the central data manager. The reporting of each patient data is done by the investigators from source documents. The interactions between the local investigators and the central data manager allows the veriﬁcation and correction of the data and the traceability of the data ﬂow. Principles established in GCP (5) and the sponsor’s July 2019 \| Volume 9 \| Article 634 Frontiers in Oncology \| www.frontiersin.org 2 Statistical Analysis of HNSCC Treatment/Outcomes Fortpied and Vinches TABLE 1 \| Responsibilities of the trial statistician in a study life cycle. Study life cycle Responsibilities of the trial statistician Study design • Contributes to the deﬁnition of the study objectives and to the selection of the primary and secondary endpoints • Proposes one or several statistical designs until ﬁnal selection • Computes the sample size and the study duration • For randomized studies, speciﬁes the method and parameters of the randomization procedure • Deﬁnes accurately the trial endpoints and the methods of assessment • Designs the early stopping rules and interim monitoring plan • Writes the statistical analysis plan During the study conduct • Monitors the assumptions underlying the statistical design • Interacts with the Independent Data Monitoring Committee, when pre-speciﬁed in the study protocol or in case of unanticipated issues • Contributes to the amendment of the study protocol in case of major changes to the statistical considerations of the trial At each interim, ﬁnal or long-term data analysis • Assesses the quality of the clinical database for the intended analysis, in terms of completeness and consistency • Develops the analysis programs to produce the required tables, graphical displays and inferential analyses • Writes the statistical analysis report • Contributes to the presentation and publication of the study results • Submits the ﬁnal study results to EMA European Clinical Trials Database (EudraCT) and to US ClinicalTrials.gov public websites TABLE 1 \| Responsibilities of the trial statistician in a study life cycle. The Clinical Data Study life cycle Responsibilities of the trial statistician Study design • Contributes to the deﬁnition of the study objectives and to the selection of the primary and secondary endpoints • Proposes one or several statistical designs until ﬁnal selection • Computes the sample size and the study duration • For randomized studies, speciﬁes the method and parameters of the randomization procedure • Deﬁnes accurately the trial endpoints and the methods of assessment • Designs the early stopping rules and interim monitoring plan • Writes the statistical analysis plan During the study conduct • Monitors the assumptions underlying the statistical design • Interacts with the Independent Data Monitoring Committee, when pre-speciﬁed in the study protocol or in case of unanticipated issues • Contributes to the amendment of the study protocol in case of major changes to the statistical considerations of the trial At each interim, ﬁnal or long-term data analysis • Assesses the quality of the clinical database for the intended analysis, in terms of completeness and consistency • Develops the analysis programs to produce the required tables, graphical displays and inferential analyses • Writes the statistical analysis report • Contributes to the presentation and publication of the study results • Submits the ﬁnal study results to EMA European Clinical Trials Database (EudraCT) and to US ClinicalTrials.gov public websites TABLE 1 \| Responsibilities of the trial statistician in a study life cycle. Study life cycle Responsibilities of the trial statistician products in 2017) (12) and/or to EMA European Clinical Trials Database—EudraCT (since 2014) (13). Keeping abreast of statistical methodology. products in 2017) (12) and/or to EMA European Clinical Trials Database—EudraCT (since 2014) (13). Keeping abreast of statistical methodology. gy Again quoting the Royal Statistical Society website (7), the medical statistician is also “part of an academic group which develops statistical methodology to be applied to medical research.” Up to the nineties, the methodology for the design and statistical analysis of clinical trials was well-established and statisticians could do their job with a limited number of tools. Since then, parallel developments, such as increased computer power, advances in biology science leading to the emergence of a new class of treatment called “targeted,” brought new opportunities for clinical development and new methodological challenges (14). Some of these methodological challenges are discussed below in the context of head and neck cancer clinical studies. Heterogeneity of the Disease In addition, as the statistician is typically concerned by bias and precision, he/she is not only involved in the pure statistical aspects of the study. But he/she will also participate in planning operational aspects that may potentially induce a bias or undesired variability aﬀecting the interpretability of the study results. As such, the statistician carefully reviews the procedures planned for the study such as selection, diagnosis and staging of patients; treatment administration; follow-up assessments; data processing (3). He also pays attention to the potential aspects of the protocol where adherence is more diﬃcult to achieve in order to minimize the incidence of violations of the entry criteria, non-compliance, withdrawals, losses to follow-up, missing data and other deviations from the protocol. Deviations may aﬀect the subsequent analyses and ultimately the interpretation and conclusions of the study. Head and neck cancers are a group of diseases characterized by phenotypic, etiological, biological and clinical heterogeneity. Squamous cell carcinoma is the predominant histology type (15). The complexity of the upper respiratory and gastro- intestinal apparatus creates a number of anatomical subdomains that are apprehended together. Still the prognosis is speciﬁc to each localization, correlated to a distinct TNM classiﬁcation (16). Historically, the most common risk factors are tobacco and alcohol consumption, responsible for up to 75% of head and neck squamous cell carcinoma (HNSCC) (17). The etiologic association of human papillomavirus (HPV) with a distinct subset of HNSCC that occur mostly in oropharynx is increasing, aﬀecting non-smokers in developed countries. HPV positive oropharyngeal tumors have better survival, particularly July 2019 \| Volume 9 \| Article 634 3 Statistical Analysis of HNSCC Treatment/Outcomes Fortpied and Vinches node(s) at level IV or V in patients with oral cavity or oropharynx carcinomas, pathological demonstration of vascular embolisms, and/or perineural disease. The RTOG study included in its selection of risk factors the presence of tumor in two or more lymph nodes, as was suggested by the analysis of the RTOG database. In 2004, the publication of the two studies established, with level I evidence, that concurrent chemoradiation was more eﬃcacious than radiation alone as adjuvant postoperative treatment, in terms of local-regional control and disease-free survival. Because of the diﬀerence in the deﬁnition of “high risk” features between the two trials, additional analyses were conducted to identify precisely which patients were more suitable for such intense treatment (25). Heterogeneity of the Disease The ﬁndings suggested that microscopically involved resection margins and extracapsular spread of tumor from neck nodes were signiﬁcant prognostic factors for poor outcome. Despite the limitations inherent to a retrospective subgroup analyses, their results are now the basis for the selection of patients in clinical trials in the postoperative setting (e.g., EORTC study 1735: NCT03673735). for locoregionally advanced disease (18). The comprehensive genomic analysis performed by The Cancer Genome Atlas (19) revealed the genomic heterogeneity of this disease with clear diﬀerences between HPV negative and positive tumors. This, in the era of personalized medicine, could lead to diﬀerent treatments depending on the plausible therapeutic targets. Selection of Patient Population A clinical research question is intrinsically deﬁned in terms of a speciﬁc population. Eligibility criteria, assumptions about the prognosis of patients and the magnitude of the treatment eﬀect in the intended population, stratiﬁcation of patients within the clinical trial are all fundamental questions when planning a clinical trial (20). These decisive elements of the design can be obtained from expert opinion and a careful review of the medical literature. Past and currently ongoing clinical studies deﬁne their target population based on the anatomical location of the disease, the classical TNM classiﬁcation and more recently on the distinction between HPV positive and negative nature of the disease. In addition, a search of medical literature reveals an abundance of articles reporting the assessment of prognostic factors in head and neck cancer and the classiﬁcation of patients according to diﬀerent risk levels of progression, recurrence, or death. These analyses are useful to circumscribe the population of interest for our research question and when searching Medline for head and neck risk classiﬁcation (see Supplementary Material for the exact Search query used), one retrieves 404 papers published in the past 10 years, 59 of them in 2018. But are all these analyses conducted adequately from a methodological point of view? Are the conclusions useful from a clinical point of view (21)? How can we separate the wheat from the chaﬀ? The statistician will do his/her best to review these articles with a critical eye to evaluate the methodology used, and to assess how applicable and generalizable these results are. More speciﬁcally the statistician scrutinizes multiple aspects of the work, including but not limited to: characteristics of patients included in the modeling; selection and deﬁnition of the outcome of interest (locoregional failure, risk of distant metastasis, or survival); treatment received by the patients included in the analysis; set of candidate prognostic factors; data analysis method (statistical model such as Cox model or machine learning tools such as neural networks or random survival forests); model performance measures; internal and external validation procedures (22). Another example of clinical trials based on a risk classiﬁcation is given by an ongoing Canadian Cancer Trials Group study (NCT03410615), testing the eﬀect of immunotherapy in intermediate-risk, HPV-positive, locoregionally advanced, oropharyngeal squamous cell cancer. Here the deﬁnition of intermediate-risk is based on data showing that HPV-related oropharyngeal cancer patients with limited neck disease (N0-N1) have a favorable prognosis, even without chemotherapy (26, 27). Frontiers in Oncology \| www.frontiersin.org Favorable Prognosis and Non-inferiority Designs g y g The majority of studies test novel treatments or combination of treatments in order to improve disease outcome and survival in patients with unfavorable prognosis, with intermediate or high risk of progression, recurrence or death. Some trials are also designed and conducted in patients with a favorable prognosis, in order to assess whether treatments reduce acute and late toxicity while preserving a similar disease outcome and survival. Since the identiﬁcation of HPV positive patients as a separate disease entity with a more favorable prognosis, a number of studies have been developed to de-intensify treatment in these patients. This is the case of RTOG 1016 study (NCT01302834) (28) and De-Escalate study (NCT01874171) (29). Both studies attempt to replace cisplatin by the epidermal growth factor receptor (EGFR) inhibitor cetuximab in patients with HPV positive oropharyngeal cancer. The objective of these studies is to maintain a similar patient survival while reducing toxicity, and as such they require a diﬀerent type of design. RTOG 1016 was designed as a classical non-inferiority trial with overall survival as primary endpoint. One point of consideration for non-inferiority studies is the value of the non-inferiority margin that is considered as an acceptable loss, in disease outcome or survival in view of the gain in toxicity. It has to be put in perspective with the prognosis of patients since a loss of 10% does not mean the same when survival rate with the standard of care is at the level of 70 or 90%. This non-inferiority margin needs to be small enough to be considered as non-clinically relevant and certainly substantially lower than diﬀerences targeted in superiority trials (30). Deﬁning the non-inferiority margin is an An example of the complexity to deﬁne the targeted patient population can be illustrated by the European Organization for Research and Treatment of Cancer (EORTC) trial 22931 (23) and the Radiation Therapy Oncology Group (RTOG) trial 9501 (24). Both trials evaluated post-operative chemoradiation vs. radiation alone in patients at high-risk of recurrence after surgery. The deﬁnition of high-risk and therefore the inclusion criteria, although sharing some common criteria, diﬀered between the two studies. While the eligibility criteria common to both trials were the presence of extracapsular extension and/or microscopic- sized tumor involvement of the surgical margins of resection, some diﬀered. Designs for Studies in Rare Cancers and Accrual Issues in Randomized Trials With a heterogeneous disease, distinct subtypes, in terms of tumor localization and biological characteristics, need to be investigated separately in small groups of patients. In case of a rare population, a classical design may just not be feasible and we need to reﬂect on the level of evidence we still wish to reach (31). One possible solution is to allow more uncertainty, that is to allow a Type I error higher than the traditional 5% two-sided that is required to reach scientiﬁc evidence for a superiority trial and/or to allow a Type II higher than the classical 10 or 20% which is equivalent to say that the study is only powered to detect large diﬀerences. In these cases, we need to be careful with the consequences of relaxing the errors/power, given that it is unlikely that another trial will be conducted to conﬁrm the results. EORTC study 1206 (NCT01969578) aims to assess the superiority of androgen deprivation therapy (ADT) over standard chemotherapy (CT) in patients with recurrent/metastatic salivary gland cancer. It has been designed with a one-sided Type I error of 10% and with a power of 80% to detect an ambitious diﬀerence between ADT and CT in progression-free survival with a hazard ratio HR = 0.56. Because our predictions indicate that the study will likely fail to recruit the total planned number of patients in a reasonable timeframe, the design has been revised recently. An analysis of the primary endpoint from a non-inferiority perspective has been added, by pre-specifying a non-inferiority margin, in case the study fails to meet the criteria of superiority. This non-inferiority test has adequate statistical power under the hypothesis of the superiority of ADT over CT. If the objective of non-inferiority is met, this is considered valuable from a clinical point of view given the favorable safety proﬁle of ADT compared to CT, except for sexual dysfunction, and the dismal prognosis of these patients. Beyond the speciﬁc case of a rare disease, accrual of patients in the clinical trial may be slower than initially expected as a result of strict eligibility criteria, over-optimism of participating institutions at the start of the study; lengthy approval of the study by competent authorities and ethic committees; patients reluctant to enter the study or to be assigned a treatment at random. Favorable Prognosis and Non-inferiority Designs The EORTC study included in its selection of risk factors stage III/IV disease, the presence of enlarged lymph July 2019 \| Volume 9 \| Article 634 July 2019 \| Volume 9 \| Article 634 Frontiers in Oncology \| www.frontiersin.org 4 Statistical Analysis of HNSCC Treatment/Outcomes Fortpied and Vinches essential part of a non-inferiority design just as the diﬀerence is essential to the design of a superiority or diﬀerence trial. Both have to be pre-deﬁned based on clinical and statistical considerations and both have a strong impact on the sample size, the trial duration and its cost. The primary endpoint of RTOG 1016 was overall survival and the study was designed to reject the null hypothesis of inferiority, with a non-inferiority margin of 9%, meaning that a decrease of 9% in 5 years overall survival was considered, by the RTOG 1016 team, acceptable. By contrast, the primary endpoint of De-Escalate study was overall severe (grade 3–5) toxicity events at 24 months from the end of treatment and the study was powered to detect a reduction in the rate of severe toxicities in cetuximab arm compared to cisplatin arm. Equivalent disease control and survival between treatment arms were hypothesized but the study was not formally powered to show non-inferiority. Interestingly, overall survival and time to recurrence were planned to be compared between the two arms using the log-rank test, which is a test aiming at detecting diﬀerences, not aiming at showing non-inferiority or equivalence; now, failing to detect a diﬀerence does not mean there is no diﬀerence as “absence of evidence is not evidence of absence.” EORTC study 1206 is one of the studies developed within the International Rare Cancers Initiative (IRCI) (32), a strategic collaboration between several academic organizations, including EORTC. IRCI’s aim is to stimulate and facilitate the development of international clinical trials for patients with rare cancers. Some of the studies from the IRCI portfolio, focusing on rare cancers, are designed using Bayesian methodology. With this methodology, the conclusion of the study is based on the combination of the study data itself together with prior knowledge based on literature review, previous studies, meta- analyses or the elicitation of expert’s opinion. Contrary to the classical (frequentist) approach, the focus of the Bayesian approach is on estimation rather than testing hypotheses, with data being used to reduce uncertainty about the size of the treatment eﬀect. Favorable Prognosis and Non-inferiority Designs However, we remain unsure regarding the advantages of this methodology as in a small trial the choice of the prior may carry heavy weight thus inﬂuencing the ﬁnal results. Moreover, in rare cancers only weak prior evidence might be available. In addition, in absence of prior information, Bayesian designs do not immediately add value over equivalent classical (frequentist) designs in terms of the statistical properties (type I error rate, type II error rate, power, sample size). However, this does not prevent that a trial designed in a frequentist setting is analyzed using Bayesian methods, and the results interpreted using the posterior distribution of the treatment eﬀect which is obtained from the combination of prior knowledge with currently observed trial data. To our knowledge, only one prospective clinical trial in head and neck cancer is designed using a Bayesian methodology: this is two-stage phase II design of Magnetic Resonance-guided radiotherapy dose adaptation in patients with HPV positive oropharyngeal cancer. This study uses Bayesian decision rules applied to loco regional control and toxicity to make the go/no-go decision for each stage (33). Frontiers in Oncology \| www.frontiersin.org Treatment Allocation These considerations about the heterogeneity of the disease are at the basis of the selection of the study population but also of the stratiﬁcation of patients within the randomized clinical trial. This stratiﬁcation is to be taken into account not only in the process of randomization, in order to produce comparable treatment arms in terms of factors that aﬀect the course of the disease, but also at the data analysis levels. Due to the association between these factors and the outcome variable, adjustment for such factors generally improves the eﬃciency of the analysis. Randomization tends to produce treatment arms in which the distributions of prognostic factors, known and unknown, are similar (3). Achieving a balanced allocation overall and for important prognostic factors allows to attribute diﬀerences in outcomes to diﬀerences in eﬃcacy of the treatments under study; this is the concept of causality. In randomized studies, the most relevant factors for stratiﬁcation need to be identiﬁed, bearing in mind that too many stratiﬁcation factors are detrimental to a balanced allocation. The number of stratiﬁcation factors and which ones to select is discussed between the research physician and the medical statistician until a compromise is found. It is particularly important to consider institution as a stratiﬁcation factor in order to account for the diﬀerences across treating sites in terms of patient selection, treatment and care, assessments, and data reporting. In EORTC study 1420 “Best-of” (NCT02984410), a randomized phase III study the main objective is to assess the patient-reported swallowing function over the ﬁrst year after treatment start with either Intensity-Modulated Radiation Therapy (IMRT) or Trans Oral Surgery (TOS) among patients with early stage oropharyngeal, supraglottic, or hypopharyngeal carcinoma. In this study, the eight disease localizations were classiﬁed into two strata, lateral vs. central lesions, thought to be an appropriate classiﬁcation taking into account that the primary endpoint was the swallowing function. Two other clinical stratiﬁcation factors were considered, N stage and the swallowing function score at baseline. Because of the relative small size of the study, 170 patients potentially accrued in more than 30 treating institutions from 8 countries, stratifying by treating institution would have resulted in too many small strata and in this study the decision was made to group them by country. Treatment Allocation g g It is worth emphasizing that comparing outcomes in patients subgroups deﬁned by some other outcomes or variables measured after treatment start, such as dose intensity, compliance to treatment or adverse events require non-standard analysis methods, as these variables are themselves aﬀected by treatment (37, 38). In particular, standard analysis methods of comparing survival between responders with non-responders are wrong and lead to biased estimates and misleading conclusions. This bias results from the fact that responders must live long enough for a response to be observed and that patients who die early without observing a response are automatically classiﬁed as non-responders. A better approach, proposed by Anderson et al. (37), is the landmark method, where each patient’s response is determined at some ﬁxed time point after treatment start and the survival estimates are calculated from that time point. This method has for example been applied in the analysis of a study of induction treatment followed by chemoradiation in advanced stage in head and neck cancer. An 8 weeks landmark analysis was carried out to compare survival between patients with positive vs. negative biopsy of the primary site done after induction. A 4 months landmark analysis was also performed to evaluate the eﬀect of maintenance therapy on survival. Survival was computed from the landmark (39). Similarly an analysis of the predictive value of cetuximab- induced skin toxicity in recurrent or metastatic head and neck cancer was conducted using the landmark method applied to PFS and OS counted from 90 days after the start of therapy (40). For each study, the statistician evaluates which treatment allocation method is most appropriate. The two most common methods are the static permuted blocks method and the dynamic minimization algorithm (34). While the minimization method is often discouraged by Regulatory Authorities due to theoretical concerns, a cancer-speciﬁc review published in 2010 (35), indicated that it becomes more common over time and is used more frequently when an academic cooperative group is involved. For both methods, it is recommended to perform computer simulations to assess the performance of the chosen method and the stratiﬁcation design, in terms of the balance of the stratiﬁcation factors over the treatment arms. When dealing with subgroup of patients, and especially in the era of personalized medicine, the question whether some patient characteristics or some biomarkers are predictive of treatment beneﬁt is of interest. Subgroup Analyses validity of such a revision, it should be done before any of the data is revealed. validity of such a revision, it should be done before any of the data is revealed. It is tempting to conduct multiple subgroup analyses in large studies of patients with heterogeneous characteristics. However, as indicated in ICH E9 and often reiterated in medical and statistical literature, such analyses carry the risks of generating false positive ﬁndings due to statistical testing in multiple subgroups. It also runs the risks of false negative ﬁndings due to the small size of the subgroups. The appropriateness of the use and interpretation of subgroup analyses on the basis of the CONSORT statement requirements (4) was investigated in 188 phase III randomized controlled trials in solid tumors, published between 2011 and 2013 (36). When focusing on the 102 articles claiming a subgroup diﬀerence, for 24% of them it was unclear whether the subgroup analyses were prespeciﬁed or post-hoc, and subgroup analyses of 36% of these trials were post-hoc only. Eighty-four percentage of these trials reported more than ﬁve subgroup analyses but only 6% cautioned about multiplicity. This review shows that despite recommendations from the CONSORT statement published more than a decade ago, the reporting of subgroup analyses is generally not adequate to provide valuable information in guiding clinical decisions. Designs for Studies in Rare Cancers and Accrual Issues in Randomized Trials Slow accrual leads to longer study duration and therefore delayed availability of the study results, possibly when the main research question posed by the study is no longer relevant given the evolution of clinical research in the ﬁeld. In order to speed up accrual, some actions are envisaged such as broadening the eligibility criteria of the study; opening the study to additional treating institutions, countries, or other research organizations. In some other cases, the ultimate decision is to close the study deﬁnitely before having reached the targeted sample size. It is then necessary to evaluate to which extent the available data can be used to assess the objectives of the trial. Sometimes the available study data allows to conduct the initially planned analyses with a decreased but still acceptable statistical power, say 70% instead of the initially stated 80%. When data are scarcer, only a mere descriptive analysis of the study data is feasible. In other cases, it is possible to rescue the study through a substantial revision of the statistical analysis plan. To ensure the July 2019 \| Volume 9 \| Article 634 July 2019 \| Volume 9 \| Article 634 5 Statistical Analysis of HNSCC Treatment/Outcomes Fortpied and Vinches Frontiers in Oncology \| www.frontiersin.org Complexity of the Treatments—Multimodality Treatment for head and neck cancer is complex and is based on diﬀerent levels of evidence as stated in the National Comprehensive Cancer Network guidelines (44). Treatment options depend on the stage of the disease: early, locally advanced or recurrent/metastatic. Surgery, radiotherapy, chemotherapy and targeted therapy are all front line options, alone or in combination, depending on the tumor characteristics and stage of the disease. New categories of treatment have been evaluated in head and neck cancer, check point inhibitors have been approved in the metastatic settings with improved survival in ﬁrst [pembrolizumab (45)] and second line [nivolumab (46)]. With new treatments available, new combinations are being tested. Still multimodality remains key. p y y In phase III studies, overall survival (OS) remains the gold standard for the demonstration of clinical beneﬁt, as it is an objective and accurate measure, its importance is unquestioned and it addresses both safety and eﬃcacy. Because overall survival analysis requires a large sample size and may require long follow-up, the investigators may power the study for an alternative endpoint. Doing so, it reduces study timeframe, and improves study feasibility while still capturing a clinical beneﬁt relevant for the patient. Alternative endpoint may be time to local or loco-regional recurrence/progression for early stage disease or to evaluate a local therapy (e.g., EORTC study 1219, NCT01880359); disease free survival in the adjuvant setting [e.g., EORTC study 1735, NCT03673735 or LUX-Head & Neck 2 (50)]; or progression-free survival in the advanced setting [e.g., LUX-Head & Neck 1 (51)]. In 2009, Michiels et al. (52) showed that progression-free survival, deﬁned as the time from randomization to locoregional relapse, distant recurrence, or death whichever comes ﬁrst, can be used as a surrogate endpoint for overall survival to assess the treatment eﬀect of radiotherapy and chemotherapy in randomized trials of locally advanced HNSCC. The surrogacy has been established based on [1] the individual-level correlation between Event Free Survival (EFS) and OS, and [2] the correlation between treatment eﬀects on EFS and OS following the methodology developed by Buyse et al. (53). However, we need to remember that, as pointed out by Michiels et al., EFS is a surrogate endpoint for OS only for chemotherapy or radiotherapy, but cannot be assumed for immunotherapy and for targeted agents, which have a diﬀerent mode of action. We will come back later on this matter. Treatment Allocation To determine whether a biomarker is potentially predictive, a formal and adequately powered statistical test of the treatment-by-biomarker interaction needs to be performed (41). For more detailed considerations on the statistical methodology required to establish predictive July 2019 \| Volume 9 \| Article 634 6 Statistical Analysis of HNSCC Treatment/Outcomes Fortpied and Vinches biomarkers, readers are referred elsewhere (42). To date, in the ﬁeld of head and neck cancer, no biological marker has been proven to be predictive (43). designed as a randomized two-arm trial, with physician choice as control treatment and with progression-free survival assessed 4 months after randomization as primary endpoint. Some early phase studies are designed with the objective to assess the feasibility of the treatment, in which case the main endpoints may be deﬁned as the proportion of patients completing therapy, the rate of patients without severe toxicity, the rate of patients compliant with protocol treatment or similar endpoints. This is the case of the EORTC study 24061 (49), a randomized phase II feasibility study of cetuximab combined with 4 cycles of Docetaxel, cisplatin, and ﬂuorouracil (TPF)followed by chemoradiation with platinum. The main objective of this study was to select the platinum compound, cisplatin or carboplatin, for the chemoradiation regimen to be evaluated in a future Phase III study. Unfortunately, the study was closed prematurely for safety reasons. Complexity of the Treatments—Multimodality Selection of Activity/Efﬁcacy Endpoints of Interest The therapeutic eﬀect of a new treatment or combination of treatments is assessed by means of endpoints selected according to the study objectives. y j In early phase studies, the main endpoint is usually selected to capture the eﬀect of the treatment on the tumor, that is whether the treatment is expected to induce a complete disappearance of the tumor, shrinkage of the tumor or a stabilization of the disease. Complete response (CR) or response (complete response CR or partial response PR), has long been selected as primary endpoint of early phase studies. However, other endpoints may be preferred, such as disease control (complete response CR or partial response PR or stable disease SD) to evaluate treatments with a mechanism of action diﬀerent from chemotherapy, such as targeted or immunotherapy, or where the response to treatment is diﬃcult to assess. Progression free survival (PFS) rate or another time to event endpoint (TTE) evaluated at a ﬁxed point in time after randomization or start of treatment may also be used so the timing of the ﬁnal analysis is ﬁxed and not dependent on a pre-speciﬁed number of events to be observed. When designing a study to evaluate treatments that induce disease stabilization rather than disease reduction, it is recommended that the study includes an internal control arm to make sure the eﬀect of treatment is not confounded with the natural course of the disease. This is especially important if historical information on the control treatment is lacking or limited due to diﬀerences in patient population (e.g., biomarker selected population), in staging system, in imaging / diagnostic tools for assessing outcome, etc. The EORTC study 1559 [NCT03088059 (47)] is an umbrella trial (48) with a platform for enrollment, screening and central proﬁling of patients who are subsequently allocated to one of the molecularly deﬁned sub studies and treated with a matched experimental treatment. Diﬀerent designs are used across the study, reﬂecting diﬀerences in study objectives and in the mechanism of action of the investigated treatment among the sub studies: in particular, a single arm design with response as primary endpoint is chosen for some sub studies, while others are Frontiers in Oncology \| www.frontiersin.org Deﬁnition of Endpoints It is not enough to select the endpoint of interest but we also need to state how exactly it is deﬁned and how it is assessed. International standards are available for measuring response in clinical trials, the most common being the Response Evaluation Criteria in Solid Tumors (RECIST) (http://www.eortc. org/investigators-area/recist). Because of the loss of information inherent to categorizing a continuous measure of tumor shrinkage into categories (progression, stable, response), more and more often waterfall plots are used to display graphically the individual numerical change in tumor size for all patients. July 2019 \| Volume 9 \| Article 634 7 Statistical Analysis of HNSCC Treatment/Outcomes Fortpied and Vinches Time to event endpoints need to be deﬁned very clearly and it is very useful that their exact deﬁnition is accurately provided in the scientiﬁc publications, as there is considerable heterogeneity in the literature regarding these deﬁnitions (54). The methodology section should describe which events are of interest for the selected endpoint, which events constitute competing risks, which events are censored and which events are ignored. In a complex disease such as head and neck cancer with multimodal treatments, for each time-to-event endpoint other than overall survival, and depending on the setting, the following events need to be considered: residual disease after curative treatment, local, regional or distant progression, second primary cancer, death due reasons other than progression. To date there is no consensus on how these and other events such as elective neck dissection and salvage surgery (with residual disease detected or not, depending on the timing of these procedures) are taken into account in the deﬁnition of endpoints. It is the purpose of the Deﬁnition for the Assessment of Time-to-event Endpoints in CANcer trials (DATECAN) project (55) to reach, by consensus among experts, a standardization of the deﬁnitions of commonly used time-to-event endpoints in cancer clinical trials. In addition, events such as treatment stop or switch before the event of interest being reported are not handled the same way by all methodologists, as some recommend ignoring the treatment switch while others recommend censoring these cases (56). The latter approach is highly problematic since it ignores the issue of informative censoring and is not recommended by the EMA (57) while it is proposed in FDA guidelines (58, 59). Schedule of disease assessments plays a critical role in the evaluation of endpoints. Deﬁnition of Endpoints The assessments should ideally match standard practice but for the purpose of the clinical trial they should be planned adequately to capture the eﬀect of treatment. In a multi-arm study, there should also be symmetry between treatment arms in order not to introduce a bias in the comparison of the treatments. With time-to-event endpoint other than overall survival, such as progression-free survival, the exact time of progression is unknown and progressions that occur in between visits are commonly assigned to the visit at which progression was detected. This leads to an over-estimation of the time to progression and a loss of statistical power (56). The analysis may become problematic and biases may arise when clinic visits are missed or delayed. In some cases, it is a challenge to reach a common schedule of assessment across arms because of the intrinsic diﬀerence between treatments: surgery vs. radiotherapy as in EORTC study 1420 “Best-of” (NCT02984410), chemotherapy vs. targeted agent as in EORTC study 1206, induction chemotherapy vs. no induction. When the schedules cannot be made symmetrical across arms, the time assessment biases inherent to the trial may be taken into account by the statistical analysis, by assigning the progressions or recurrences to a speciﬁc point in time (e.g., the next planned visit). This technique was used in a study comparing three nonsurgical treatment strategies to preserve the larynx in patients with locally advanced larynx cancer (60). Patients were randomized between induction cisplatin/ﬂuorouracil followed by radiotherapy, concomitant cisplatin and radiotherapy, or radiotherapy alone. The primary endpoint was a composite endpoint of laryngectomy-free survival. In this study, to account for diﬀerences between treatment arms in the timing of protocol-speciﬁed disease assessments, patients with recurrence or censored before 6 months after random assignment were counted as having treatment failure or censored at 6 months, for eﬃcacy endpoints other than overall survival. g In EORTC study 1219 (NCT01880359), a blind randomized multicenter study of accelerated fractionated chemo- radiotherapy with or without the hypoxic cell radiosensitizer nimorazole, the primary eﬃcacy endpoint is time to locoregional recurrence. This is counted from the day of randomization to the day of ﬁrst record of appearance of local or regional progression, assessed via clinical, imaging or pathological exam. Distant recurrence/progression and second cancers diagnosed before locoregional recurrence and death in absence of locoregional recurrence are not considered events of interest. Deﬁnition of Endpoints But these events are considered as competing risk events in the analysis of the primary endpoint, because they may alter or even preclude the onset of locoregional progression. Therefore, during the design phase, the statistician together with the research physician needs to make assumptions, not only regarding the risk of locoregional progressions but also the risk of distant recurrence/progression, second cancers and death in absence of locoregional recurrence. It is also recommended to monitor these assumptions regularly as they have the potential to directly impact the sample size of the trial, the timelines for the analyses and possibly the statistical power. When a marked departure from the original statistical design assumptions, such as the ones described above, is observed, the consequences need to be evaluated as well as the need for a modiﬁcation of the study design. In order to maintain trial integrity, an Independent Data Monitoring Committee (IDMC) is consulted and the study design is revised, based on the IDMC recommendations, by a statistician not directly involved in the conduct of the study. July 2019 \| Volume 9 \| Article 634 Frontiers in Oncology \| www.frontiersin.org Deﬁnition of Analysis Populations If all subjects enrolled into a clinical trial satisﬁed all entry criteria, completed treatment, followed all trial procedures perfectly with no losses to follow-up, and provided complete data records, then the set of subjects to be included in the analysis would be self-evident. But, in practice, it is doubtful if it can ever be fully achieved specially in the setting of a life-threatening disease, when dealing with complex treatments, administered concomitantly or sequentially. The intention-to-treat principle requires that the primary analysis should include all enrolled subjects. In many clinical trials, this principle provides a conservative strategy and estimates of treatment eﬀects that are more likely to mirror those observed in subsequent practice (3). However, in speciﬁc cases, such as early phase trials, the primary analysis is conducted in the per-protocol population, that is, in the subset of patients who are more compliant with the protocol in order to maximize the opportunity for a new treatment to show activity. For trials with a non-inferiority objective, it is recommended to conduct the main analysis on the per-protocol population in addition to the intention-to-treat population as the latter one may be biased July 2019 \| Volume 9 \| Article 634 Frontiers in Oncology \| www.frontiersin.org 8 Statistical Analysis of HNSCC Treatment/Outcomes Fortpied and Vinches by the hazard ratio, evolves over time. Alternative analysis methods should therefore be considered and are currently being proposed (63). Models assuming a diﬀerent hazard ratio for diﬀerent follow-up times are one possible option. An alternative measure to quantify the treatment eﬀect can be Restricted Mean Survival Time (RMST), which represents the area between the two survival curves up to a predeﬁned follow-up time (69). Simulations are required to evaluate the impact of non- standard patterns on the statistical power using classical or alternative methods of analysis. A delayed treatment eﬀect has also implications on the design of interim analyses for eﬃcacy or futility (63). An interim look for eﬃcacy performed too soon will unlikely result in stopping earlier for a positive outcome, while a futility interim look for futility planned too soon will likely increase the chance of erroneously terminate early the development of an active agent. Altogether this shows how critical is the assumption of proportionality of hazards for the design and analysis of clinical trials with immunotherapy agents. toward demonstrating non-inferiority. Immunotherapy: Impact on Trial Endpoints With the advent of immunotherapies, because of the diﬀerent mechanism of action, how eﬃcacy/activity endpoints are deﬁned and evaluated poses a number of new methodological challenges (63). Although overall survival remains the gold standard endpoint to evaluate the eﬃcacy of treatments in oncology, a number of studies select progression/recurrence free survival as primary endpoint mainly in order to reduce the size and the duration of the studies. As indicated above, it cannot be extrapolated from the work of Michiels et al. that progression-free survival is a surrogate endpoint for overall survival to assess the treatment eﬀect of immunotherapy agents. In addition, as the criteria for progression would be adapted following iRECIST, the issue of surrogacy might be impacted. Surrogate endpoints for immunotherapy trials are currently under investigation (70–72). Novel criteria for the evaluation of antitumor responses with immunotherapeutic agents were ﬁrst developed and published in 2009 by Wolchok et al (immune-related response criteria:irRC) (64), as an attempt to capture new response patterns observed with immune therapy in advanced melanoma beyond those described by RECIST. These criteria, based on bidimensional measurements, were adapted in 2013 by Nishino et al. (65) to only consider unidimensional measurements. In 2017, a consensus guideline for modiﬁed RECIST for immune-based therapeutics (termed iRECIST) was published by a multidisciplinary group including academic, commercial and regulatory members for the use of modiﬁed RECIST (V1.1) in cancer immunotherapy trials (66). The guideline takes into account distinctive behaviors linked to these types of drugs, such as delayed responses and pseudoprogressions. This guideline is consensus based but is not yet validated. It deﬁnes the minimum data to be collected for future and currently in development trials, in order to facilitate the compilation of a data warehouse needed to validate iRECIST. In the meantime, it is recommended that RECIST 1.1 continues to be used as the primary criteria for response based endpoints for randomized studies planned for licensing applications. iRECIST should be considered exploratory in such trials, although earlier phase trials may consider using primarily iRECIST. Frontiers in Oncology \| www.frontiersin.org Deﬁnition of Analysis Populations It is to be noted that the per protocol analysis may lead to biased results when adherence to the study protocol is related to treatment and outcome. y p A textbook example of such bias is given by study TTCC 2002 (grupo español de Tratamiento de Tumores de Cabeza y Cuello), a randomized phase III trial comparing induction chemotherapy followed by chemoradiotherapy vs. chemoradiotherapy alone as treatment of unresectable head and neck cancer (61). The intention-to-treat analysis including all randomized patients showed no advantage of induction chemotherapy followed by chemoradiotherapy over chemoradiotherapy alone; while the analysis excluding patients from the induction arm who did not reach the chemoradiotherapy part of the study resulted in a beneﬁt in favor of the induction arm. The latter analysis, which was the one ﬁrst published (62), was obviously biased because of the selection of the “best” patients from the induction arm. Immunotherapy: Impact on Trial Endpoints Comorbidities Comorbidity is frequent in HNSCC patients (73, 74). The main risk factors associated to this cancer are tobacco and alcohol use, so the comorbid illnesses in these patients are largely related to these habits. The most prevalent comorbidities in this population will be cardiovascular, respiratory or neurological aﬀections. Due to their high prevalence of comorbidities head and neck cancer patients are less often included in early phase trials because of their higher risk of complications. Clinical trials severely select patients and requirenormal organ function, whether of the heart, lungs, kidneys, liver or bone marrow at baseline. It is important to bear this in mind when generalizing trials results to the clinical practice population. Impact on Adherence to Treatment Comorbidities have an inﬂuence on adherence to planned treatment (treatment missed or delayed), to protocol procedures (e.g., visits missed or delayed) and may induce loss to follow-up. The increasing complexity of treatment strategies and of trial designs with complex protocols which entail multiple procedures, adds an additional layer of diﬃculty for patients to adhere to treatment and protocol procedures. Oral medications and self-administered subcutaneous therapies oﬀer the patient convenience over intravenous infusions but the responsibility of administration of these critical medications has been transferred to the patient, potentially increasing the risk of non-adherence. For some studies, a quality of life score has been selected as the primary endpoint. This is the case of the EORTC study 1420 “Best-of” (NCT02984410). Because the techniques that have been developed in parallel in the radiotherapy and surgical ﬁelds both have an excellent oncological control, the main focus of this prospective randomized trial is to assess which one of the two modalities provides better functional outcome and more speciﬁcally better swallowing function. This is assessed using the M. D. Anderson dysphagia inventory (MDADI), a validated and reliable self-administered questionnaire designed speciﬁcally for evaluating the impact of dysphagia on the quality of life of patients with head and neck cancer (79). A retrospective analysis of comorbidities and adherence to treatment in patients with oropharyngeal carcinoma has been reported by Hess (76), suggesting a poorer adherence to treatment in patients with HPV-negative status as compared HPV-positive, as a result of the higher comorbidities in the former patient group due to alcohol and tobacco consumption. These results add to the recognition that HPV-positive and HPV-negative oropharyngeal cancer represent distinct entities and the authors recommend to take this additional diﬀerence into account in the design of clinical trials addressing these populations. There is to date no consensus on how quality of life data in cancer clinical trials are analyzed. A variety of statistical techniques are available to handle the longitudinal nature of the data, to adjust for multiple scales and items, to deal with missing data (80). Currently the methods range from simple descriptive analyses up to complex modeling approaches. CONCLUSION The medical statistician is responsible for a wide variety of tasks covering the design and the analysis of a clinical study, which requires speciﬁc competencies in terms of statistical methodology and programming skills. It is particularly important to use eﬃcient communication, in order that the medical statistician gets some understanding of the medical ﬁeld and that the research physician gets fairly acquainted with the principles of statistical methodology. Only a ﬂuid interaction between the two ﬁelds enables that the study design addresses adequately the research question that is at the basis of the clinical trial and that the results of the analysis are interpreted appropriately. Impact on Primary Endpoints p y p Studies evaluating the impact of comorbidities in head and neck cancer patients show that it is an important feature of these patients, which has a detrimental impact on overall survival. Patients with head and neck cancer are concurrently at risk for other events, including second malignancies and mortality due to adverse treatment eﬀects or comorbid diseases (75). Overall survival and progression/recurrence-free survival are composite endpoints, constituted of events of diﬀerent nature, directly linked to the primary cancer (disease progression/recurrence or death due to the disease) or not (second malignancies, deaths due to treatment toxicity or comorbidities). Analyzed as composite endpoints, they are not suﬃcient for a complete Another issue is the delayed treatment eﬀect leading to a separation of PFS or OS curves between treatment arms only after a lag time of several months. This phenomenon has been observed, particularly in melanoma studies (67, 68). This pattern has also been observed for OS in the phase 3 trial comparing nivolumab to standard systemic therapy in patients with recurrent HNSCC (46). Such a late separation is indicative of non-proportional hazards. This pattern may invalidate the use of classical statistical analysis methods to estimate and test treatment eﬀects such as the Cox model, which is based on the assumption of proportional hazards. Such analyses become diﬃcult to interpret since the treatment eﬀect, expressed July 2019 \| Volume 9 \| Article 634 9 Statistical Analysis of HNSCC Treatment/Outcomes Fortpied and Vinches developed and validated tools for the assessment of quality of life in cancer patients, using high standards of methodology. These questionnaires are meant to be used primarily in clinical trials. Speciﬁcally for head and neck cancer, patients are asked to complete a list of 60 head and neck cancer-speciﬁc items comprising the recently updated EORTC head and neck module (EORTC QLQ-HN43) as well as the core questionnaire (EORTC QLQ-C30) (77, 78). interpretation of the results of the trial. It is then useful to analyze the components as individual time-to-event endpoint, via cumulative incidence functions, in order to distinguish and characterize the weight of the diﬀerent components on the observed outcomes. Impact on Adherence to Treatment The consortium SISAQOL (Setting International Standards in Analyzing Patient- Reported Outcomes and Quality of Life Endpoints Data for Cancer Clinical Trials) has been created with the aim to develop guidelines and recommendations for the statistical analyses of quality of life data and more generally of patient-reported outcome data in cancer clinical trials (81, 82). Poor compliance does aﬀect the analysis and interpretation of clinical trial data and represents a potential source of bias in a clinical trial. The data related to treatment exposure, the frequency and reasons for treatment interruptions or deﬁnitive withdrawals, the frequency and nature of severe protocol violations, the frequency of patients lost to follow up need to be analyzed as well as their relationship to outcome in order to identify these potential biases. Sensitivity analyses conducted in diﬀerent analyses populations, i.e., intention-to-treat vs. per-protocol population, may be useful to assess the robustness of the ﬁndings. Frontiers in Oncology \| www.frontiersin.org July 2019 \| Volume 9 \| Article 634 REFERENCES 15. Rettig EM, D’Souza G. Epidemiology of head and neck cancer. Surg Oncol Clin N Am. (2015) 24:379–96. doi: 10.1016/j.soc.2015.03.001 1. EEC note for guidance: good clinical practice for trials on medicinal products in the European Community. CPMP working party on eﬃcacy of medicinal products. Pharmacol Toxicol. (1990) 67:361–72. doi: 10.1111/j.1600-0773.1990.tb00847.x 16. 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International council for harmonisation of technical requirements for pharmaceuticals for human use (ICH). ICH harmonised guideline. Integrated addendum to ich e6(r1): guideline for good clinical practice e6(r2) (2016). 20. Fortpied C, Liberatoscioli C, Bogaerts J. Design issues in head and neck clinical trials. Anticancer Drugs. (2010) 22:682–7. doi: 10.1097/CAD.0b013e3283417a0d 6. Senn S. Dicing With Death : Chance, Risk, and Health. Cambridge: Cambridge University Press (2003). p. 251. 21. Vickers AJ, Cronin AM. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc. 2019.00634/full#supplementary-material ACKNOWLEDGMENTS We thank Laurence Collette, Head of Biostatistics Department at the EORTC, for her valuable suggestions to improve the manuscript. FUNDING MV work as fellow at EORTC headquarters was supported by a grant from by Fonds Cancer (FOCA) from Belgium. MV work as fellow at EORTC headquarters was supported by a grant from by Fonds Cancer (FOCA) from Belgium. Impact on Quality of Life and Assessment of Quality of Life in Clinical Trials The symptoms and treatments associated with advanced head and neck cancer often have a devastating impact on quality of life. Head and neck cancer can disrupt many life essential functions. It can impact on breathing, swallowing, and speaking, and treatment can even increase the physical impairment. These consequences aﬀect multiple spheres of daily functioning. As one consequence head and neck cancer patients have a higher risk of depression and suicide. In particular head and neck cancer is a complex ﬁeld: a heterogeneous disease, with multimodality treatment and associated comorbidities. We have set out how these speciﬁcities raise a number of methodological challenges with some examples of approaches that current and future clinical researchers and medical statisticians may altogether consider useful in order to generate valuable information to guide clinical decisions and ultimately make progress in the treatment of this disease. Quality of Life is thus an important outcome to be considered in routine treatment but also to evaluate new treatments in clinical trials, even in early stage disease. The EORTC has July 2019 \| Volume 9 \| Article 634 Frontiers in Oncology \| www.frontiersin.org 10 Statistical Analysis of HNSCC Treatment/Outcomes Fortpied and Vinches AUTHOR’S NOTE All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. There is abundant literature in the ﬁeld of head and neck cancer as well as abundant literature in statistical methodology. There is abundant literature in the ﬁeld of head and neck cancer as well as abundant literature in statistical methodology. The present article makes the bridge between the two ﬁelds hopefully encouraging and enabling ﬂuid communication between the research physician and the medical statistician involved in clinical trials in head and neck cancer. The methodological challenges resulting from the heterogeneity of the head and neck cancer, the complexity of the treatments and the associated comorbidities are presented with examples. A formal literature search for this review was not performed. This review is based on the authors’ work and expertise in designing, monitoring and analysing clinical trials as well as reading and reviewing clinical and statistical literature. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 72. Buyse M, Burzykowski T, Saad ED. The search for surrogate endpoints for immunotherapy trials. Ann Transl Med. (2018) 6:231. doi: 10.21037/atm.2018.05.16 73. Paleri V, Wight RG, Silver CE, Haigentz M, Takes RP, Bradley PJ, et al. Comorbidity in head and neck cancer: a critical appraisal July 2019 \| Volume 9 \| Article 634 Frontiers in Oncology \| www.frontiersin.org 13
https://openalex.org/W3039271871	https://europepmc.org/articles/pmc7334002?pdf=render	English	null	Chronic inducible urticaria: confirmation through challenge tests and response to treatment	Einstein	2,020	cc-by	5,885	How to cite this article: Pereira AR, Motta AA, Kalil J, Agondi RC. Chronic inducible urticaria: confirmation through challenge tests and response to treatment. einstein (São Paulo). 2020;18:eAO5175. http://dx.doi.org/10.31744/ einstein_journal/2020AO5175 ORIGINAL ARTICLE ORIGINAL ARTICLE ❚❚RESUMO Objetivo: Avaliar a positividade dos testes de provocação de pacientes com suspeita de urticária crônica induzida e sua resposta ao tratamento. Métodos: Estudo retrospectivo de prontuários eletrônicos de pacientes com suspeita de urticária crônica induzida. Todos os pacientes foram submetidos aos testes de provocação com estímulos desencadeantes, conforme história clínica e, posteriormente, foi avaliada a resposta ao tratamento medicamentoso. Resultados: Foram incluídos 191 pacientes com suspeita de urticária crônica induzida, a qual foi confirmada em 118 pacientes e 122 testes positivos (4 pacientes com 2 testes positivos diferentes). A maioria apresentava urticária dermográfica (70,3%), seguida de urticária colinérgica (17,8%). Em relação ao tratamento, 28% responderam ao anti-histamínico em doses licenciadas, 34,7% em doses aumentadas e 9,3% responderam à adição de outro medicamento. A concomitância de urticária crônica induzida com urticária crônica espontânea foi encontrada em 35,3% dos pacientes, sendo mais frequente no sexo feminino, com tempo mais prolongado para controle dos sintomas e maior frequência de urticária colinérgica. Conclusão: A confirmação de urticária crônica induzida nos pacientes com suspeita da doença foi elevada. Houve boa resposta ao anti-histamínico. Na concomitância com urticária crônica espontânea, observou-se maior tempo para o controle dos sintomas e maior frequência de urticária colinérgica. Keywords: Urticaria; Angioedema; Diagnosis; Drug therapy; Dose-response relationship, drug Keywords: Urticaria; Angioedema; Diagnosis; Drug therapy; Dose-response relationship, drug Chronic inducible urticaria: confirmation through challenge tests and response to treatment Urticária crônica induzida: confirmação por testes de provocação e resposta ao tratamento Official Publication of the Instituto Israelita de Ensino e Pesquisa Albert Einstein ISSN: 1679-4508 \| e-ISSN: 2317-6385 Amanda Rocha Firmino Pereira1, Antônio Abílio Motta2, Jorge Kalil2, Rosana Câmara Agondi1 1 Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil. 2 Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil. DOI: 10.31744/einstein_journal/2020AO5175 ❚❚ABSTRACT Objective: To evaluate the positivity of challenge tests of patients suspected of chronic inducible urticaria and the response to treatment. Methods: A retrospective study of electronic medical records of patients suspected of chronic inducible urticaria. All patients were submitted to challenge tests with triggering stimuli, according to the clinical history and, subsequently, the response to drug treatment was evaluated. Results: A total of 191 patients with suspected chronic inducible urticaria were included. It was confirmed in 118 patients and 122 positive tests (4 patients with 2 different positive tests). Most had dermographic urticaria (70.3%), followed by cholinergic urticaria (17.8%). Regarding treatment, 28% responded to antihistamine in licensed doses, 34.7% with increased doses, 9.3% responded to the addition of another medication. The concomitance of chronic inducible urticaria and chronic spontaneous urticaria was found in 35.3% of patients, being more frequent in females, with longer time to control symptoms and higher frequency of cholinergic urticaria. Conclusion: The confirmation of chronic inducible urticaria in patients with this suspicion, after challenge tests, was high. There was a good response to antihistamine. In the concomitance of chronic spontaneous urticaria, longer time to control symptoms and higher frequency of cholinergic urticaria were observed. How to cite this article: P i AR M AA K lil J Keywords: Urticaria; Angioedema; Diagnosis; Drug therapy; Dose-response relationship, drug ❚❚INTRODUCTION Chronic urticaria including spontaneous and inducible, has been associated with a negative impact on different aspects of patients’ quality of life.(9,10) Despite the prevalence of chronic urticaria, the evidence on the associated economic and humanistic load in the Brazilian population is still limited. A Brazilian study showed that adults with chronic urticaria present with substantially worse results than do people who live without chronic urticaria relative to quality of life, anxiety, and sleep difficulties. Chronic urticaria, has also been associated with significant losses in work, and with a high-level usage of health resources. The disease has been associated with significantly higher chances of any medical or emergency consultation, or hospitalization.(11) The degree to which the quality of life is hindered varies according to the etiology and severity of the chronic urticaria, and chronic delayed pressure urticaria has affected quality of life more significantly that isolated chronic urticaria.(12) Urticaria is characterized by the presence of weals, angioedema, or both, and is classified as acute or chronic, according to its duration. Chronic urticaria is characterized by the presence of symptoms, daily or on most days of the week, for more than 6 weeks. chronic urticaria can be classified as spontaneous or inducible, as per the identification of a specific stimulus.(1) Chronic inducible urticaria (CIndU) is characterized by the need for a specific trigger, such as physical stimuli (dermographic urticaria, hot contact urticaria, cold urticaria, delayed pressure urticaria, solar urticaria, and vibratory angioedema) and non-physical stimuli (cholinergic urticaria, contact urticaria, and aquagenic urticaria).(1,2) Chronic inducible urticaria is common, with an estimated prevalence between 0.1 and 0.5% of general population. The most affected age group is between 20 and 40 years.(3) Silpa-archa et al.,(4) observed that 7.2% of patients with chronic urticaria presented with the inducible type, and symptomatic dermographism or dermographic urticaria was the most prevalent among CIndU. The association among the various types of chronic urticaria is common. Sánchez et al.,(5) observed that up to 36% of patients with chronic spontaneous urticaria (CSU) reported concomitant physical triggers.(5) The diagnosis of CIndU is based on the clinical history and challenge tests. Corresponding author: p g Amanda Rocha Firmino Pereira Ambulatório de Imunologia Clínica e Alergia Avenida Dr. Enéas Carvalho Aguiar, 155, 5o floor, building 4B – Cerqueira César Zip code: 05401-050 – São Paulo, SP, Brazil Phone: (55 11) 2661-9571 E-mail: amandafirpe@yahoo.com.br Copyright 2020 This content is licensed under a Creative Commons Attribution 4.0 International License. Received on: May 18, 2019 Accepted on: Nov 3, 2019 Conflict of interest: none. Copyright 2020 This content is licensed under a Creative Commons Attribution 4.0 International License. Received on: May 18, 2019 Accepted on: Nov 3, 2019 Conflict of interest: none. Descritores: Urticária; Angioedema; Diagnóstico; Tratamento farmacológico; Relação dose-resposta a droga einstein (São Paulo). 2020;18:1-8 1 Pereira AR, Motta AA, Kalil J, Agondi RC ❚❚INTRODUCTION The objectives of the challenge tests are to determine the relevant stimulus and evaluate the threshold for that stimulus.(2) Its management involves avoiding the triggering factors, and the symptomatic treatment is the same as recommended for the treatment of CSU, that is, to use the first line of treatment, such as second generation anti-histamines (AH1) at licensed doses. If there is no response, the second line of treatment should be used, increasing the dose of the second generation AH1 up to four times a day. When the patient does not respond to the AH1, omalizumab is the third line of treatment and, after 6 months, when the patient does not respond to this medication, the fourth line of treatment is cyclosporine.(1) The pathogenesis of CIndU depends on the release of histamine and other active mastocyte mediators. Many studies suggested the participation of autoantigens formed by environmental stimuli, leading to the formation of IgEs that would recognize them.(3,6) The most common CIndU is dermographic, which is characterized by the appearance of linear erythematous and pruritic weals, after friction (scratches or skin friction).(6,7) Delayed pressure urticaria is characterized by the delayed appearance of edema, erythema, and pruritus, a burning sensation, and pain, located in the skin areas exposed to a vertical pressure. Cold urticaria is characterized by the appearance of erythema, weals and pruritus or angioedema when there is exposure to cold air, liquids, or an object.(8) ❚❚OBJECTIVE To evaluate the positivity of challenge tests carried out in patients with a presumptive diagnosis of chronic inducible urticaria and response to drug treatment. Cholinergic urticaria is a frequent CIndU comprising up to 7% of all inducible urticaria, characterized by the appearance of punctiform erythematous and itchy weals, measuring from 1 to 5mm. Lesions appear with the increase in body temperature, such as with physical exercise, hot baths, and emotional stress.(3,8) einstein (São Paulo). 2020;18:1-8 Figure 1. Positive test for cold urticaria, with presence of erythematous and edematous plaque in the volar region of the patient’s forearm Department of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (USP), a public tertiary care service, were evaluated considering confirmation of this diagnosis, its progression, and response to treatment. To confirm diagnosis, the challenge tests were performed in all patients. The demographic data, results of challenge tests, concomitance of CSU, the medications used, medication that led to control of CIndU, and time of treatment for the control of the disease were evaluated. The disease was considered controlled when the patient remained asymptomatic for more than 1 month. Results of the challenge tests are influenced by various factors, including treatment of patients. Therefore, the symptomatic treatment of the patients was discontinued before the test. The AH1 were interrupted for at least 3 days before, and the corticosteroids, when in use, 7 days before the tests.(2) Figure 1. Positive test for cold urticaria, with presence of erythematous and edematous plaque in the volar region of the patient’s forearm Figure 2. Positive test for delayed pressure urticaria, with the presence of edema and erythema on the patient’s left shoulder , y Patients were submitted to tests according to the clinical history reported. ❚❚METHODS This was a retrospective and descriptive study based on electronic patient records, carried out during years 2003 to 2018. Enrolled patients were those with clinical suspicion of CIndU, based on international consensus,(1,2,13) adults (aged >18 years), and of both sexes. Patients with a clinical suspicion of CIndU who had been referred to the Clinical Immunology and Allergy Other rarer CIndU types include solar, heat contact, and vibratory angioedema, triggered by solar radiation, contact with heat, and vibration, respectively.(3,8) Patients with a clinical suspicion of CIndU who had been referred to the Clinical Immunology and Allergy 2 Chronic inducible urticaria: confirmation through challenge tests and response to treatment The dermographism test was done on the volar surface of the patients’ forearm, with moderate pressure, using a smooth flat object and/or FricTest®, a dermographometer with reading in 10 minutes, which is considered positive in the presence of linear weal and itching.(1) The test for heat contact urticaria was performed by placing the patient’s forearm into a tub of water at 45°C, for 5 minutes, with reading 10 minutes later, and is considered positive in the presence of weal and itching at the site.(1) The test for cold urticaria was carried out by placing an ice cube in a plastic bag on the volar region of the forearm, for 5 minutes, with reading after 10 minutes, and is positive in the presence of weal and itching (Figure 1).(1) For the delayed pressure urticaria test, the Warin test was used by applying 4kg for 5 minutes over the skin of the upper third of the forearm or test with 7kg of pressure divided into 3.5kg each weight, connected by a strip 3cm wide by 15 minutes over the shoulder or thigh, both reading after 6 hours, and considered positive with the presence of local edema and erythema (Figure 2).(14) In the solar urticaria test, a small dorsal area was exposed at 10cm away from the visible light from a slide projector for 10 minutes, with readings at 10, 20, and 30 minutes after the end of the test; it was considered positive if there were weals, erythema, and itching, or a burning sensation.(1) The vibratory test was not performed since there were no patients suspected to have this condition at our service. The test for cholinergic urticaria was carried out by submitting the patient to walk on the treadmill until sweating began, and then 15 minutes of the test were counted out and the reading was done immediately after the test ended and 10 minutes later (Figure 3).(13) Figure 2. Positive test for delayed pressure urticaria, with the presence of edema and erythema on the patient’s left shoulder Figure 3. Positive test for cholinergic urticaria, with punctiform erythematous weals on the patient’s dorsum Figure 3. Positive test for cholinergic urticaria, with punctiform erythematous weals on the patient’s dorsum einstein (São Paulo). 2020;18:1-8 3 Pereira AR, Motta AA, Kalil J, Agondi RC Table 1. Demographic characteristics of patients with chronic inducible urticaria Patient characteristics (n=118)* Results Female sex 81.4 Current age, years 41.2±13.1 Age at onset, years 36.5±14.7 Time of disease, years 4.9±6.0 Results expressed by % or mean±standard deviation. Mann-Whitney test. Table 1. Demographic characteristics of patients with chronic inducible urticaria Aquagenic urticaria was assessed by applying gauzes wetted with room temperature water on the patient’s dorsum for 20 minutes; it was considered positive in the presence of papules formed in up to 10 minutes.(15) The project was approved by the Research Ethics Committee, CAAE: 79655117.1.0000.0068 and official opinion 2.391.902. Before performing each test, the Informed Consent Form was applied. For the statistical analysis, all the data analyzed used nonparametric tests. Fisher’s test was used to compare the frequencies of the female sex, response to AH1 (licensed and increased doses), frequency of refractoriness to AH1, and frequency of the type of CIndU among the groups, CIndU, and CIndU associated with CSU. The Mann-Whitney test was used to compare demographic data of the patients, such as current age, age at onset of the disease, and time of the disease. The Kruskal-Wallis test was used to evaluate the clinical comparison between the primary subtypes of inducible urticaria. Diagnosis was confirmed as per the clinical suspicion in 83/133 patients (62.4%) with suspected symptomatic dermographism; 21/43 patients (48.8%) with suspected cholinergic urticaria; 9/23 patients (39.1%) with suspected cold urticaria; 6/15 patients (40.0%) with suspected delayed pressure urticaria, 2/7 patients (28.6%) with suspected solar urticaria, and 1/15 patient (6.7%) confirmed the suspicion of heat contact urticaria. No patient with suspected aquagenic urticaria presented with a positive test, and four patients presented with two concomitant CIndU. Figure 4 shows the frequency of positivity of the challenge tests, as per clinical suspicion. einstein (São Paulo). 2020;18:1-8 Clinical comparison among the primary subtypes of inducible urticaria Table 2. Clinical comparison among the primary subtypes of inducible urticaria p=0.02. CIndU: chronic inducible urticaria. Figure 5. Frequency of chronic inducible urticaria according to concomitance or not of chronic spontaneous urticaria p=0.02. CIndU: chronic inducible urticaria. Figure 5. Frequency of chronic inducible urticaria according to concomitance or not of chronic spontaneous urticaria Figure 6. Crystalline miliaria confirmed after challenge tests for cholinergic urticaria. (A) Significant sweating and vesicles with crystalline content on the limbs. (B) Significant sweating and vesicles with crystalline content on the trunk A B Concomitance of CIndU and CSU was present in 35.3% of patients. When the patients who only had CIndU were compared to those with a concomitant diagnosis of CSU, the second group presented with a higher frequency of females, longer time needed for symptom control (Table 3, Figure 5), and greater frequency of cholinergic urticaria. B Figure 6. Crystalline miliaria confirmed after challenge tests for cholinergic urticaria. (A) Significant sweating and vesicles with crystalline content on the limbs. (B) Significant sweating and vesicles with crystalline content on the trunk Two patients with suspected cholinergic urticaria and negative challenge tests presented with significant sudoresis and the presence of microvesicles on the trunk and limbs minutes after the end of the test; the presumptive diagnosis was crystalline miliaria (Figures 6A and 6B). ❚❚RESULTS A total of 191 patients was enrolled in this study. Of these, 158 patients (82.7%) were female, mean age of 41.8 years (standard deviation – SD of 13.6 years), mean age at onset of symptoms was 36.1 years (SD of 15.0 years), and mean time of disease of 5.8 years (SD of 7.8 years). Figure 4. Positivity of challenge tests according to clinical suspicion of chronic inducible urticaria As to the suspected CIndU, 133 patients (69.6%) presented with a hypothesis of symptomatic dermographism, 43 (22.5%) had a suspicion of cholinergic urticaria, 23 (12.0%) to cold, 15 (7.9%) to heat, 15 (7.9%) to delayed pressure, 7 (3.7%) solar, and 5 (2.6%) aquagenic urticaria. Of these patients, 41 (21.5%) were suspected of having two or more associated CIndU. Seventy-three (38.2%) patients presented with a history of concomitant CSU. Figure 4. Positivity of challenge tests according to clinical suspicion of chronic inducible urticaria Of the total 118 patients (and 122 positive tests) with CIndU confirmed by challenge tests, symptomatic dermographism was present in 70.3%; cholinergic urticaria in 17.8%; cold contact in 7.6%; delayed pressure in 5.1%; solar in 1.7%; and heat contact in 0.8%. All the patients were referred to challenge tests, according to the suspected stimulus. Although instructed about the preparation for the tests, 33 patients (17.3%) were not submitted to the challenge tests due to some contradiction at the time. Moreover, two other patients referred with suspected two or more types of CIndU, were not submitted to at least one of the specific tests. As to response to treatment, considering that omalizumab was not available for these patients in our outpatient service of the 118 patients with CIndU confirmed by the challenge tests, most responded to AH1; in that, 32 patients (27.1%) responded to licensed doses of AH1, 41 (34.7%) to increased doses of AH1, and 11 (9.3%) to the addition of another medication to AH1. At the end of this study, 34 patients (28.9%) with no response to AH1 had also not responded to the addition of another medication to AH1. After referral to challenge tests, CIndU was confirmed in 118 patients (74.7%), and four patients (2.5%) confirmed positivity for two subtypes. The demographic data of patients with CIndU, confirmed by means of challenge tests, are shown on table 1. einstein (São Paulo). 2020;18:1-8 4 Chronic inducible urticaria: confirmation through challenge tests and response to treatment Table 3. Comparison between patients with isolated chronic inducible urticaria and those with chronic inducible urticaria associated with chronic spontaneous urticaria Demographic characteristics Chronic inducible urticaria (n=76) Chronic inducible urticaria + chronic spontaneous urticaria (n=42) p value Female sex* 73.7 95.2 0.03 Current age, years† 41.3±13.5 41.0±12.5 NS Age at onset, years† 36.9±14.7 35.6±14.6 NS Time of disease, years† 4.5±5.2 5.7±7.2 NS Response to treatment* Response to AH1 at licensed dose 32.9 19.0 NS Response to AH1 at a dose up to 4-fold higher than the licensed dose 30.3 42.9 NS Refractory to AH1 36.8 38.1 NS Time to response to treatment, months† 15.0 19.8 0.02 * Fisher’s test. † Mann-Whitney test. Results expressed by % or mean±standard deviation. AH1: antihistamine; NS: not significant. Table 3. Comparison between patients with isolated chronic inducible urticaria and those with chronic inducible urticaria associated with chronic spontaneous urticaria The primary subtypes of induced urticaria were dermographic, cholinergic, cold, and delayed pressure. The primary clinical differences among them were that dermographic urticaria was the most frequent; in cholinergic urticaria, the male sex was more frequently involved than in other subtypes, as well as a lower response to treatment (AH1 and others) until the time this study was finalized (38%); cold contact urticaria was the one that presented with the shortest history of urticaria, greater frequency of response to AH1 at licensed doses (once a day), and shorter time to control the urticaria; in delayed pressure urticaria, all patients were female, none presented with control of urticaria with the use of AH1 at licensed doses, and time to reach clinical control was longer. These data can be observed on table 2. Table 2. Clinical comparison among the primary subtypes of inducible urticaria Subtypes of urticaria versus clinical characteristics Dermographic urticaria Cholinergic urticaria Cold urticaria Delayed pressure urticaria p value Female sex 85.5 61.9 88.9 100 NS Current age, years 42.6±12.7 36.9±13.9 37.7±16.7 41.3±11.2 NS Time of urticaria, years 4.0±4.7 7.0±7.1 2.3±1.5 10.5±13.0 NS Response to AH1 at a licensed dose 28.9 14.3 44.4 0 0.012 Response to AH1 at a dose 4-fold higher than the licensed dose 36.1 28.6 22.2 66.7 0.012 Refractory to AH1 8.4 19.1 0 16.7 0.012 No response to treatment 26.6 38 33.4 16.6 0.012 Months to response to treatment 17.0±15.8 17.8±17.7 10.2±4.5 19.2±6.6 NS Kruskal-Wallis Test. Results expressed as % or mean±standard deviation. NS: not significant. AH1: antihistamine. Table 2. ❚❚DISCUSSION Chronic inducible urticaria, in contrast with chronic spontaneous, is characterized by the need for specific einstein (São Paulo). 2020;18:1-8 5 Pereira AR, Motta AA, Kalil J, Agondi RC triggers for the development of weals, angioedema, or both. The signs and symptoms are generally confined to areas exposed to the specific trigger.(2) Management should be concentrated on avoiding the trigger and on symptomatic treatment, with the objective of reaching complete control of signs and symptoms. To avoid the triggering stimuli is desirable, but, in most cases, it is very difficult to be attained. In addition, for many patients, the threshold for triggering symptoms is low. Detailed information on the properties of the stimulus should allow the patient to recognize and control its expression in normal daily living.(1,2) Our study evaluated 191 patients referred to the service with suspected CIndU. Excluding patients with contraindications (33 patients); all were referred for challenge tests with the trigger suggested by the clinical history. Chronic inducible urticaria was confirmed in 118 patients (74.7%), and 25.3% of patients had their clinical suspicions not confirmed, demonstrating the importance of performing the challenge tests. The second generation AH1 should be considered as first line symptomatic treatment for urticaria, due to their good safety profile. If the symptoms persist with the standard dose, it is recommended to use the same treatment algorithm as for CSU, which is to increase the doses of these AH1 up to four times, as second line of treatment. However, about 50% of patients did not respond to AH1.(1) Chronic inducible urticaria are diagnosed based on the patient’s history and on challenge test results. It is important to identify and precisely characterize the trigger stimulus and the triggering thresholds of symptoms in these patients, since CIndU can cause severe compromise to quality of life and even have important occupational implications.(1,2) Studies have demonstrated that patients with CIndU presented with a worse response to AH1 at licensed doses that did those with CSU.(17-19) Kocatürk et al.,(17) observed that 20.9% of patients with CIndU showed symptom control using AH1 at licensed doses, and 37.9% of patients with CSU presented with this response. On the other hand, the rates of response to the quadruplicated dose of AH1 in the two groups were not significantly different between them. It is important to know that a combination of CIndU and CSU is frequently observed. ❚❚DISCUSSION Curto-Barredo et al.,(16) noted this concomitance in 20% of patients. Our study observed such a concomitance in 35.3% of patients evaluated. These patients were mostly female, took a longer time to control their symptoms, and had a greater frequency of cholinergic urticaria than did the group with isolated CIndU. This study also noted that the occurrence of two or more forms of CIndU, confirmed by challenge tests, was observed in four patients (3.4%). Nonetheless, in our study, 73 patients obtained control with AH1 (61.9%), and of these, 32 patients (27.1%) needed only licensed doses of AH1 to control the disease. Although a larger portion of the patients with concomitant CSU had controlled the clinical picture only with AH1 at doses superior to those licensed, there was no difference as compared to the group with only CIndU. Data on prevalence, incidence, and duration of CIndU found in literature, are based on observational studies of small populations.(2) Silpa-archa et al.,(4) observed that the frequency of the female sex in patients with physical urticaria was 74.4%. In our sample, we found an even greater frequency in the cases of CIndU confirmed by challenge tests, i.e., of 81.4% of women. Studies have shown that omalizumab was effective in the treatment of CIndU refractory to AH1,(20,21) but for our study, this medication was not available. The other medications utilized for patients refractory to AH1 in our study were montelukast, cyclosporine A, and ranitidine. With these medications, control of the disease was achieved in 11 patients (9.3%). The rest of them (34 patients; 28.8%) did not reach control of the disease until the end of this study. The prevalence of symptomatic dermographism was between 50% and 78% of patients with CIndU, which is consistent with our study that found 70.3%. Cold urticaria was reported in 8% to 37%, and in our study, it was confirmed in 7.6%; for delayed pressure urticaria, from 3% to 20%, and 5.1% in our patients. Cholinergic urticaria has a prevalence of 6% to 13% among CIndU cases, diverging from our study that showed a higher prevalence of 17.8%. ❚❚REFERENCES Zuberbier T, Aberer W, Asero R, Abdul Latiff AH, Baker D, Ballmer-Weber B, Bernstein JA, Bindslev-Jensen C, Brzoza Z, Buense Bedrikow R, Canonica GW, Church MK, Craig T, Danilycheva IV, Dressler C, Ensina LF, Giménez- Arnau A, Godse K, Gonçalo M, Grattan C, Hebert J, Hide M, Kaplan A, Kapp A, Katelaris CH, Kocatürk E, Kulthanan K, Larenas-Linnemann D, Leslie TA, Magerl M, Mathelier-Fusade P, Meshkova RY, Metz M, Nast A, Nettis E, Oude- Elberink H, Rosumeck S, Saini SS, Sánchez-Borges M, Schmid-Grendelmeier P, Staubach P, Sussman G, Toubi E, Vena GA, Vestergaard C, Wedi B, Werner RN, Zhao Z, Maurer M; Endorsed by the following societies: AAAAI, AAD, AAIITO, ACAAI, AEDV, APAAACI, ASBAI, ASCIA, BAD, BSACI, CDA, CMICA, CSACI, DDG, DDS, DGAKI, DSA, DST, EAACI, EIAS, EDF, EMBRN, ESCD, GA²LEN, IAACI, IADVL, JDA, NVvA, MSAI, ÖGDV, PSA, RAACI, SBD, SFD, SGAI, SGDV, SIAAIC, SIDeMaST, SPDV, TSD, UNBB, UNEV and WAO. The EAACI/GA²LEN/EDF/WAO guideline for the definition, classification, diagnosis and management of urticaria. Allergy. 2018;73(7):1393-414. 2. Magerl M, Altrichter S, Borzova E, Giménez-Arnau A, Grattan CE, Lawlor F, et al. The definition, diagnostic testing, and management of chronic inducible urticarias - The EAACI/GA(2) LEN/EDF/UNEV consensus recommendations 2016 update and revision. Allergy. 2016;71(6):780-802. 3. Maurer M, Fluhr JW, Khan DA. How to approach chronic inducible urticaria. J Allergy Clin Immunol Pract. 2018;6(4):1119-30. Review. One limitation of our study was the impossibility of performing inducible urticaria tests in a large portion of patients (28%). Often, this situation was a consequence of non-collaboration of the patient, but on other occasions, the tests were not done because of the patient’s compromised physical condition, such as, for example, testing for cholinergic urticaria on a treadmill. Another deficiency of our study referred to the availability of medications indicated for treatment of inducible urticaria. This was because, even though supported by medical literature, some were not licensed for use, and other times, it was due to contraindications for their use in our patients. 4. Silpa-archa N, Kulthanan K, Pinkaew S. Physical urticaria: prevalence, type and natural course in a tropical country. J Eur Acad Dermatol Venereol. 2011;25(10):1194-9. 5. Sánchez J, Amaya E, Acevedo A, Celis A, Caraballo D, Cardona R. Prevalence of inducible urticaria in patients with chronic spontaneous urticaria: associated risk factors. J Allergy Clin Immunol Pract. 2017;5(2):464-70. 6. Abajian M, Schoepke N, Altrichter S, Zuberbier T, Maurer M. Physical urticarias and cholinergic urticaria. ❚❚REFERENCES Immunol Allergy Clin North Am. 2014;34(1):73- 88. Review. Erratum in: Immunol Allergy Clin North Am. 2014;34(2):xix. Zuberbier, H C Torsten [corrected to Zuberbier, Torsten]. 7. Schoepke N, Młynek A, Weller K, Church MK, Maurer M. Symptomatic dermographism: an inadequately described disease. J Eur Acad Dermatol Venereol. 2015;29(4):708-12. 8. Kontou-Fili K, Borici-Mazi R, Kapp A, Matjevic LJ, Mitchel FB. Physical urticaria: classification and diagnosis guidelines. An EAACI position paper. Allergy. 1997;52(5):504-13. 9. Grob JJ, Revuz J, Ortonne JP, Auquier P, Lorette G. Comparative study of the impact of chronic urticaria, psoriasis and atopic dermatitis on the quality of life. Br J Dermatol. 2005;152(2):289-95. ❚❚AUTHORS’ INFORMATION ❚❚AUTHORS INFORMATION Pereira AR: http://orcid.org/0000-0003-1536-5291 Motta AA: http://orcid.org/0000-0003-0276-3362 Kalil J: http://orcid.org/0000-0001-8415-4274 Agondi RC: http://orcid.org/0000-0002-0503-5179 A Brazilian review article mentioned that physical weals are skin conditions resulting from the presence of mastocyte with decreased threshold for degranulation, induced by environmental physical triggering factors, whether located or diffuse, classic or atypical, acquired or familial, with or without the participation of IgE, with variable durations, that can worsen with stress and disappear spontaneously.(27) The clinical pictures of urticaria that, in general, involve complex pathogenesis, clinical progression and complex therapies, can, in some situations, be accompanied by the risk of death; in fact, systemic symptoms can occur during severe episodes. Finally, inducible urticaria has a deep impact on the life of patients, making it important that every healthcare professional have at least a basic knowledge about this disease.(28) A precise diagnosis is needed, in addition to quantification of the clinical expression, establishing specific drug therapy accompanied by a global prevention method, and whenever possible, of physical tolerance induced by desensitization process.(27) ❚❚DISCUSSION Solar urticaria, heat contact, aquagenic, contact, and vibratory urticaria are very rare, and data are limited, as well as those found in our study that observed 1.7% for solar urticaria, 0.8% for heat contact urticaria, and no patient was confirmed as aquagenic urticaria.(3) Another therapeutic option would be desensitization, which is indicated only for cold urticaria,(22,23) heat contact urticaria,(24) and solar urticaria.(25) Nonetheless, this induction of tolerance is not long lasting, and daily exposure to the triggering stimulus is necessary. In our study, no patient was submitted to desensitization protocols. Our study noted that the patients with CIndU presented with disease duration of 4.9 years, consistent with the literature.(3) In the national literature, we found no studies on the prevalence of inducible urticaria, in general. There einstein (São Paulo). 2020;18:1-8 6 Chronic inducible urticaria: confirmation through challenge tests and response to treatment is publication about a Brazilian study conducted by our service about isolated dermographic urticaria related to autoimmune diseases.(26) Additionally, two studies have shown that chronic urticaria seriously compromises the quality of life of patients, due to the debilitating symptoms, which can last for years.(23,25) In one of the studies, 59.8% of patients needed ongoing treatment with AH1.(10) Concomitance of chronic spontaneous urticaria was high and was associated with a longer time to the control of symptoms and to the higher frequency of cholinergic urticaria. ❚❚AUTHORS’ INFORMATION Pereira AR: http://orcid.org/0000-0003-1536-5291 Motta AA: http://orcid.org/0000-0003-0276-3362 Kalil J: http://orcid.org/0000-0001-8415-4274 Agondi RC: http://orcid.org/0000-0002-0503-5179 ❚❚REFERENCES 1. Zuberbier T, Aberer W, Asero R, Abdul Latiff AH, Baker D, Ballmer-Weber B, Bernstein JA, Bindslev-Jensen C, Brzoza Z, Buense Bedrikow R, Canonica GW, Church MK, Craig T, Danilycheva IV, Dressler C, Ensina LF, Giménez- Arnau A, Godse K, Gonçalo M, Grattan C, Hebert J, Hide M, Kaplan A, Kapp A, Katelaris CH, Kocatürk E, Kulthanan K, Larenas-Linnemann D, Leslie TA, Magerl M, Mathelier-Fusade P, Meshkova RY, Metz M, Nast A, Nettis E, Oude- Elberink H, Rosumeck S, Saini SS, Sánchez-Borges M, Schmid-Grendelmeier P, Staubach P, Sussman G, Toubi E, Vena GA, Vestergaard C, Wedi B, Werner RN, Zhao Z, Maurer M; Endorsed by the following societies: AAAAI, AAD, AAIITO, ACAAI, AEDV, APAAACI, ASBAI, ASCIA, BAD, BSACI, CDA, CMICA, CSACI, DDG, DDS, DGAKI, DSA, DST, EAACI, EIAS, EDF, EMBRN, ESCD, GA²LEN, IAACI, IADVL, JDA, NVvA, MSAI, ÖGDV, PSA, RAACI, SBD, SFD, SGAI, SGDV, SIAAIC, SIDeMaST, SPDV, TSD, UNBB, UNEV and WAO. The EAACI/GA²LEN/EDF/WAO guideline for the definition, classification, diagnosis and management of urticaria. Allergy. 2018;73(7):1393-414. 1. Zuberbier T, Aberer W, Asero R, Abdul Latiff AH, Baker D, Ballmer-Weber B, Bernstein JA, Bindslev-Jensen C, Brzoza Z, Buense Bedrikow R, Canonica GW, Church MK, Craig T, Danilycheva IV, Dressler C, Ensina LF, Giménez- Arnau A, Godse K, Gonçalo M, Grattan C, Hebert J, Hide M, Kaplan A, Kapp A, Katelaris CH, Kocatürk E, Kulthanan K, Larenas-Linnemann D, Leslie TA, Magerl M, Mathelier-Fusade P, Meshkova RY, Metz M, Nast A, Nettis E, Oude- Elberink H, Rosumeck S, Saini SS, Sánchez-Borges M, Schmid-Grendelmeier P, Staubach P, Sussman G, Toubi E, Vena GA, Vestergaard C, Wedi B, Werner RN, Zhao Z, Maurer M; Endorsed by the following societies: AAAAI, AAD, AAIITO, ACAAI, AEDV, APAAACI, ASBAI, ASCIA, BAD, BSACI, CDA, CMICA, CSACI, DDG, DDS, DGAKI, DSA, DST, EAACI, EIAS, EDF, EMBRN, ESCD, GA²LEN, IAACI, IADVL, JDA, NVvA, MSAI, ÖGDV, PSA, RAACI, SBD, SFD, SGAI, SGDV, SIAAIC, SIDeMaST, SPDV, TSD, UNBB, UNEV and WAO. The EAACI/GA²LEN/EDF/WAO guideline for the definition, classification, diagnosis and management of urticaria. Allergy. 2018;73(7):1393-414. 1. ❚❚CONCLUSION Confirmation of chronic inducible urticaria in patients with suspicion of the disease is necessary by means of specific and standardized challenge tests, since a large portion of the patients did not show this confirmation. The frequency of response to antihistamine was high. 10. Dias GA, Pires GV, Valle SO, Dortas Júnior SD, Levy S, França AT, et al. Impact of chronic urticaria on the quality of life of patients followed up at a university hospital. An Bras Dermatol. 2016;91(6):754-9. 11. Balp MM, Lopes da Silva N, Vietri J, Tian H, Ensina LF. Erratum to: The Burden of Chronic Urticaria from Brazilian Patients’ Perspective. Dermatol Ther (Heidelb). 2017;7(4):547. einstein (São Paulo). 2020;18:1-8 7 Pereira AR, Motta AA, Kalil J, Agondi RC 12. O’Donnell BF, Lawlor F, Simpson J, Morgan M, Greaves MW. The impact of chronic urticaria on the quality of life. Br J Dermatol. 1997;136(2):197-201. 20. Maurer M, Metz M, Brehler R, Hillen U, Jakob T, Mahler V, et al. Omalizumab treatment in patients with chronic inducible urticaria: a systematic review of published evidence. J Allergy Clin Immunol. 2018;141(2):638-49. 13. Black AK, Lawlor F, Greaves MW. Consensus meeting on the definition of physical urticarias and urticarial vasculitis. Clin Exp Dermatol. 1996;21(6):424-6. Review. 21. Dressler C, Werner RN, Eisert L, Zuberbier T, Nast A, Maurer M. Chronic inducible urticaria: a systematic review of treatment options. J Allergy Clin Immunol. 2018;141(5):1726-34. 14. Warin RP. A simple out-patient test for delayed pressure urticaria. Br J Dermatol. 1987;116(5):742-3. 22. Leigh IM, Ramsay CA, Calnan CD. Cold urticaria-‘desensitisation’. Trans St Johns Hosp Dermatol Soc. 1974;60(1):40-2. 15. Trevisonno J, Balram B, Netchiporouk E, Ben-Shoshan M. Physical urticaria: review on classification, triggers and management with special focus on prevalence including a meta-analysis. Postgrad Med. 2015;127(6):565-70. Review. 23. Black AK, Sibbald RG, Greaves MW. Cold urticaria treated by induction of tolerance. Lancet. 1979;2(8149):964. 16. Curto-Barredo L, Archilla LR, Vives GR, Pujol RM, Giménez-Arnau AM. Clinical features of chronic spontaneous urticaria that predict disease prognosis and refractoriness to standard treatment. Acta Derm Venereol. 2018;98(7):641-7. 24. Leigh IM, Ramsay CA. Localized heat urticaria treated by inducing tolerance to heat. Br J Dermatol. 1975;92(2):191-4. 25. Ramsay CA. Solar urticaria treatment by inducing tolerance to artificial radiation and natural light. Arch Dermatol. 1977;113(9):1222-5. 17. Kocatürk E, Can PK, Akbas PE, Copur M, Degirmentepe EN, Kızıltac K, et al. Management of chronic inducible urticaria according to the guidelines: a prospective controlled study. ❚❚CONCLUSION J Dermatol Sci. 2017;87(1):60-9. 26. Duarte RA, Castro RB, Almonfrey FB, Kalil J, Motta AA, Agondi RC. Características clínicas e autoimunidade nos pacientes com urticária dermográfica. Arq Asma Alerg Imunol. 2018;2(4):434-40. 18. Guillén-Aguinaga S, Jáuregui Presa I, Aguinaga-Ontoso E, Guillén-Grima F, Ferrer M. Updosing nonsedating antihistamines in patients with chronic spontaneous urticaria: a systematic review and meta-analysis. Br J Dermatol. 2016;175(6):1153-65. Review. 27. Geller M. Physical urticarias: mast cell disfunction. Preventive, diagnostic and therapeutical approach. einstein (São Paulo). 2007;5(3):273-80. Review. 19. Metz M, Altrichter S, Ardelean E, Kessler B, Krause K, Magerl M, et al. Anti- immunoglobulin E treatment of patients with recalcitrant physical urticaria. Int Arch Allergy Immunol. 2011;154(2):177-80. 28. Lima SO, Rodrigues CS, Camelo-Nunes IC, Solé D. Urticárias físicas: revisão. Rev Bras Alerg Imunopatol. 2008;31(6):220-6. Review. einstein (São Paulo). 2020;18:1-8 einstein (São Paulo). 2020;18:1-8 8
https://openalex.org/W2990116699	https://europepmc.org/articles/pmc6950393?pdf=render	English	null	EWI-2 Inhibits Cell–Cell Fusion at the HIV-1 Virological Presynapse	Viruses	2,019	cc-by	19,784	Received: 14 October 2019; Accepted: 16 November 2019; Published: 20 November 2019 Abstract: Cell-to-cell transfer of virus particles at the Env-dependent virological synapse (VS) is a highly eﬃcient mode of HIV-1 transmission. While cell–cell fusion could be triggered at the VS, leading to the formation of syncytia and preventing exponential growth of the infected cell population, this is strongly inhibited by both viral (Gag) and host (ezrin and tetraspanins) proteins. p p g y y g p p Here, we identify EWI-2, a protein that was previously shown to associate with ezrin and tetraspanins, as a host factor that contributes to the inhibition of Env-mediated cell–cell fusion. Using quantitative ﬂuorescence microscopy, shRNA knockdowns, and cell–cell fusion assays, we show that EWI-2 accumulates at the presynaptic terminal (i.e., the producer cell side of the VS), where it contributes to the fusion-preventing activities of the other viral and cellular components. We also ﬁnd that EWI-2, like tetraspanins, is downregulated upon HIV-1 infection, most likely by Vpu. Despite the strong inhibition of fusion at the VS, T cell-based syncytia do form in vivo and in physiologically relevant culture systems, but they remain small. In regard to that, we demonstrate that EWI-2 and CD81 levels are restored on the surface of syncytia, where they (presumably) continue to act as fusion inhibitors. This study documents a new role for EWI-2 as an inhibitor of HIV-1-induced cell–cell fusion and provides novel insight into how syncytia are prevented from fusing indeﬁnitely. Keywords: EWI-2; IGSF8; tetraspanin; HIV; cell–cell fusion; virological synapse; T cell; syncytia viruses viruses www.mdpi.com/journal/viruses EWI-2 Inhibits Cell–Cell Fusion at the HIV-1 Virological Presynapse Emily E. Whitaker 1,2, Nicholas J. Matheson 3,4, Sarah Perlee 1,† , Phillip B. Munson 2,5,‡, Menelaos Symeonides 1,2,§ and Markus Thali 1,2,,§ 1 Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA; emily.e.whitaker@uvm.edu (E.E.W.); perlees@sloankettering.edu (S.P.); msymeoni@uvm.edu (M.S.) 1 Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA; emily.e.whitaker@uvm.edu (E.E.W.); perlees@sloankettering.edu (S.P.); msymeoni@uvm.edu (M.S.) 2 Graduate Program in Cellular, Molecular, and Biomedical Sciences, University of Vermont, B li t VT 05405 USA 1@ h h d d 1 Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405 emily.e.whitaker@uvm.edu (E.E.W.); perlees@sloankettering.edu (S.P.); msymeoni@uvm.edu (M.S.) p gy y g emily.e.whitaker@uvm.edu (E.E.W.); perlees@sloankettering.edu (S.P.); msymeoni@uvm.edu (M.S.) 2 Graduate Program in Cellular, Molecular, and Biomedical Sciences, University of Vermont, Burlington, VT 05405, USA; pmunson1@mgh.harvard.edu y ( ); p g ( ); y ( ) 2 Graduate Program in Cellular, Molecular, and Biomedical Sciences, University of Vermont, Burlington, VT 05405, USA; pmunson1@mgh.harvard.edu 3 Department of Medicine, University of Cambridge, Cambridge CB2 0QQ, UK; njm25@cam.ac.uk 4 Cambridge Institute for Therapeutic Immunology and Infectious Disease (CITIID), University of Cambridge, Cambridge CB2 0AW, UK 5 Department of Pathology and Laboratory Medicine, University of Vermont, Burlington, VT 05405, USA Correspondence: markus.thali@uvm.edu † Current aﬃliation: Memorial Sloan Kettering Cancer Center, Louis V. Gerstner, Jr. † Current aﬃliation: Memorial Sloan Kettering Cancer Center, Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, New York, NY 10065, USA. † Current aﬃliation: Memorial Sloan Kettering Cancer Center, Louis V. Graduate School of Biomedical Sciences, New York, NY 10065, USA. Graduate School of Biomedical Sciences, New York, NY 10065, USA. ‡ Current aﬃliation: Massachusetts General Hospital, Cutaneous Biology Research Center, Ch l MA 02129 USA ‡ Current aﬃliation: Massachusetts General Hospital, Cutaneous Biology Research Center, Charlestown, MA 02129, USA. § Co-senior authors. Received: 14 October 2019; Accepted: 16 November 2019; Published: 20 November 2019 Received: 14 October 2019; Accepted: 16 November 2019; Published: 20 November 2019 viruses viruses 1. Introduction HIV-1 spreads between T cells primarily through two modes of transmission: the release of cell-free virus particles followed by their uptake by (more or less distantly located) cells expressing the viral receptor/co-receptor, and the cell-to-cell transmission of particles to an adjacent cell via the virological synapse (VS), i.e., when infected and uninfected cells transiently align. The formation of the HIV-1 VS is initiated by the viral envelope glycoprotein (Env) on the surface of productively infected cells binding to its receptor, CD4, on target T cells [1] and is followed by the polarization of Gag at Viruses 2019, 11, 1082; doi:10.3390/v11121082 www.mdpi.com/journal/viruses 2 of 23 Viruses 2019, 11, 1082 the cell–cell contact site [1,2]. Virus particles are then released in high concentrations towards the target cell [3], facilitating eﬃcient infection while also possibly shielding virus particles from some neutralizing antibodies ([4] and recently reviewed in [5]). Indeed, as demonstrated in a recent study using physiologically relevant cell culture systems [6], it is possible that virus that is not released in close proximity to a target cell is rapidly inactivated, emphasizing the importance of VS-mediated transmission. However, given that Env is fusogenic at neutral pH, it would seem likely at ﬁrst that VS-mediated contacts should frequently result in cell–cell fusion, thus forming a multinucleated infected cell (syncytium). While we now know that small, T cell-based syncytia arise early in HIV-1 infection and can spread the virus by cell–cell contact [7–12], the majority of infected T cells observed in lymphoid tissue are mononucleated, documenting that most HIV-1 VSs ultimately result in complete cell separation and the generation of a new, productively infected cell. This is likely due to tight regulation at the VS that acts to prevent excessive syncytium formation (reviewed in [13,14]). Multiple independent studies have identiﬁed viral and host functions which, together, prevent excessive HIV-1-induced cell–cell fusion at the VS. Firstly, Env is rapidly downregulated from the surface of infected cells in the absence of Gag [15,16]. Secondly, upon Gag multimerization at the plasma membrane, Env is trapped by immature Gag through Env’s cytoplasmic tail and maintained in a poorly fusogenic state [17]. This trapping by Gag ends only after Env’s incorporation into virus particles when Gag precursor gets cleaved, i.e., upon maturation [18–21]. 1. Introduction The residual fusion activity of Gag-trapped Env on infected cells has been shown to be inhibited by several host membrane proteins that accumulate at the producer cell side of the VS, including tetraspanins and phosphorylated ezrin (p-ezrin) [22–24]. Tetraspanins inhibit HIV-1-induced cell–cell fusion at a post-hemifusion stage [23], while ezrin is implicated in F-actin organization and the recruitment of the tetraspanin CD81 to the VS [24]. It remains unclear how and whether these protein functions are coordinated, though based on other cell–cell fusion regulation paradigms (discussed below), additional host proteins are likely required to mediate the eﬃcient inhibition of HIV-1-induced fusion by tetraspanins and ezrin. EWI-F (CD9P-1/FPRP) is an immunoglobulin superfamily (IgSF) member and partner of tetraspanins CD9 and CD81 [25]. EWI-F was shown to be a potent inhibitor of cell–cell fusion in myoblasts, where EWI-F knockout resulted in more frequent fusion than CD9/CD81 double knockout [26]. However, EWI-F is poorly expressed in T cells [27], the primary host cell type for HIV-1. A related protein, EWI-2 (IGSF8/PGRL) [28,29], which also associates with tetraspanins and is expressed in T cells [25,27], has been documented to play a role in hepatitis C virus (HCV) entry [30,31] and T cell immunological synapse (IS) formation [32]. The latter study also suggested that EWI-2 has a yet undetermined involvement in HIV-1 particle production [32]. Furthermore, both EWI-F and EWI-2 interact with ezrin to organize the cytoskeleton in concert with tetraspanins [27]. EWI-2 thus lies at the nexus of tetraspanins, ezrin, and the actin cytoskeleton (which can also inhibit cell–cell fusion) [33]. 2.3. Plasmids and Virus Strains Respectively, pcDNA3, pCDNA3.1, and pCMV SPORT6 (Invitrogen, Carlsbad, CA, USA) were vectors for EWI-2, CD81, and L6 overexpression (EWI-2 was a kind gift from Dr. Eric Rubinstein; Université Paris-Sud, Villejuif, France). Proviral plasmids pNL4-3 and pNL4-3 ∆Env (KFS) were kind gifts from Dr. Eric Freed (National Cancer Institute, Frederick, MD, USA) [47]. NL4-3-derived ﬂuorescent protein-tagged proviral plasmids pNL-sfGI, pNL-sfGI ∆Env, pNL-CI, and pNL-CI ∆Env [10] were kind gifts from Dr. Benjamin Chen (Mount Sinai School of Medicine, New York, NY, USA). Vesicular stomatitis virus glycoprotein (VSV-G) was used to pseudotype viral stocks produced in HEK 293T cells. The lentiviral vector FG12 [48], previously modiﬁed to include a puromycin resistance cassette [24], was further modiﬁed to remove the GFP reporter cassette by digestion with AfeI and PshAI and subsequent blunt-end relegation. 2.2. Antibodies Mouse monoclonal antibody (mAb) to EWI-2 (8A12) was a kind gift from Dr. Eric Rubinstein [25]. Mouse mAb to HIV-1 p24 (AG3.0) was obtained through the NIH AIDS Reagent Program (Germantown, MD, USA), Division of AIDS, NIAID, NIH, from Dr. Jonathan Allan [46]. Rabbit antiserum to HIV-1 p6 was a kind gift from David E. Ott. Rabbit polyclonal antibody (pAb) to HIV-1 p24 was obtained from Advanced Biotechnologies (Eldersburg, MD, USA, Cat. #13-203-000). Secondary antibodies were as follows: Alexa Fluor 488-conjugated donkey pAb to mouse IgG (#A21202), Alexa Fluor 488-conjugated donkey pAb to rabbit IgG (Cat. #A21206), Alexa Fluor 594-conjugated donkey pAb to mouse IgG (Cat. #R37115), Alexa Fluor 594-conjugated donkey pAb to rabbit IgG (Cat. #A21207), Alexa Fluor 647-conjugated donkey pAb to mouse IgG (Cat. #A31571), and Alexa Fluor 647-conjugated goat pAb to mouse IgG (Cat. #A21235), all from Invitrogen (Carlsbad, CA, USA). Zenon labeling of primary antibodies with either Alexa Fluor 488 or Alexa Fluor 594 was carried out using Zenon Labeling Kits according to the manufacturer’s instructions (Molecular Probes, Eugene, OR, USA, Cat. #Z25002 and #Z25007). 2.1. Cell Lines and Cell Culture The following cells were obtained through the NIH AIDS Reagent Program (Germantown, MD, USA), Division of AIDS, NIAID, NIH: HeLa cells from Dr. Richard Axel [34], TZM-bl cells from Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme Inc. [35–39], CEM.NKR CCR5+Luc+ (CEM-luc) cells from Dr. John Moore and Dr. Catherine Spenlehauer [40,41], CEM-T4 cells from Dr. J.P. Jacobs [42], and CEM-SS cells from Dr. Peter L. Nara [34,43,44]. HEK 293T, HeLa, and TZM-bl cells were maintained in Dulbecco’s Modiﬁcation of Eagle’s Medium (DMEM) (Corning, Corning, NY, USA, Cat. #10-017-CV) containing 10% fetal bovine serum (FBS; Corning, Corning, NY, USA, Cat. #35-010-CV) and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin; Invitrogen, Carlsbad, CA, USA). CEM-luc cells were maintained in RPMI 1640 medium (Corning, Corning, NY, USA, Cat. #10-104-CV) supplemented with 10% FBS and 0.8 mg/mL geneticin 3 of 23 Viruses 2019, 11, 1082 sulfate (G418). CEM2n, a kind gift from R. Harris [45], and CEM-SS cells were maintained in RPMI medium supplemented with 10% FBS and antibiotics. Human primary blood mononuclear cells (PBMCs) were isolated as buﬀy coats from the whole blood of healthy donors by Ficoll density centrifugation. CD4+ T cells were enriched from PBMCs by negative selection using the MACS CD4+ T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA, USA, Cat. #130-096-533) or the EasySep Human CD4+ T Cell Isolation Kit (STEMCELL Technologies, Vancouver, BC, Canada, Cat. #17952) according to manufacturer’s instructions. Primary CD4+ T cells were activated in RPMI containing 10% FBS, 50 units/mL IL-2, antibiotics, and 5 µg/mL phytohemagglutinin. After 48 h of activation, cells were washed and subsequently maintained and expanded in the same medium but without phytohemagglutinin. Cells were used for infections at 4–7 days post isolation. 2.4. Virus Stocks and Infections VSV-G-pseudotyped virus stocks of NL4-3, NL4-3 ∆Env, NL-sfGI, NL-CI, and NL-CI ∆Env were produced in HEK 293T cells transfected with the proviral plasmid and pVSV-G (at 17:3 ratio) using calcium phosphate precipitation. For shRNA encoding lentiviruses, shEWI-2 and shScramble, stocks were produced in HEK 293T cells transfected with FG12-shRNA vector, ∆R8.2 packaging vector, and pVSV-G (at a ratio of 3:7:1. Supernatants were harvested 2 days after transfection, cleared by centrifugation at 2000 rcf for 10 min, ﬁltered, and stored at −80 ◦C. To infect CEM2n cells by spinoculation, two million cells were incubated with RPMI/10% FBS containing 90 µL of virus stock (resulting in ~3% of the cells being infected) or medium alone (for uninfected controls), for 20 min at 37 ◦C, followed by centrifugation at 1200 rcf for 2 h at 37 ◦C. Cell pellets were allowed to recover at 37 ◦C for 15 min, centrifuged at 300 rcf for 2 min, and resuspended Viruses 2019, 11, 1082 4 of 23 in fresh RPMI/10% FBS. Cells were incubated at 37 ◦C, the medium was refreshed 2 days post infection, and the cells were used 1 day later for all subsequent experiments. in fresh RPMI/10% FBS. Cells were incubated at 37 ◦C, the medium was refreshed 2 days post infection, and the cells were used 1 day later for all subsequent experiments. To infect primary CD4+ T cells, 1 or 2 million cells were incubated in RPMI/10% FBS/IL-2 containing 200 or 400 µL of virus, respectively, and spinoculated as described above. Cells were resuspended in fresh RPMI/10% FBS/PS/IL-2 and incubated at 37 ◦C/5% CO2. Cells were used 2-3 days post infection for all subsequent experiments. To infect CEM-SS cells by shaking, one or two million cells suspended in CO2-independent medium (Gibco, Grand Island, NY, USA, Cat #18045088) supplemented with 10% FBS were mixed with VSV-G-pseudotyped virus stocks and shaken at 220 rpm for 2 h at 37 ◦C. Cells were then washed and plated in fresh RPMI/10% FBS, and used for experiments as described. For CEM-SS infection by spinoculation, the procedure was performed as described above with some modiﬁcations; one or two million cells were incubated in RPMI/10% FBS containing 40–50 µL (analyzing surface expression and post-synapse enrichment, respectively) of virus stock or medium alone (for uninfected controls). Following spinoculation, cells were incubated at 37 ◦C for 2 days before being used for subsequent experiments. 2.5. Imaging and Quantiﬁcation of EWI-2 Accumulation at the VS CEM-SS and primary CD4+ T cells were infected by shaking or spinoculation, respectively, with VSV-G-pseudotyped WT or ∆Env virus then treated as follows: For CEM-SS cells, two days post infection, uninfected CEM-SS target cells were labeled with CMAC (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions, mixed with infected cells at a 1:1 or 1:2 ratio (infected:target), seeded onto the microwell of a 35 mm glass-bottom dish (MatTek Corporation, Ashland, MA, USA, Cat. #P35G-1.5-14-C) coated with poly-L-Lysine (Trevigen, Gaithersburg, MD, USA), and incubated at 37◦C for 3 to 4.5 h. Cells were then chilled on ice and surface-labeled with 1:200 mouse anti-EWI-2 mAb in RPMI/10% FBS for 45 min at 4 ◦C. Surface-labeled cells were ﬁxed with 4% PFA in PBS at 4 ◦C for 10 min, and blocked and permeabilized overnight with 1% BSA and 0.2% Triton X-100 in PBS (block/perm buﬀer). All CEM-SS conditions were labeled with Alexa Fluor 647-conjugated anti-mouse secondary pAb in block/perm buﬀer at 1:500 dilution. Cells were subsequently stained with Alexa Fluor 594 Zenon-labeled anti-p24 AG3.0 mouse mAb and ﬁxed again with 4% PFA in PBS. Cells were kept in PBS for imaging. p g g For primary cells, uninfected cells were mixed with infected cells at a 1:1 ratio (infected:target), seeded onto 8-well glass-bottom plates (CellVis, Mountain View, CA, USA, Cat. #C8-1.5H-N) coated with 1:10 poly-L-Lysine in double-distilled water (ddH2O), and incubated for 2 to 2.5 h at 37 ◦C. Cells were surface-labeled for EWI-2 and ﬁxed as above, then blocked and permeabilized with block/perm buﬀer for 10 min. Cells were then labeled with a mixture of rabbit anti-p24 and anti-p6 antibodies, each at 1:1000 dilution, in PBS with 1% BSA (block) for 45 min. Subsequently, cells were labeled with Alexa Fluor-conjugated secondary pAbs as indicated. Cells were kept in PBS for imaging. To visualize the only producer cell-associated EWI-2 at the VS, 10,000 target TZM-bl cells (which have nearly-undetectable levels of EWI-2) were seeded onto 8-well glass-bottom plates coated with 1:10 poly-L-Lysine in ddH2O. The next day, those TZM-bl cells were labeled with CMAC at 1:250 dilution in serum-free DMEM, and then co-cultured with 150,000 CEM-SS cells (either uninfected or infected with NL-CI or NL-CI ∆Env 2 days prior as described above) per well for 2.5 h at 37 ◦C in RPMI/10% FBS. The cells were then surface-labeled with 1:200 mouse anti-EWI-2 mAb in RPMI/10% FBS on ice for 45 min. 2.5. Imaging and Quantiﬁcation of EWI-2 Accumulation at the VS Cells were subsequently ﬁxed with 4% PFA in PBS and permeabilized with block/perm for 10 min. After permeabilization, the cells were labeled using a mixture of rabbit anti-p24 and anti-p6 antibodies, each at 1:1000 dilution, in block for 45 min. Cells were subsequently labeled using Alexa Fluor-conjugated secondary pAbs (anti-mouse-Alexa Fluor 647 and anti-rabbit-Alexa Fluor 488) each at 1:500 in block for 45 min. Cells were kept in PBS for imaging. For primary cells, uninfected cells were mixed with infected cells at a 1:1 ratio (infected:target), seeded onto 8-well glass-bottom plates (CellVis, Mountain View, CA, USA, Cat. #C8-1.5H-N) coated with 1:10 poly-L-Lysine in double-distilled water (ddH2O), and incubated for 2 to 2.5 h at 37 ◦C. Cells were surface-labeled for EWI-2 and ﬁxed as above, then blocked and permeabilized with block/perm buﬀer for 10 min. Cells were then labeled with a mixture of rabbit anti-p24 and anti-p6 antibodies, each at 1:1000 dilution, in PBS with 1% BSA (block) for 45 min. Subsequently, cells were labeled with Alexa Fluor-conjugated secondary pAbs as indicated. Cells were kept in PBS for imaging. To visualize the only producer cell-associated EWI-2 at the VS, 10,000 target TZM-bl cells (which have nearly-undetectable levels of EWI-2) were seeded onto 8-well glass-bottom plates coated with 1:10 poly-L-Lysine in ddH2O. The next day, those TZM-bl cells were labeled with CMAC at 1:250 dilution in serum-free DMEM, and then co-cultured with 150,000 CEM-SS cells (either uninfected or infected with NL-CI or NL-CI ∆Env 2 days prior as described above) per well for 2.5 h at 37 ◦C in RPMI/10% FBS. The cells were then surface-labeled with 1:200 mouse anti-EWI-2 mAb in RPMI/10% FBS on ice for 45 min. Cells were subsequently ﬁxed with 4% PFA in PBS and permeabilized with block/perm for 10 min. After permeabilization, the cells were labeled using a mixture of rabbit anti-p24 and anti-p6 antibodies, each at 1:1000 dilution, in block for 45 min. Cells were subsequently labeled using Alexa Fluor-conjugated secondary pAbs (anti-mouse-Alexa Fluor 647 and anti-rabbit-Alexa Fluor 488) each at 1:500 in block for 45 min. Cells were kept in PBS for imaging. To visualize only target cell-associated EWI-2 at the VS, HeLa producer cells (which have nearly-undetectable levels of EWI-2) were plated (10,000 cells per well) in 8-well glass-bottom plates 5 of 23 Viruses 2019, 11, 1082 coated with 1:10 poly-L-Lysine in ddH2O. 2.5. Imaging and Quantiﬁcation of EWI-2 Accumulation at the VS Twenty-four hours later, cells were transfected with NL-sfGI, NL-sfGI ∆Env, or empty vector, using FuGENE6 transfection reagent at a ratio of 3:1 (FuGENE6:DNA) according to manufacturer’s instructions (Promega, Madison, WI, USA, Cat. #E2691). Twenty-four hours post-transfection, 100,000–150,000 uninfected CEM-SS cells (labeled with CMAC at a 1:250 dilution in serum-free RPMI) were added to form VSs with provirus-transfected HeLa cells. After 2-2.5 h of coculture, cells were surface-labeled with 1:200 mouse anti-EWI-2 mAb in RPMI/10% FBS for 45 min at 4 ◦C. Surface-labeled cells were ﬁxed with 4% PFA in PBS at 4 ◦C for 10 min, and then incubated with block/perm for 10 min, before labeling with a mixture of rabbit anti-p24 and anti-p6 antibodies, each at 1:1000 dilution, in block for 45 min. Subsequently, cells were labeled with secondary pAbs (anti-mouse-Alexa Fluor 647 and anti-rabbit-Alexa Fluor 594), each at 1:500 in block. Cells were kept in PBS for imaging. Images were acquired on a DeltaVision epiﬂuorescence microscope (GE/Applied Precision, Issaquah, WA, USA) with an Olympus IX-70 base using an Olympus 60× PlanApo 1.42 NA objective and equipped with a CoolSNAP HQ CCD camera (Photometrics, Tucson, AZ, USA). Images were imported into Fiji Version 2.0.0-rc-69/1.52p [49] for analysis following deconvolution and cropping using Softworx software (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The VS was identiﬁed using the Gag channel and the level of EWI-2 accumulation was determined by measuring its signal intensity at the VS. For ∆Env controls, cell–cell contacts were identiﬁed using the diﬀerential interference contrast (DIC) channel and treated analogous to a VS. The EWI-2-associated signal intensity at non-contact sites was determined by manually outlining the surface of the cell, excluding any regions that were in contact with an adjacent cell, and calculating the mean EWI-2 intensity within the selected area. To determine the level of enrichment at the VS (or cell–cell contact for ∆Env controls), an “unbiased” approach was applied to account for the EWI-2 signal contributed by both the target and producer cell at each VS/contact. Enrichment was calculated as the EWI-2 signal intensity at the VS/contact divided by the sum of the EWI-2 signal at non-contact sites of the producer and target cell in that particular VS/contact. 2.7. Determining Surface Levels of EWI-2 by Microscopy To compare EWI-2 surface expression between infected and uninfected cells, CEM-SS, CEM2n cells, and primary CD4+ T cells were infected with VSV-G-pseudotyped NL-sfGI as described above. Two to three days post infection, 3 × 105 infected cells were plated onto each well of 8-well glass-bottom plates coated with 1:10 poly-L-Lysine in ddH2O. Two additional wells were used for uninfected controls. After 2 h of incubation at 37 ◦C, the medium was replaced with ice cold RPMI/10% FBS containing mouse anti-EWI-2 mAb at 1:200 dilution for surface labeling, and incubated for 45 min at 4 ◦C. Following the primary antibody incubation, cells were washed with RPMI/10% FBS and ﬁxed with 4% PFA in PBS for 10 min at 4 ◦C, blocked and permeabilized with PBS containing 1% BSA and 100 µg/mL digitonin for 10 min, and incubated with the indicated secondary antibody in block for 45 min at room temperature. Cells were washed with block and imaged in PBS. At least 50 ﬁelds containing infected cells were selected for each biological replicate and imaged, deconvolved, and cropped using the DeltaVision microscope and Softworx software described above. After deconvolution, Fiji was used to manually select the cell surface at the midline of each cell and the mean intensity of EWI-2-associated signal was quantiﬁed and subsequently subtracted by the mean intensity of an area that did not contain cells. Cell–cell contact sites were excluded from the quantiﬁcation. Background subtracted intensity values of all cells were normalized to the average surface associated intensity of the entire uninfected cell population, internal controls contained in the same wells as infected cells, contained within respective biological replicates. This normalization allowed for the direct comparison of surface expression trends between biological replicates that accounts for potential variation in protein labeling eﬃciency between replicates. The virus-associated ﬂuorescent reporter channel was used to segregate measurements into uninfected and infected. The data shown in Figure 3B are pooled from 2–3 independent biological replicates, each consisting of two technical replicates, all of which were sampled randomly until a minimum of 50 infected cells were quantiﬁed. To compare EWI-2 surface expression levels between mononucleated infected cells and HIV-1-induced syncytia, primary CD4+ T cells were infected with VSV-G-pseudotyped virus as described above. Three days post infection, 3 × 105 infected cells were plated onto each well of 8-well glass-bottom plates coated with 1:10 poly-L-Lysine in ddH2O alongside two wells of uninfected cells as controls. 2.6. Proteomic Analysis of EWI-2 Levels in HIV-1 Infected Cells To identify HIV-1-dependent changes in the abundance of total EWI-2, we re-analysed data from two previous studies [50,51]. In brief, primary human CD4+ T cells were infected with pNL4-3-∆Env-Nef-P2A-SBP-∆LNGFR (HIV-AFMACS) at MOI≤0.5, enriched by antibody-free magnetic cell sorting (AFMACS) [52] and analysed 48 h after infection [51]. CEM-T4 T cells were infected with pNL4-3-∆Env-EGFP at MOI=1.5 and analysed 48 h after infection [50]. TMT-labeled tryptic peptides from whole cell lysates were subjected to oﬀ-line high pH reversed-phase (HpRP)-HPLC fractionation and analysed using an Orbitrap Fusion Tribrid mass spectrometer (ThermoFisher Scientiﬁc, Waltham, MA, USA) coupled to a Dionex UltiMate 3000 UHPLC (Thermo Scientiﬁc, Waltham, MA, USA). Details of sample processing and data analysis have been previously described [50,51] and proteomic data from primary human CD4+ T cells are available from the ProteomeX-change Consortium using dataset identiﬁer PXD012263 (http://proteomecentral.proteomexchange.org). To characterise HIV-1-dependent changes in the abundance of plasma membrane EWI-2, we re-analysed data from a previous study [53]. In brief, for the TMT-based time course experiment, CEM-T4 T cells were infected with pNL4-3-∆Env-EGFP at MOI=10 and analysed at the indicated time points after infection. For the SILAC-based single time point experiments, cells were pre-labeled with light, medium or heavy lysine and arginine and either infected with WT or Vpu-/Nef-deﬁcient pNL4-3-∆Env-EGFP at MOI=10 and analysed 72 h after infection, or transduced with GFP or Vpu/Nef and selected with puromycin. Sialylated cell surface glycoproteins were enriched by selective aminooxy-biotinylation followed by immunoaﬃnity puriﬁcation using streptavidin-conjugated beads (plasma membrane proﬁling). Tryptic peptides were labeled with TMT reagents (time course experiment only), subjected to oﬀ-line High pH Reversed-Phase (HpRP)-HPLC fractionation and analysed using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientiﬁc, Waltham, MA, 6 of 23 Viruses 2019, 11, 1082 USA) coupled to a Dionex UltiMate 3000 UHPLC (Thermo Scientiﬁc, Waltham, MA, USA). Details of sample processing and data analysis have been previously described [53] and time course proteomic data are available from the ProteomeX-change Consortium using dataset identiﬁer PXD002934 (http://proteomecentral.proteomexchange.org). 2.7. Determining Surface Levels of EWI-2 by Microscopy Cells were incubated at 37 ◦C for 2 h and surface labeled as described above using either mouse anti-EWI-2 or mouse anti-CD81 mAb at 1:200 or 1:100, respectively. Samples were ﬁxed, permeabilized, and labeled with appropriate AlexaFluor conjugated antibodies and DAPI as described above. Cells were imaged in PBS and at least 50 ﬁelds containing 10–20 cells each and containing at least some infected cells with multinucleated appearance (determined by DAPI and GFP signal) were selected for each biological replicate and imaged, deconvolved, and cropped as described above. Fiji was then used to analyze the surface expression of each protein of interest as described above. The virus-associated ﬂuorescent reporter channel (GFP) was used to segregate measurements into infected and uninfected populations, and nuclear staining (DAPI) was used to further segregate infected cells into mononucleated and multinucleated infected cells. The EWI-2/CD81 channel was not viewed at all during imaging and ﬁeld selection, or throughout image processing. The data shown in Figure 6 are pooled from 2–3 biological replicates, with two technical replicates each, all of which were sampled randomly until a minimum of 15 syncytia per biological replicate were quantiﬁed. 2.9. HeLa-based HIV-1-Induced Cell–Cell Fusion Assay First, 50,000 HeLa cells were plated in each well of a 24-well plate and, the next day, transfected (using FuGENE6; see Section 2.5) in duplicate with 100 ng of either pNL-sfGI or pNL-sfGI ∆Env along with 500 ng total expression vector carrying CD81 or EWI-2. L6, a tetraspanin-like protein that does not inhibit cell–cell fusion, was co-transfected instead of CD81 or EWI-2 as a positive control for maximum fusion activity, For dose response assays, 125, 250, or 500 ng of either EWI-2 or CD81 plasmid was ”stuﬀed” with L6 expression plasmid to maintain 500 ng of total protein expression plasmid in each condition. No cytotoxicity was observed upon transfection for any of the experimental conditions. Then, 24 h post-transfection, producer HeLa cells were co-cultured with 106 TZM-bl target cells (which, upon producer-target cell fusion, express ﬁreﬂy luciferase under control of the HIV-1 LTR) per well for 3 h before unattached target cells were washed oﬀand the medium was refreshed. 14-18 h later, cells were lysed for at least 30 min on ice using 1% Triton X-100, 2 mM EDTA, 50 mM Tris-HCl, 200 mM NaCl, with 1% protease inhibitor cocktail. Lysates were precleared by centrifugation at 20,000 rcf for 5 min at 4 ◦C and stored at –80 ◦C until use for luciferase activity assays. Note that the timepoints used here ensure that there is not enough time for the development of any luciferase signal resulting from productive infection of target TZM-bl cells through virus transmission and that only cell–cell fusion contributes to the luciferase activity measured. Each lysate was incubated with an equal volume of ﬁreﬂy luciferase reagent (Promega, Madison, WI, USA, Cat. #E1500) for 1 min in a 96-well white-walled plate (ThermoFisher Scientiﬁc, Waltham, MA, USA, Cat. #7571) before collecting luminescence signal intensity on a microplate reader (BioTek Synergy 2, BioTek, Winooski, VT, USA). Background luminescence was determined using a lysis buﬀer blank and subtracted from all experimental samples. Luminescence intensity was used as a quantitative measurement of relative HeLa-TZM syncytium formation against the non-fusogenic (therefore incapable of forming syncytia) ∆Env control by dividing each value by the ∆env value (which eﬀectively corresponds to any leaky expression of luciferase in TZM-bl cells as no cell–cell fusion occurs at all in this condition). To then determine relative fusion activity of cells transfected with EWI-2 and CD81, those values were normalized to the L6 condition. 2.9. HeLa-based HIV-1-Induced Cell–Cell Fusion Assay Normalized fusion is therefore the fold diﬀerence of cell–cell fusion activity taking place when cells were co-transfected with the indicated amount of either CD81 or EWI-2 plasmid, compared to the activity taking place when cells were co-transfected with L6. The data shown are the collection of four independent biological replicates. 2.8. Determining Surface EWI-2 Signal on Infected Cells by Flow Cytometry .8. Determining Surface EWI-2 Signal on Infected Cells by Flow Cytometry 2.8. Determining Surface EWI-2 Signal on Infected Cells by Flow Cytometry CEM2n cells infected as described above were harvested after three days and incubated in cold PBS with 5 mM EDTA for 15 min (3.0 × 105 cells/tube). Cells were pelleted at 400 rcf for 7 min at 4 ◦C 7 of 23 Viruses 2019, 11, 1082 and resuspended in cold RMPI/10% FBS containing mouse anti-EWI-2 mAb at 1:200 dilution. After a 45 min incubation at 4 ◦C, cells were washed with cold RPMI/10% FBS and resuspended in ice cold PBS with 5 mM EDTA. To ﬁx, an equal volume of PBS with 8% PFA was added and samples were incubated on ice for 10 min. Cells were washed and stained with Alexa Fluor 594-conjugated secondary antibody at 1:500 in block for 45 min at room temperature, before being washed, resuspended in PBS, and analyzed using a BD LSRII ﬂow cytometer. Data were analyzed using FlowJo V10 (Becton, Dickinson & Company, Franklin Lakes, NJ, USA). Samples were gated for infected and uninfected populations by GFP expression. EWI-2high and EWI-2low gates were set based in part on controls lacking primary antibody, and in part by adjusting the gates to reﬂect the number of uninfected EWI-2high cells as measured by microscopy. The data shown are the collection of three independent biological replicates, each consisting of two technical replicates. 2.10. Establishment of EWI-2 Knockdown CEM-SS Cells The shRNA-encoding sequences targeting either EWI-2 (modiﬁed from previously described EWI-2-targeting siRNA [27] or a scrambled control, were introduced to the lentiviral vector FG12 (as described in 2.3) using oligos containing shRNA sequences, a loop sequence, and an AgeI site, ﬂanked by BbsI and XhoI restriction site overhangs, as previously described [24], (EWI-2 sense, 5′- ACCGGGGCTTCGAAAACGGTGATCTTCAAGAGAGATCACCGTTTTCGAAGCCCTTTTTTACCGG TC-3′, and anti-sense, 5′- TCGAGACCGGTAAAAAAGGGCTTCGAAAACGGTGATCTCTCTTGAAGA 8 of 23 Viruses 2019, 11, 1082 TCACCGTTTTCGAAGCCC-3′; scramble sense, 5′- ACCGGGCAGATGCGTCCAGTTAGATTCAAGAG ATCTAACTGGACGCATCTGCCTTTTTTACCGGTC-3′, and anti-sense, 5′- TCGAGACCGGT AAAAAAGGCAGATGCGTCCAGTTAGATCTCTTGAATCTAACTGGACGCATCTGCC-3′). A PolII promoter was ﬁrst obtained by ligating the oligo with PBS-hU6 digested with BbsI and XhoI restriction endonucleases (New England BioLabs, Ipswich, MA, USA). The PolII-shRNA constructs were obtained by digesting the resulting PBS-hU6 vector with XbaI and XhoI, and the insert was subsequently ligated into the FG12 vector digested with the same enzymes. g y VSV-G pseudotyped FG12-shRNA lentiviruses were used to transduce CEM-SS cells by spinoculating one million cells with 500 µL of lentiviral supernatant (either shEWI-2 or shScramble). Cells were incubated at 37 ◦C for 2 days in RPMI/10% FBS and positively transduced cells were then selected for puromycin resistance by supplementing the media with 0.5 µg/mL of puromycin for 8 days. Subsequently, shEWI-2 and shScramble CEM-SS cells were maintained in RPMI/10% FBS/0.25 µg/mL puromycin. µg/ p y EWI-2 knockdown was analyzed by ﬂow cytometry and microscopy. For ﬂow cytometry analysis, 3.0 × 10 5 shScramble and shEWI-2 cells, alongside parental CEM-SS controls, were pelleted at 400 rcf for 7 min, resuspended in 1:1000 Live/Dead Fixable Near-IR stain (Invitrogen, Carlsbad, CA, USA, Cat. #L10119) in PBS for 30–45 min, washed with RPMI/10% FBS and ﬁxed for 10 min in 4% PFA in PBS by resuspending the cells in PBS and then adding an equal volume of 8% PFA in PBS. Fixed samples were washed with 1 mL of PBS, blocked and permeabilized in 100 µL of block/perm buﬀer for 10 min, and washed with PBS containing 1% BSA. EWI-2 was labeled using mAb 8A12 diluted 1:200 in block for 45 min, washed with block, and stained with Alexa Fluor 488-conjugated secondary antibody in block for 45 min. Cells were then washed and resuspended in PBS for ﬂow cytometry analysis using a BD LSRII ﬂow cytometer. Data were analyzed using FlowJo V10. 2.10. Establishment of EWI-2 Knockdown CEM-SS Cells Samples were gated for live cells, and EWI-2 expression was measured by the mean ﬂuorescence intensity of EWI-2 signal in the live cell population and normalized to the parental control expression within each biological replicate. The data are the result of three independent biological replicates with two technical replicates each. For microscopy, 2.5 × 105 shScramble and shEWI-2 cells, alongside parental CEM-SS controls, were plated on 8-well glass bottom plates coated with 1:10 poly-L-lysine in ddH2O. After 2 h at 37 ◦C, cells were ﬁxed for 10 min using 4% PFA in PBS, washed, and incubated with block/perm for 10 min. Cells were washed with block and incubated with 1:200 mAb 8A12 for 45 min, washed, and stained with 1:500 Alexa Fluor 647-conjugated secondary antibody and 1:2500 DAPI in block for 45 min. Cells were washed with block and imaged in PBS using a 60× objective as described above. Images were deconvolved and cropped by DeltaVision microscope and Softworx software described above and imported into Fiji for analysis. 2.11. CEM-luc-based HIV-1-Induced Cell–Cell Fusion Assay Two million shScramble or shEWI-2 cells were spinoculated as described above with 1.7 or 2 µL of VSV-G pseudotyped NL4-3, alongside parental CEM-SS cells spinoculated with 25 µL of VSV-G pseudotyped NL4-3 ∆Env to achieve an infection rate of ~30% for each condition. Cells were incubated at 37 ◦C for 2 days and then co-cultured with uninfected CEM-luc cells in RPMI/10% FBS containing the following drug treatments; 1:1000 DMSO for vehicle control, 1 µM Efavirenz (EFV) (NIH AIDS Reagent Program, Cat. #4624) to inhibit transmission, or 1 µM EFV with 0.5 µM HIV-1 IIIB C34 peptide (C34) (NIH AIDS Reagent Program, Cat. #9824) to inhibit both transmission and cell–cell fusion. After 24 h, the co-culture medium was refreshed, and all conditions were incubated at 37 ◦C in RPMI/10% FBS containing 1 µM EFV and 0.5 µM C34. 24 h later, cells were pelleted at 1000 rcf for 5 min at 4 ◦C and resuspended in luciferase reporter lysis buffer (Promega, Cat. #E4530) with 1% protease inhibitor cocktail (Millipore Sigma, Darmstadt, Germany, Cat. #P8340) to lyse on ice for 15 min. Lysates were cleared by centrifugation at 20,000 rcf for 5 min at 4 ◦C and stored at –80 ◦C until use for luciferase activity assays. In parallel, infected cells were prepared for ﬂow cytometry analysis alongside uninfected controls, to determine the infection rate across each condition at the start of the co culture with uninfected 9 of 23 Viruses 2019, 11, 1082 CEM-luc cells. Cells were pelleted and resuspended in 1:1000 Live/Dead Fixable Near-IR stain in PBS as described above, washed and resuspended in PBS. An equal volume of 8% PFA in PBS was added to ﬁx the cells in a ﬁnal concentration of 4% PFA in PBS for 10 min. Cells were washed and resuspended in block/perm, incubated for 10 min, washed with block, and resuspended for an overnight incubation in 1:100 AG3.0 in block. Cells were washed and stained with 1:500 Alexa Fluor 488-conjugated secondary antibody for 45 min followed by a wash with block. Cells were resuspended in PBS and analyzed by ﬂow cytometry using a BD LSRII ﬂow cytometer. Data was analyzed using FlowJo V10. Live cells were gated using the Live/Dead signal, and the percentage of infected cells in the live population was determined by gating on the AG3.0 associated signal. 2.11. CEM-luc-based HIV-1-Induced Cell–Cell Fusion Assay y g g g Each lysate was incubated with an equal volume of ﬁreﬂy luciferase reagent for 1 min in a 96-well white-walled plate before collecting luminescence signal intensity on a microplate reader as described above (2.9). Background luminescence was determined using a lysis buﬀer blank and subtracted from all experimental samples. Relative luminescence units (RLUs) were normalized based on the infection level of each cell type determined by ﬂow cytometry analysis, and the average RLU value from the ∆Env infected, DMSO treated condition was subtracted from all conditions. All samples treated with both EFV and C34 had RLU values below that of the ∆Env DMSO condition (data not shown), validating the eﬃcacy of the inhibitors for complete inhibition of transmission to target CEM-luc cells. To determine the proportion of luciferase expression due to cell–cell fusion, the average RLU value from the EFV-treated condition (syncytium formation-dependent signal) was divided by that of the DMSO-treated (signal from both transmission and syncytium formation) and multiplied by 100. Data represent the percentage of luciferase signal due to syncytium formation between infected shScramble or shEWI-2 cells and uninfected CEM-luc cells from three independent biological replicates each consisting of 1–2 technical replicates. 3. Results 3.1. EWI-2 Accumulates at the Virological Presynapse in HIV-1-Infected Cells 2.12. Statistical Analysis All statistical analyses were carried out in GraphPad Prism 8 (GraphPad Software, San Diego, CA) as indicated in Figure legends. 3.1. EWI-2 Accumulates at the Virological Presynapse in HIV-1-Infected Cells The data quantiﬁed are from one biological replicate consisting of two technical replicates. Similar trends were observed in a second dataset; not shown. (B) Primary CD4+ T cells infected with NL-sfGI WT or NL-CI ∆Env were co-cultured with uninfected target primary cells for 2 h and stained for EWI-2 (magenta) and Gag (yellow), followed by secondary pAbs (Alexa Fluor 647-conjugated for EWI-2, and either Alexa Fluor 594 or Alexa Fluor 488-conjugated for Gag in the case of WT and ∆Env, respectively). Because diﬀerent secondary antibodies were used for Gag in either condition, the scaling shown for that channel is not the same across the two conditions and was based on corresponding primary and uninfected controls done alongside each dataset. Enrichment of EWI-2 at Env-dependent (WT) or Env-independent (∆Env) infected-uninfected cell contacts was quantiﬁed as described in (A). The data quantiﬁed are pooled from two independent biological replicates, each consisting of two technical replicates. Scale bars = 10 µm. In both data plots, each data point represents one cell–cell contact site (as opposed to one cell). The dotted horizontal line indicates a theoretical fold enrichment value of 1, which indicates no enrichment. Error bars = standard deviation of the mean (SD). p-values are the result of two-tailed non-parametric Mann-Whitney U tests. T d t i h th EWI 2 i h t t th VS t k l ithi th i f t d ll i t 1 Figure 1. EWI-2 co-accumulates with Gag at the HIV-1 VS in T cells. (A) CEM-SS cells infected with HIV-1 NL4-3 WT or ∆Env were co cultured with uninfected CEM-SS target cells for 5 h, and subsequently stained for surface EWI-2 (magenta) and Gag (yellow). The EWI-2-associated ﬂuorescence intensity at cell–cell contacts either enriched with Gag (WT) or not Gag-enriched but identiﬁed by DIC (∆Env) was measured. This value was then divided by the sum of the EWI-2-associated ﬂuorescence intensity on non-contact sites on the producer and target cell in each VS/contact to yield EWI-2 enrichment (i.e., the values shown here). The data quantiﬁed are from one biological replicate consisting of two technical replicates. Similar trends were observed in a second dataset; not shown. 3.1. EWI-2 Accumulates at the Virological Presynapse in HIV-1-Infected Cells Because EWI-2 is known to associate with ezrin and CD81 [25,27], two cellular factors that accumulate at the producer cell side of the virological synapse (VS) [24,54], we ﬁrst sought to determine whether this protein also localizes to the VS. CEM-SS cells were infected with (VSV-G-pseudotyped) NL4-3 WT or NL4-3 ∆Env (virus that does not express Env) and mixed with target CEM-SS cells (labeled with a cytoplasmic dye). Upon imaging with a 60× objective, the VS was identiﬁed and deﬁned by region selection as clusters of immunolabeled Gag present at producer-target cell contact sites. DIC was used to identify and region-select cell–cell contacts between ∆Env producers and uninfected target cells as Gag will not accumulate at these contacts in the absence of Env [1]. The EWI-2 channel was not viewed during the process of deﬁning VS/contact regions to eliminate possible bias. To calculate the enrichment at the VS/contact, we divided the EWI-2 signal intensity within the deﬁned VS/contact site by the sum of the EWI-2 surface intensity at non-contact sites on the producer and target cell at each VS/contact. This unbiased approach prevents potential inﬂation of the enrichment value that could occur if we assumed that EWI-2 was solely contributed by either the target or producer cell. Similarly to p-ezrin and CD81 [24,54], EWI-2 was observed to co-accumulate with Gag at the VS in an Env-dependent manner (Figure 1A). EWI-2 signal intensity was ~4-fold enriched at the VS in CEM-SS cells infected with NL4-3 WT, while no EWI-2 enrichment was seen at cell–cell contacts in cells expressing NL4-3 ∆Env (Figure 1A). EWI-2 signal intensity was also enriched ~1.6-fold at the VS in infected primary CD4+ T cells at Env-dependent VSs and was again not enriched at non-VS contact sites (∆Env) (Figure 1B). 10 of 23 Viruses 2019, 11, 1082 1 Figure 1. EWI-2 co-accumulates with Gag at the HIV-1 VS in T cells. (A) CEM-SS cells infected with HIV-1 NL4-3 WT or ∆Env were co cultured with uninfected CEM-SS target cells for 5 h, and subsequently stained for surface EWI-2 (magenta) and Gag (yellow). The EWI-2-associated ﬂuorescence intensity at cell–cell contacts either enriched with Gag (WT) or not Gag-enriched but identiﬁed by DIC (∆Env) was measured. This value was then divided by the sum of the EWI-2-associated ﬂuorescence intensity on non-contact sites on the producer and target cell in each VS/contact to yield EWI-2 enrichment (i.e., the values shown here). 3.1. EWI-2 Accumulates at the Virological Presynapse in HIV-1-Infected Cells The data quantified are pooled from two independent biological replicates, each consisting of two technical replicates. Scale bars = 10 μm. In both data plots, each data point represents one cell–cell contact site (as opposed to one cell). The dotted horizontal line indicates a theoretical fold enrichment value of 1, which indicates no enrichment. Error bars = standard deviation of the mean (SD). p-values are the result of two-tailed non-parametric Mann-Whitney U tests. Figure 2. EWI-2 accumulation takes place on the producer cell side of the VS. (A) To evaluate presynaptic accumulation of EWI-2, CEM-SS cells infected with HIV-1 NL-CI WT or ΔEnv were co cultured with CMAC (cyan) labeled TZM-bl target cells (which have nearly-undetectable EWI-2 surface levels compared to CEM-SS cells) for 2.5 h, and subsequently stained for surface EWI-2 (magenta) and Gag (yellow). EWI-2 enrichment was quantified as described in Figure 1. Quantification is the result of pooled VS/contacts from two independent biological replicates. (B) To evaluate postsynaptic accumulation of EWI-2, HeLa cells (which, like TZM-bl cells, also have nearly- undetectable EWI-2 surface levels) were transfected with HIV-1 NL-sfGI or NL-sfGI ΔEnv and cocultured with uninfected CEM-SS target cells (cyan) for 2–2.5 h. Cells were stained for surface EWI- 2 (magenta) and Gag (yellow). Note that Gag expression in the ΔEnv condition was quite low, since Gag expression in this virus is already expected to be considerably reduced [55]. EWI-2 enrichment was calculated as described in Figure 1. Quantification is the result of pooled VSs/contacts from two independent biological replicates. Scale bars = 10 μm. In both data plots, each dot represents the EWI- 2 enrichment value of one VS/contact. The dotted horizontal line indicates a theoretical fold Figure 2. EWI-2 accumulation takes place on the producer cell side of the VS. (A) To evaluate presynaptic accumulation of EWI-2, CEM-SS cells infected with HIV-1 NL-CI WT or ∆Env were co cultured with CMAC (cyan) labeled TZM-bl target cells (which have nearly-undetectable EWI-2 surface levels compared to CEM-SS cells) for 2.5 h, and subsequently stained for surface EWI-2 (magenta) and Gag (yellow). EWI-2 enrichment was quantiﬁed as described in Figure 1. Quantiﬁcation is the result of pooled VS/contacts from two independent biological replicates. 3.1. EWI-2 Accumulates at the Virological Presynapse in HIV-1-Infected Cells (B) To evaluate postsynaptic accumulation of EWI-2, HeLa cells (which, like TZM-bl cells, also have nearly-undetectable EWI-2 surface levels) were transfected with HIV-1 NL-sfGI or NL-sfGI ∆Env and cocultured with uninfected CEM-SS target cells (cyan) for 2–2.5 h. Cells were stained for surface EWI-2 (magenta) and Gag (yellow). Note that Gag expression in the ∆Env condition was quite low, since Gag expression in this virus is already expected to be considerably reduced [55]. EWI-2 enrichment was calculated as described in Figure 1. Quantiﬁcation is the result of pooled VSs/contacts from two independent biological replicates. Scale bars = 10 µm. In both data plots, each dot represents the EWI-2 enrichment value of one VS/contact. The dotted horizontal line indicates a theoretical fold enrichment of 1, which indicates no enrichment. Error bars = standard deviation of the mean (SD). p-values are the result of two-tailed non-parametric Mann Whitney U tests Figure 2. EWI-2 accumulation takes place on the producer cell side of the VS. (A) To evaluate presynaptic accumulation of EWI-2, CEM-SS cells infected with HIV-1 NL-CI WT or ΔEnv were co cultured with CMAC (cyan) labeled TZM-bl target cells (which have nearly-undetectable EWI-2 surface levels compared to CEM-SS cells) for 2.5 h, and subsequently stained for surface EWI-2 (magenta) and Gag (yellow). EWI-2 enrichment was quantified as described in Figure 1. Quantification is the result of pooled VS/contacts from two independent biological replicates. (B) To evaluate postsynaptic accumulation of EWI-2, HeLa cells (which, like TZM-bl cells, also have nearly- undetectable EWI-2 surface levels) were transfected with HIV-1 NL-sfGI or NL-sfGI ΔEnv and cocultured with uninfected CEM-SS target cells (cyan) for 2–2.5 h. Cells were stained for surface EWI- 2 (magenta) and Gag (yellow). Note that Gag expression in the ΔEnv condition was quite low, since Gag expression in this virus is already expected to be considerably reduced [55]. EWI-2 enrichment was calculated as described in Figure 1. Quantification is the result of pooled VSs/contacts from two independent biological replicates. Scale bars = 10 μm. In both data plots, each dot represents the EWI- 2 enrichment value of one VS/contact. The dotted horizontal line indicates a theoretical fold Figure 2. EWI-2 accumulation takes place on the producer cell side of the VS. 3.1. EWI-2 Accumulates at the Virological Presynapse in HIV-1-Infected Cells (B) Primary CD4+ T cells infected with NL-sfGI WT or NL-CI ∆Env were co-cultured with uninfected target primary cells for 2 h and stained for EWI-2 (magenta) and Gag (yellow), followed by secondary pAbs (Alexa Fluor 647-conjugated for EWI-2, and either Alexa Fluor 594 or Alexa Fluor 488-conjugated for Gag in the case of WT and ∆Env, respectively). Because diﬀerent secondary antibodies were used for Gag in either condition, the scaling shown for that channel is not the same across the two conditions and was based on corresponding primary and uninfected controls done alongside each dataset. Enrichment of EWI-2 at Env-dependent (WT) or Env-independent (∆Env) infected-uninfected cell contacts was quantiﬁed as described in (A). The data quantiﬁed are pooled from two independent biological replicates, each consisting of two technical replicates. Scale bars = 10 µm. In both data plots, each data point represents one cell–cell contact site (as opposed to one cell). The dotted horizontal line indicates a theoretical fold enrichment value of 1, which indicates no enrichment. Error bars = standard deviation of the mean (SD). p-values are the result of two-tailed non-parametric Mann-Whitney U tests. To determine whether EWI-2 enrichment at the VS takes place within the infected cell, i.e., at the presynaptic terminal (rather than the apposed uninfected target cell), HIV-1-infected CEM-SS cells were co-cultured with uninfected target TZM-bl cells (which have nearly-undetectable levels of EWI-2 on their surface) and imaged as described above. Signiﬁcant EWI-2 enrichment (~5.3-fold) was observed at the VS as before (Figure 2A), demonstrating that the observed EWI-2 accumulation in 11 of 23 Viruses 2019, 11, 1082 CEM-SS-CEM-SS co-cultures takes place at least partially within the producer cell. To evaluate the relative contribution of any postsynaptic (i.e., target cell-side) accumulation of EWI-2, HIV-1-producing HeLa cells (which, like TZM-bl cells, also exhibit nearly undetectable levels of EWI-2 on their surface) were cocultured with uninfected target CEM-SS cells. In this case, minimal EWI-2 accumulation was detected at synapses (~1.1-fold; Figure 2B), showing that EWI-2 enrichment seen at T cell-T cell VSs takes place (almost) exclusively at the presynaptic terminal of the VS, i.e., in the producer cell. Together, these results conclusively document that EWI-2 is recruited to the virological presynapse during HIV-1 cell-to-cell transmission. Viruses 2019, 11, 1082 11 of 23 Env-independent (ΔEnv) infected-uninfected cell contacts was quantified as described in (A). 3.2. Overall Surface Levels of EWI-2 Are Decreased upon HIV-1 Infection 3.2. Overall Surface Levels of EWI-2 Are Decreased upon HIV-1 Infection Despite its enrichment at the virological presynapse, the EWI-2 partner protein CD81 (as well as other tetraspanins) is overall downregulated in HIV-1-infected cells [54,56,57]. We previously used Tandem Mass Tag (TMT)-based quantitative proteomics to map global changes in whole cell protein abundances in HIV-infected T cells [50,51]. Like CD81, EWI-2 was decreased in abundance in both CEM-T4 T cells and primary human CD4+ T cells (Figure 3A). To conﬁrm these data using an orthogonal approach, we tested whether surface levels of EWI-2 are decreased in lymphocytes infected with HIV-1 NL-sfGI, a strain in which superfolder GFP (sfGFP) replaces the Nef gene and Nef expression is restored using an IRES [10]. We chose to utilize this GFP reporter virus, rather than immunolabeling Gag after ﬁxation, because Gag-negative (or undetectable) cells still in the early phase of infection may exhibit host protein downregulation due to early Nef expression (reviewed in [58]). y p g y p HIV-1-infected cells adhered to glass-bottom dishes were surface-labeled with EWI-2 primary antibody on ice and ﬁxed before incubation with ﬂuorescent secondary antibody. Uninfected and HIV-1-infected cells were imaged with a 60× objective and the resulting images were deconvolved. The mean ﬂuorescence intensity (MFI) of EWI-2 on the surface of each cell was determined by measuring the EWI-2-associated signal intensity of manually-selected regions of the cell surface (representative images shown in Figure 3B) and normalizing the raw MFI of each cell to the average EWI-2 signal from uninfected cells within the same imaging set. After measuring surface MFI, on average across three independent biological replicates, infected (GFP-expressing) cells had signiﬁcantly lower (~2-fold) EWI-2-associated signal than uninfected (GFP-negative) cells, after subtracting background signal (Figure 3B). This phenomenon was consistent across CEM-SS, CEM2n, and primary CD4+ T cells. We also sought to quantify EWI-2 surface expression by ﬂow cytometry as a means of high-throughput analysis. HIV-1 NL-sfGI-infected CEM2n cells, surface-labeled for EWI-2 and analyzed by ﬂow cytometry, were gated for high or low levels of EWI-2 using appropriate controls (representative histogram plots shown in Figure 3D). These data showed that a much lower proportion of infected cells (identiﬁed as GFP+) had high levels of EWI-2 surface expression than of uninfected cells (identiﬁed as GFP-) in the same culture (Figure 3E). 3.1. EWI-2 Accumulates at the Virological Presynapse in HIV-1-Infected Cells (A) To evaluate presynaptic accumulation of EWI-2, CEM-SS cells infected with HIV-1 NL-CI WT or ∆Env were co cultured with CMAC (cyan) labeled TZM-bl target cells (which have nearly-undetectable EWI-2 surface levels compared to CEM-SS cells) for 2.5 h, and subsequently stained for surface EWI-2 (magenta) and Gag (yellow). EWI-2 enrichment was quantiﬁed as described in Figure 1. Quantiﬁcation is the result of pooled VS/contacts from two independent biological replicates. (B) To evaluate postsynaptic accumulation of EWI-2, HeLa cells (which, like TZM-bl cells, also have nearly-undetectable EWI-2 surface levels) were transfected with HIV-1 NL-sfGI or NL-sfGI ∆Env and cocultured with uninfected CEM-SS target cells (cyan) for 2–2.5 h. Cells were stained for surface EWI-2 (magenta) and Gag (yellow). Note that Gag expression in the ∆Env condition was quite low, since Gag expression in this virus is already expected to be considerably reduced [55]. EWI-2 enrichment was calculated as described in Figure 1. Quantiﬁcation is the result of pooled VSs/contacts from two independent biological replicates. Scale bars = 10 µm. In both data plots, each dot represents the EWI-2 enrichment value of one VS/contact. The dotted horizontal line indicates a theoretical fold enrichment of 1, which indicates no enrichment. Error bars = standard deviation of the mean (SD). p-values are the result of two-tailed non-parametric Mann-Whitney U tests. Viruses 2019, 11, 1082 12 of 23 3.2. Overall Surface Levels of EWI-2 Are Decreased upon HIV-1 Infection Additionally, the mean ﬂuorescence intensity of EWI-2-associated signal was lower within the total population of infected cells compared to that of the uninfected cells (Figure 3F). Like other cell surface proteins downregulated by HIV-1, depletion of CD81 (as well as other tetraspanins) is mediated by the accessory proteins Vpu (predominantly) and Nef [56,57]. We have previously shown that substrates of diﬀerent HIV-1 accessory proteins may be distinguished by their characteristic patterns of temporal regulation in HIV-1-infected T cells [50,51,53]. Accordingly, the temporal expression proﬁle of plasma membrane EWI-2 was strikingly similar to that of BST2 (Tetherin), a canonical Vpu target (Figure 4A). Furthermore, like BST2, depletion of cell surface EWI-2 by HIV-1 infection was abrogated in the presence of reverse transcriptase inhibitors, and when cells were infected with Vpu-deﬁcient HIV-1 (Figure 4B). Taken together, our proteomic data therefore strongly suggest that Vpu is primarily responsible for HIV-1-dependent EWI-2 downregulation. As with the tetraspanins however, the incomplete rescue in the presence of Vpu-deﬁcient virus, and relatively modest depletion when Vpu was expressed as a single gene (Figure 4B), suggest that Nef may also contribute to the depletion of cell surface EWI-2 in the context of HIV-1 infection. uses 2019, 11, 1082 13 o , p ( ) p g Nef expression is restored using an IRES [10]. We chose to utilize this GFP reporter virus, rather than immunolabeling Gag after fixation, because Gag-negative (or undetectable) cells still in the early phase of infection may exhibit host protein downregulation due to early Nef expression (reviewed in [58]). Figure 3. EWI-2 is downregulated from the surface of infected cells. (A) Abundance of EWI-2 in mock- infected (grey) versus WT HIV-infected (yellow) CEM-T4 T cells or primary human CD4+ T cells. Experiments were conducted in triplicate and whole cell lysates subjected to Tandem Mass Tag (TMT)-based quantitative proteomics 48 h after infection (reanalysis of data from [50] and [51]). Seven Figure 3. EWI-2 is downregulated from the surface of infected cells. (A) Abundance of EWI-2 in mock-infected (grey) versus WT HIV-infected (yellow) CEM-T4 T cells or primary human CD4+ T cells. Experiments were conducted in triplicate and whole cell lysates subjected to Tandem Mass Tag (TMT)-based quantitative proteomics 48 h after infection (reanalysis of data from [50,51]). Seven (CEM-T4 T cells) or six (primary human CD4+ T cells) unique peptides were used for EWI-2 quantitation. 3.2. Overall Surface Levels of EWI-2 Are Decreased upon HIV-1 Infection Experiments were conducted in triplicate and whole cell lysates subjected to Tandem Mass Tag (TMT)-based quantitative proteomics 48 h after infection (reanalysis of data from [50,51]). Seven (CEM-T4 T cells) or six (primary human CD4+ T cells) unique peptides were used for EWI-2 quantitation. Mean relative abundances (fraction of maximum TMT reporter ion intensity) shown. (B) Cells were infected with NL-sfGI and surface-labeled for EWI-2, ﬁxed, stained with DAPI (shown in cyan) and Alexa Fluor 594-conjugated secondary antibody, and imaged. GFP signal (yellow) was used to identify infected cells, and EWI-2-associated signal is shown pseudocolored in magenta. Representative cells are shown. Scale bars = 10 µm. (C) Cells were prepared as in (B) and EWI-2 levels at the plasma membrane in infected (Inf) and uninfected (Uninf) cells were measured by manually selecting the plasma membrane at the midline of each cell and quantifying the mean EWI-2-associated ﬂuorescence intensity. Fluorescence intensity of each cell was normalized to the average intensity value of uninfected cells within the same imaging set. Data shown are pooled from two to three biological replicates, each consisting of two technical replicates. Only non-contact sites were quantiﬁed. Error bars = SD. p-values are the result of a two-tailed non-parametric Mann-Whitney U test. (C-E) CEM2n cells were infected with NL-sfGI and surface-labeled for EWI-2, ﬁxed, and stained with Alexa Fluor 647-conjugated secondary antibody, and analyzed by ﬂow cytometry. (D) Representative histogram normalized to mode of the EWI-2 signal intensity at the cell surface for unstained controls (black outline), infected cells (yellow), and uninfected cells (cyan). The gates deﬁning EWI-2high and EWI-2low cells are shown. (E) Data represent the percentage of uninfected and infected cells that fell into the EWI-2high gate shown in (d) from 3 independent biological replicates, averaged across two technical replicates within each. (F) EWI-2 surface expression was measured by mean ﬂuorescence intensity (MFI) of EWI-2-associated signal. In both panels, lines connect paired data points, i.e., infected cells and uninfected cells (within an infected tube) from the same biological replicate. Figure 3. EWI-2 is downregulated from the surface of infected cells. (A) Abundance of EWI-2 in mock- infected (grey) versus WT HIV-infected (yellow) CEM-T4 T cells or primary human CD4+ T cells. Experiments were conducted in triplicate and whole cell lysates subjected to Tandem Mass Tag (TMT)-based quantitative proteomics 48 h after infection (reanalysis of data from [50] and [51]). Seven Figure 3. 3.2. Overall Surface Levels of EWI-2 Are Decreased upon HIV-1 Infection Mean relative abundances (fraction of maximum TMT reporter ion intensity) shown. (B) Cells were infected with NL-sfGI and surface-labeled for EWI-2, ﬁxed, stained with DAPI (shown in cyan) and Alexa Fluor 594-conjugated secondary antibody, and imaged. GFP signal (yellow) was used to identify infected cells, and EWI-2-associated signal is shown pseudocolored in magenta. Representative cells are shown. Scale bars = 10 µm. (C) Cells were prepared as in (B) and EWI-2 levels at the plasma membrane in infected (Inf) and uninfected (Uninf) cells were measured by manually selecting the plasma membrane at the midline of each cell and quantifying the mean EWI-2-associated ﬂuorescence intensity. Fluorescence intensity of each cell was normalized to the average intensity value of uninfected cells within the same imaging set. Data shown are pooled from two to three biological replicates, each consisting of two technical replicates. Only non-contact sites were quantiﬁed. Error bars = SD. p-values are the result of a two-tailed non-parametric Mann-Whitney U test. (C-E) CEM2n cells were infected with NL-sfGI and surface-labeled for EWI-2, ﬁxed, and stained with Alexa Fluor 647-conjugated secondary antibody, and analyzed by ﬂow cytometry. (D) Representative histogram normalized to mode of the EWI-2 signal intensity at the cell surface for unstained controls (black outline), infected cells (yellow), and uninfected cells (cyan). The gates deﬁning EWI-2high and EWI-2low cells are shown. (E) Data represent the percentage of uninfected and infected cells that fell into the EWI-2high gate shown in (d) from 3 independent biological replicates, averaged across two technical replicates within each. (F) EWI-2 surface expression was measured by mean ﬂuorescence intensity (MFI) of EWI-2-associated signal. In both panels, lines connect paired data points, i.e., infected cells and uninfected cells (within an infected tube) from the same biological replicate. 13 of 23 Viruses 2019, 11, 1082 p immunola h f i Figure 3. EWI-2 is downregulated from the surface of infected cells. (A) Abundance of EWI-2 in mock- infected (grey) versus WT HIV-infected (yellow) CEM-T4 T cells or primary human CD4+ T cells. Experiments were conducted in triplicate and whole cell lysates subjected to Tandem Mass Tag (TMT)-based quantitative proteomics 48 h after infection (reanalysis of data from [50] and [51]). Seven Figure 3. EWI-2 is downregulated from the surface of infected cells. (A) Abundance of EWI-2 in mock-infected (grey) versus WT HIV-infected (yellow) CEM-T4 T cells or primary human CD4+ T cells. 3.2. Overall Surface Levels of EWI-2 Are Decreased upon HIV-1 Infection EWI-2 is downregulated from the surface of infected cells. (A) Abundance of EWI-2 in mock-infected (grey) versus WT HIV-infected (yellow) CEM-T4 T cells or primary human CD4+ T cells. Experiments were conducted in triplicate and whole cell lysates subjected to Tandem Mass Tag (TMT)-based quantitative proteomics 48 h after infection (reanalysis of data from [50,51]). Seven (CEM-T4 T cells) or six (primary human CD4+ T cells) unique peptides were used for EWI-2 quantitation. Mean relative abundances (fraction of maximum TMT reporter ion intensity) shown. (B) Cells were infected with NL-sfGI and surface-labeled for EWI-2, ﬁxed, stained with DAPI (shown in cyan) and Alexa Fluor 594-conjugated secondary antibody, and imaged. GFP signal (yellow) was used to identify infected cells, and EWI-2-associated signal is shown pseudocolored in magenta. Representative cells are shown. Scale bars = 10 µm. (C) Cells were prepared as in (B) and EWI-2 levels at the plasma membrane in infected (Inf) and uninfected (Uninf) cells were measured by manually selecting the plasma membrane at the midline of each cell and quantifying the mean EWI-2-associated ﬂuorescence intensity. Fluorescence intensity of each cell was normalized to the average intensity value of uninfected cells within the same imaging set. Data shown are pooled from two to three biological replicates, each consisting of two technical replicates. Only non-contact sites were quantiﬁed. Error bars = SD. p-values are the result of a two-tailed non-parametric Mann-Whitney U test. (C-E) CEM2n cells were infected with NL-sfGI and surface-labeled for EWI-2, ﬁxed, and stained with Alexa Fluor 647-conjugated secondary antibody, and analyzed by ﬂow cytometry. (D) Representative histogram normalized to mode of the EWI-2 signal intensity at the cell surface for unstained controls (black outline), infected cells (yellow), and uninfected cells (cyan). The gates deﬁning EWI-2high and EWI-2low cells are shown. (E) Data represent the percentage of uninfected and infected cells that fell into the EWI-2high gate shown in (d) from 3 independent biological replicates, averaged across two technical replicates within each. (F) EWI-2 surface expression was measured by mean ﬂuorescence intensity (MFI) of EWI-2-associated signal. In both panels, lines connect paired data points, i.e., infected cells and uninfected cells (within an infected tube) from the same biological replicate. 3.2. Overall Surface Levels of EWI-2 Are Decreased upon HIV-1 Infection 14 of 23 letion of Viruses 2019, 11, 1082 was expressed as uses 2019, 11, 1082 14 as expressed as a single gene (Figure 4B), suggest that Nef may also contribute to the depletio ll surface EWI-2 in the context of HIV-1 infection. Figure 4. Plasma membrane EWI-2 is downregulated by Vpu. (A) Temporal expression profiles of cell surface EWI-2 (red, upper panel) or indicated control proteins (blue/green/gold, lower panels) in WT HIV-1-infected CEM-T4 T cells (reanalysis of data from [53]). Plasma membrane proteins were subjected to TMT-based quantitative proteomics 0 (uninfected), 6, 24, 48, and 72 h after infection, or 72 h after infection in the presence of reverse transcriptase inhibitors (RTi). Twelve unique peptides were used for EWI-2 quantitation. Relative abundances (fraction of maximum TMT reporter ion intensity) are shown. (B) Abundance of EWI-2 (red, upper panel) or indicated control proteins (blue/green/gold, lower panels) in control CEM-T4 T cells or CEM-T4 T cells infected with WT HIV-1 in the presence/absence of RTi, infected with Vpu- or Nef-deficient HIV-1, or transduced with Vpu or Nef as single genes (reanalysis of data from [53]). Plasma membrane proteins were subjected to Stable Isotope Labelling with Amino acids in Cell culture (SILAC)-based quantitative proteomics 72 h after infection (3 × 3-way comparisons). Twelve (WT HIV-1 +/− RTi), nine (∆Vpu/∆Nef HIV-1) or 14 (Vpu/Nef) unique peptides were used for EWI-2 quantitation. Ratios of abundances to mock-infected CEM-T4 T cells (WT HIV-1 +/- RTi and ∆Vpu/∆Nef HIV-1) or GFP-transduced CEM-T4 T cells (Vpu/Nef) are shown. Note that (A) and (B) show data from two different quantitative proteomic Figure 4. Plasma membrane EWI-2 is downregulated by Vpu. (A) Temporal expression proﬁles of cell surface EWI-2 (red, upper panel) or indicated control proteins (blue/green/gold, lower panels) in WT HIV-1-infected CEM-T4 T cells (reanalysis of data from [53]). Plasma membrane proteins were subjected to TMT-based quantitative proteomics 0 (uninfected), 6, 24, 48, and 72 h after infection, or 72 h after infection in the presence of reverse transcriptase inhibitors (RTi). Twelve unique peptides were used for EWI-2 quantitation. Relative abundances (fraction of maximum TMT reporter ion intensity) are shown. 3.2. Overall Surface Levels of EWI-2 Are Decreased upon HIV-1 Infection (B) Abundance of EWI-2 (red, upper panel) or indicated control proteins (blue/green/gold, lower panels) in control CEM-T4 T cells or CEM-T4 T cells infected with WT HIV-1 in the presence/absence of RTi, infected with Vpu- or Nef-deﬁcient HIV-1, or transduced with Vpu or Nef as single genes (reanalysis of data from [53]). Plasma membrane proteins were subjected to Stable Isotope Labelling with Amino acids in Cell culture (SILAC)-based quantitative proteomics 72 h after infection (3 × 3-way comparisons). Twelve (WT HIV-1 +/−RTi), nine (∆Vpu/∆Nef HIV-1) or 14 (Vpu/Nef) unique peptides were used for EWI-2 quantitation. Ratios of abundances to mock-infected CEM-T4 T cells (WT HIV-1 +/- RTi and ∆Vpu/∆Nef HIV-1) or GFP-transduced CEM-T4 T cells (Vpu/Nef) are shown. Note that (A) and (B) show data from two diﬀerent quantitative proteomic methods, as described above and in Materials and Methods ll surface EWI-2 in the context of HIV-1 infection. ace EWI-2 in the context of HIV-1 infection Figure 4. Plasma membrane EWI-2 is downregulated by Vpu. (A) Temporal expression profiles of cell surface EWI-2 (red, upper panel) or indicated control proteins (blue/green/gold, lower panels) in WT HIV-1-infected CEM-T4 T cells (reanalysis of data from [53]). Plasma membrane proteins were subjected to TMT-based quantitative proteomics 0 (uninfected), 6, 24, 48, and 72 h after infection, or 72 h after infection in the presence of reverse transcriptase inhibitors (RTi). Twelve unique peptides were used for EWI-2 quantitation. Relative abundances (fraction of maximum TMT reporter ion intensity) are shown. (B) Abundance of EWI-2 (red, upper panel) or indicated control proteins (blue/green/gold, lower panels) in control CEM-T4 T cells or CEM-T4 T cells infected with WT HIV-1 in the presence/absence of RTi, infected with Vpu- or Nef-deficient HIV-1, or transduced with Vpu or Nef as single genes (reanalysis of data from [53]). Plasma membrane proteins were subjected to Stable Isotope Labelling with Amino acids in Cell culture (SILAC)-based quantitative proteomics 72 h after infection (3 × 3-way comparisons). Twelve (WT HIV-1 +/− RTi), nine (∆Vpu/∆Nef HIV-1) or 14 (Vpu/Nef) unique peptides were used for EWI-2 quantitation. Ratios of abundances to mock-infected CEM-T4 T cells (WT HIV-1 +/- RTi and ∆Vpu/∆Nef HIV-1) or GFP-transduced CEM-T4 T cells (Vpu/Nef) are shown. Note that (A) and (B) show data from two different quantitative proteomic methods, as described above and in Materials and Methods. Figure 4. Plasma membrane EWI-2 is downregulated by Vpu. 3.3. EWI-2 Inhibits HIV-1-Induced Syncytium Formation 3.3. EWI-2 Inhibits HIV-1-Induced Syncytium Formation 3.3. EWI-2 Inhibits HIV-1-Induced Syncytium Formation Likely through their accumulation at the producer cell side of the VS, the EWI-2 partner proteins CD81 and ezrin repress the fusion of infected and uninfected cells, i.e., syncytium formation [22–24]. Given that EWI-2 also accumulates at the VS (Figure 1), we sought to test whether it also contributes to the inhibition of HIV-1-induced syncytium formation by both overexpressing EWI-2 and reducing its expression using RNA interference. p g As we have done previously to examine the fusion-inhibitory capacity of tetraspanins [22,23], we tested whether EWI-2 inhibits HIV-1-induced syncytium formation in a dose-dependent manner by overexpressing EWI-2 in HeLa cells (which have nearly-undetectable endogenous levels of EWI-2). NL-sfGI-producing HeLa cells overexpressing either EWI-2, CD81, or L6 (a tetraspanin-like surface protein that does not repress HIV-1-induced cell–cell fusion; [23,59]) were co-cultured with uninfected target TZM-bl cells. As a negative control for HIV-1-induced cell–cell fusion, Env-deleted (∆Env) NL-sfGI-expressing HeLa cells were also co-cultured with target TZM-bl cells. HIV-1-induced HeLa-TZM-bl syncytia express ﬁreﬂy luciferase under control of the HIV-1 LTR [22]. After 3 h of co-culture (and another 14–18 h to allow for reporter expression), cells were lysed, the lysates were incubated with luciferase substrate, and luminescence was measured using a microplate reader. Overall, the overexpression of increasing amounts of either CD81 or EWI-2 (125, 250, or 500 ng of plasmid) in NL-sfGI-producing cells resulted in a dose-dependent decrease of cell–cell fusion (Figure 5A). In parallel, we established an EWI-2 knockdown CEM-SS cell line by lentiviral transduction using a targeting vector (FG12) that directs expression of a short hairpin RNA (shRNA) targeting EWI-2 (shEWI-2), using the same targeting sequence as in a previous report [32]. As a control, this targeting sequence was scrambled several times, all resulting sequences were tested against the human genome by BLASTn, and the sequence with the least homology to any human transcript was selected (shScramble, or shScr). This modiﬁed FG12 vector also carries a puromycin resistance cassette, while the GFP reporter cassette (as used in [24]) was removed to allow use of GFP reporter viruses. The puromycin-resistant shEWI-2 CEM-SS cells were analyzed by microscopy (Figure 5B) and by ﬂow cytometry (Figure 5C–D) and were found to have ~3-fold reduced EWI-2 surface levels, compared to both the shScramble control and the parental non-transduced CEM-SS cells. 3.2. Overall Surface Levels of EWI-2 Are Decreased upon HIV-1 Infection (A) Temporal expression proﬁles of cell surface EWI-2 (red, upper panel) or indicated control proteins (blue/green/gold, lower panels) in WT HIV-1-infected CEM-T4 T cells (reanalysis of data from [53]). Plasma membrane proteins were subjected to TMT-based quantitative proteomics 0 (uninfected), 6, 24, 48, and 72 h after infection, or 72 h after infection in the presence of reverse transcriptase inhibitors (RTi). Twelve unique peptides were used for EWI-2 quantitation. Relative abundances (fraction of maximum TMT reporter ion intensity) are shown. (B) Abundance of EWI-2 (red, upper panel) or indicated control proteins (blue/green/gold, lower panels) in control CEM-T4 T cells or CEM-T4 T cells infected with WT HIV-1 in the presence/absence of RTi, infected with Vpu- or Nef-deﬁcient HIV-1, or transduced with Vpu or Nef as single genes (reanalysis of data from [53]). Plasma membrane proteins were subjected to Stable Isotope Labelling with Amino acids in Cell culture (SILAC)-based quantitative proteomics 72 h after infection (3 × 3-way comparisons). Twelve (WT HIV-1 +/−RTi), nine (∆Vpu/∆Nef HIV-1) or 14 (Vpu/Nef) unique peptides were used for EWI-2 quantitation. Ratios of abundances to mock-infected CEM-T4 T cells (WT HIV-1 +/- RTi and ∆Vpu/∆Nef HIV-1) or GFP-transduced CEM-T4 T cells (Vpu/Nef) are shown. Note that (A) and (B) show data from two diﬀerent quantitative proteomic methods, as described above and in Materials and Methods. 15 of 23 Viruses 2019, 11, 1082 3.3. EWI-2 Inhibits HIV-1-Induced Syncytium Formation (B–D) EWI-2 expression in Figure 5. EWI-2 inhibits infected-uninfected cell fusion. (A) HeLa-TZM-bl fusion assays were performed using producer HeLa cells that were co-transfected with either pNL-sfGI ΔEnv (ΔEnv) or pNL-sfGI (WT) in combination with overexpression plasmid totaling 500 ng (using the indicated amount of CD81 or EWI-2 supplemented with L6). Luminescence readings (across 4 independent biological replicates, each with two technical replicates) were divided by the ΔEnv condition to obtain the fold increase in fusion, and then normalized to the WT co-transfected with only L6 (i.e., 0 ng of CD81 or EWI-2) condition, making it have a value of 1. Deviation from the dashed line at 1 thus Figure 5. EWI-2 inhibits infected-uninfected cell fusion. (A) HeLa-TZM-bl fusion assays were performed using producer HeLa cells that were co-transfected with either pNL-sfGI ∆Env (∆Env) or pNL-sfGI (WT) in combination with overexpression plasmid totaling 500 ng (using the indicated amount of CD81 or EWI-2 supplemented with L6). Luminescence readings (across 4 independent biological replicates, each with two technical replicates) were divided by the ∆Env condition to obtain the fold increase in fusion, and then normalized to the WT co-transfected with only L6 (i.e., 0 ng of CD81 or EWI-2) condition, making it have a value of 1. Deviation from the dashed line at 1 thus indicates an eﬀect on Figure 5. EWI-2 inhibits infected-uninfected cell fusion. (A) HeLa-TZM-bl fusion assays were performed using producer HeLa cells that were co-transfected with either pNL-sfGI ΔEnv (ΔEnv) or pNL-sfGI (WT) in combination with overexpression plasmid totaling 500 ng (using the indicated amount of CD81 or EWI-2 supplemented with L6). Luminescence readings (across 4 independent biological replicates, each with two technical replicates) were divided by the ΔEnv condition to obtain the fold increase in fusion, and then normalized to the WT co-transfected with only L6 (i.e., 0 ng of CD81 or EWI-2) condition, making it have a value of 1. Deviation from the dashed line at 1 thus indicates an effect on fusion. Values from the same biological replicate are linked by a grey line. (B– D) EWI-2 expression in shScramble (shScr) and shEWI-2 CEM-SS cells was analyzed by microscopy (B) and flow cytometry (C–D). (B) For microscopy, cells were plated onto poly-L-lysine-coated glass, fixed, permeabilized, labeled for EWI-2, and stained using fluorescent secondary antibody (magenta) and DAPI (cyan). Scale bars = 10 μm. 3.3. EWI-2 Inhibits HIV-1-Induced Syncytium Formation (C–D) For flow cytometry analysis, cells were labeled with Live/Dead Fixable Near-IR, fixed, permeabilized, labeled for EWI-2, and stained with fluorescent secondary antibody. (C) Representative histogram of the EWI-2 signal intensity normalized to mode in live cells for unstained controls (black line), shEWI-2 (blue), and shScr (red) cells. (D) EWI-2 MFI Figure 5. EWI-2 inhibits infected-uninfected cell fusion. (A) HeLa-TZM-bl fusion assays were performed using producer HeLa cells that were co-transfected with either pNL-sfGI ∆Env (∆Env) or pNL-sfGI (WT) in combination with overexpression plasmid totaling 500 ng (using the indicated amount of CD81 or EWI-2 supplemented with L6). Luminescence readings (across 4 independent biological replicates, each with two technical replicates) were divided by the ∆Env condition to obtain the fold increase in fusion, and then normalized to the WT co-transfected with only L6 (i.e., 0 ng of CD81 or EWI-2) condition, making it have a value of 1. Deviation from the dashed line at 1 thus indicates an eﬀect on fusion. Values from the same biological replicate are linked by a grey line. (B–D) EWI-2 expression in shScramble (shScr) and shEWI-2 CEM-SS cells was analyzed by microscopy (B) and ﬂow cytometry (C–D). (B) For microscopy, cells were plated onto poly-L-lysine-coated glass, ﬁxed, permeabilized, labeled for EWI-2, and stained using ﬂuorescent secondary antibody (magenta) and DAPI (cyan). Scale bars = 10 µm. (C–D) For ﬂow cytometry analysis, cells were labeled with Live/Dead Fixable Near-IR, ﬁxed, permeabilized, labeled for EWI-2, and stained with ﬂuorescent secondary antibody. (C) Representative histogram of the EWI-2 signal intensity normalized to mode in live cells for unstained controls (black line), shEWI-2 (blue), and shScr (red) cells. (D) EWI-2 MFI in live shScr (red) and shEWI-2 (blue) cells from three independent biological replicates, normalized to EWI-2-labeled parental CEM-SS cells (represented at a value of 1 with a dashed line). (E) CEM-luc fusion assays were performed using shScr or shEWI-2 producer cells infected with NL4-3, which were co-cultured with CEM-luc target cells in the presence of DMSO (vehicle control, luciferase signal from transmission and cell–cell fusion) or EFV (luciferase signal resulting exclusively from cell–cell fusion). Luminescence readings (across three independent biological replicates) from the EFV-treated condition were divided by the DMSO reading from the same producer cell type and multiplied by 100 to determine the percentage of luciferase expression dependent on cell–cell fusion (syncytium formation) between either shScr or shEWI-2 producer and CEM-luc target cells. 3.3. EWI-2 Inhibits HIV-1-Induced Syncytium Formation (B) For microscopy, cells were plated onto poly-L-lysine-coated glass, ﬁxed, permeabilized, labeled for EWI-2, and stained using ﬂuorescent secondary antibody (magenta) and DAPI (cyan). Scale bars = 10 µm. (C–D) For ﬂow cytometry analysis, cells were labeled with Live/Dead Fixable Near-IR, ﬁxed, permeabilized, labeled for EWI-2, and stained with ﬂuorescent secondary antibody. (C) Representative histogram of the EWI-2 signal intensity normalized to mode in live cells for unstained controls (black line), shEWI-2 (blue), and shScr (red) cells. (D) EWI-2 MFI in live shScr (red) and shEWI-2 (blue) cells from three independent biological replicates, normalized to EWI-2-labeled parental CEM-SS cells (represented at a value of 1 with a dashed line). (E) CEM-luc fusion assays were performed using shScr or shEWI-2 producer cells infected with NL4-3, which were co-cultured with CEM-luc target cells in the presence of DMSO (vehicle control, luciferase signal from transmission and cell–cell fusion) or EFV (luciferase signal resulting exclusively from cell–cell fusion). Luminescence readings (across three independent biological replicates) from the EFV-treated condition were divided by the DMSO reading from the same producer cell type and multiplied by 100 to determine the percentage of luciferase expression dependent on cell–cell fusion (syncytium formation) between either shScr or shEWI-2 producer and CEM-luc target cells. Values from the same biological replicate are linked by a black line. Figure 5. EWI-2 inhibits infected-uninfected cell fusion. (A) HeLa-TZM-bl fusion assays were performed using producer HeLa cells that were co-transfected with either pNL-sfGI ΔEnv (ΔEnv) or pNL-sfGI (WT) in combination with overexpression plasmid totaling 500 ng (using the indicated amount of CD81 or EWI-2 supplemented with L6). Luminescence readings (across 4 independent biological replicates, each with two technical replicates) were divided by the ΔEnv condition to obtain the fold increase in fusion, and then normalized to the WT co-transfected with only L6 (i.e., 0 ng of CD81 or EWI-2) condition, making it have a value of 1. Deviation from the dashed line at 1 thus indicates an effect on fusion. Values from the same biological replicate are linked by a grey line. (B– D) EWI-2 expression in shScramble (shScr) and shEWI-2 CEM-SS cells was analyzed by microscopy (B) and flow cytometry (C–D). (B) For microscopy, cells were plated onto poly-L-lysine-coated glass, fixed, permeabilized, labeled for EWI-2, and stained using fluorescent secondary antibody (magenta) and DAPI (cyan). Scale bars = 10 μm. 3.3. EWI-2 Inhibits HIV-1-Induced Syncytium Formation (C–D) For flow cytometry analysis, cells were labeled with Live/Dead Fixable Near-IR, fixed, permeabilized, labeled for EWI-2, and stained with fluorescent secondary antibody. (C) Representative histogram of the EWI-2 signal intensity normalized to mode in live cells for unstained controls (black line), shEWI-2 (blue), and shScr (red) cells. (D) EWI-2 MFI Figure 5. EWI-2 inhibits infected-uninfected cell fusion. (A) HeLa-TZM-bl fusion assays were performed using producer HeLa cells that were co-transfected with either pNL-sfGI ∆Env (∆Env) or pNL-sfGI (WT) in combination with overexpression plasmid totaling 500 ng (using the indicated amount of CD81 or EWI-2 supplemented with L6). Luminescence readings (across 4 independent biological replicates, each with two technical replicates) were divided by the ∆Env condition to obtain the fold increase in fusion, and then normalized to the WT co-transfected with only L6 (i.e., 0 ng of CD81 or EWI-2) condition, making it have a value of 1. Deviation from the dashed line at 1 thus indicates an eﬀect on fusion. Values from the same biological replicate are linked by a grey line. (B–D) EWI-2 expression in shScramble (shScr) and shEWI-2 CEM-SS cells was analyzed by microscopy (B) and ﬂow cytometry (C–D). (B) For microscopy, cells were plated onto poly-L-lysine-coated glass, ﬁxed, permeabilized, labeled for EWI-2, and stained using ﬂuorescent secondary antibody (magenta) and DAPI (cyan). Scale bars = 10 µm. (C–D) For ﬂow cytometry analysis, cells were labeled with Live/Dead Fixable Near-IR, ﬁxed, permeabilized, labeled for EWI-2, and stained with ﬂuorescent secondary antibody. (C) Representative histogram of the EWI-2 signal intensity normalized to mode in live cells for unstained controls (black line), shEWI-2 (blue), and shScr (red) cells. (D) EWI-2 MFI in live shScr (red) and shEWI-2 (blue) cells from three independent biological replicates, normalized to EWI-2-labeled parental CEM-SS cells (represented at a value of 1 with a dashed line). (E) CEM-luc fusion assays were performed using shScr or shEWI-2 producer cells infected with NL4-3, which were co-cultured with CEM-luc target cells in the presence of DMSO (vehicle control, luciferase signal from transmission and cell–cell fusion) or EFV (luciferase signal resulting exclusively from cell–cell fusion). Luminescence readings (across three independent biological replicates) from the EFV-treated condition were divided by the DMSO reading from the same producer cell type and multiplied by 100 to determine the percentage of luciferase expression dependent on cell–cell fusion (syncytium formation) between either shScr or shEWI-2 producer and CEM-luc target cells. 3.3. EWI-2 Inhibits HIV-1-Induced Syncytium Formation Values from the same biological replicate are linked by a black line. 3.3. EWI-2 Inhibits HIV-1-Induced Syncytium Formation Values from the same biological replicate are linked by a black line. 4. EWI-2 and CD81 Surface Expression is Restored on HIV-1-Induced Syncytia HIV 1 infected cells have been well documented to have altered surface expression proﬁ Figure 5. EWI-2 inhibits infected-uninfected cell fusion. (A) HeLa-TZM-bl fusion assays were performed using producer HeLa cells that were co-transfected with either pNL-sfGI ΔEnv (ΔEnv) or pNL-sfGI (WT) in combination with overexpression plasmid totaling 500 ng (using the indicated amount of CD81 or EWI-2 supplemented with L6). Luminescence readings (across 4 independent biological replicates, each with two technical replicates) were divided by the ΔEnv condition to obtain the fold increase in fusion, and then normalized to the WT co-transfected with only L6 (i.e., 0 ng of CD81 or EWI-2) condition, making it have a value of 1. Deviation from the dashed line at 1 thus indicates an effect on fusion. Values from the same biological replicate are linked by a grey line. (B– D) EWI-2 expression in shScramble (shScr) and shEWI-2 CEM-SS cells was analyzed by microscopy (B) and flow cytometry (C–D). (B) For microscopy, cells were plated onto poly-L-lysine-coated glass, fixed, permeabilized, labeled for EWI-2, and stained using fluorescent secondary antibody (magenta) and DAPI (cyan). Scale bars = 10 μm. (C–D) For flow cytometry analysis, cells were labeled with Live/Dead Fixable Near-IR, fixed, permeabilized, labeled for EWI-2, and stained with fluorescent secondary antibody. (C) Representative histogram of the EWI-2 signal intensity normalized to mode in live cells for unstained controls (black line), shEWI-2 (blue), and shScr (red) cells. (D) EWI-2 MFI Figure 5. EWI-2 inhibits infected-uninfected cell fusion. (A) HeLa-TZM-bl fusion assays were performed using producer HeLa cells that were co-transfected with either pNL-sfGI ∆Env (∆Env) or pNL-sfGI (WT) in combination with overexpression plasmid totaling 500 ng (using the indicated amount of CD81 or EWI-2 supplemented with L6). Luminescence readings (across 4 independent biological replicates, each with two technical replicates) were divided by the ∆Env condition to obtain the fold increase in fusion, and then normalized to the WT co-transfected with only L6 (i.e., 0 ng of CD81 or EWI-2) condition, making it have a value of 1. Deviation from the dashed line at 1 thus indicates an eﬀect on fusion. Values from the same biological replicate are linked by a grey line. (B–D) EWI-2 expression in shScramble (shScr) and shEWI-2 CEM-SS cells was analyzed by microscopy (B) and ﬂow cytometry (C–D). 3.3. EWI-2 Inhibits HIV-1-Induced Syncytium Formation Subsequently, shEWI-2 and shScramble cells were assayed for their ability to support HIV-1-induced cell–cell fusion with CEM-luc cells as target cells, using a previously reported assay that discriminates between the luciferase signal derived from active virus transmission and signal from cell–cell fusion [24,60]. Across three independent biological replicates, HIV-1-infected shEWI-2 cells were found to consistently form syncytia more frequently (between 1.5 and 2.3-fold) than HIV-1-infected shScramble cells (Figure 5E). Taken together, the accumulation of EWI-2 at the presynaptic terminal of the HIV-1 VS (Figures 1 and 2), the concomitant overall downregulation of EWI-2 in infected T cells (Figure 3), and the requirement for high EWI-2 expression for eﬃcient control of Env-induced cell–cell fusion (Figure 5) establish EWI-2 as a host fusion-inhibitory protein harnessed by HIV-1 during cell-to-cell virus transmission. 16 of 23 d) in NL- 16 of 23 d) in NL- Viruses 2019, 11, 1082 the overexpression p g p ( g ) Figure 5. EWI-2 inhibits infected-uninfected cell fusion. (A) HeLa-TZM-bl fusion assays were performed using producer HeLa cells that were co-transfected with either pNL-sfGI ΔEnv (ΔEnv) or pNL-sfGI (WT) in combination with overexpression plasmid totaling 500 ng (using the indicated amount of CD81 or EWI-2 supplemented with L6). Luminescence readings (across 4 independent biological replicates, each with two technical replicates) were divided by the ΔEnv condition to obtain the fold increase in fusion, and then normalized to the WT co-transfected with only L6 (i.e., 0 ng of CD81 or EWI-2) condition, making it have a value of 1. Deviation from the dashed line at 1 thus indicates an effect on fusion. Values from the same biological replicate are linked by a grey line. (B– Figure 5. EWI-2 inhibits infected-uninfected cell fusion. (A) HeLa-TZM-bl fusion assays were performed using producer HeLa cells that were co-transfected with either pNL-sfGI ∆Env (∆Env) or pNL-sfGI (WT) in combination with overexpression plasmid totaling 500 ng (using the indicated amount of CD81 or EWI-2 supplemented with L6). Luminescence readings (across 4 independent biological replicates, each with two technical replicates) were divided by the ∆Env condition to obtain the fold increase in fusion, and then normalized to the WT co-transfected with only L6 (i.e., 0 ng of CD81 or EWI-2) condition, making it have a value of 1. Deviation from the dashed line at 1 thus indicates an eﬀect on fusion. Values from the same biological replicate are linked by a grey line. 3.4. EWI-2 and CD81 Surface Expression is Restored on HIV-1-Induced Syncytia HIV-1-infected cells have been well documented to have altered surface expression proﬁles compared to uninfected cells (reviewed in [61]). However, previous analyses (including ours) were performed using bulk populations of HIV-1 infected cells, and thus could not or did not discriminate between mono- and multinucleated HIV-1-infected cells. HIV-1-induced syncytia likely have altered surface expression compared to mononucleated infected cells, as the process of syncytium formation (infected-uninfected cell fusion) provides a sudden inﬂux of yet-to-be downregulated host proteins 17 of 23 Viruses 2019, 11, 1082 contributed by the uninfected target cell upon membrane merger and cytoplasm mixing. Therefore, we chose to use microscopy to analyze the surface expression of EWI-2 and CD81 on HIV-1-infected cells in order to, for the ﬁrst time, conﬁdently discriminate between mononucleated infected cells and multinucleated HIV-1-induced syncytia. HIV-1-infected primary CD4+ T cells were cultured for three days post infection to allow time for syncytium formation. Infected cells were plated, surface-labeled for EWI-2 or CD81 on ice and ﬁxed prior to incubation with secondary antibody and imaging as before. The surface expression of each cell was quantiﬁed, normalized to internal uninfected controls, and data were segregated into populations of uninfected cells, mononucleated infected cells, and multinucleated infected cells (syncytia, identiﬁed as multinucleated by DAPI nuclear staining and positive for the viral reporter (GFP), as shown in representative images; Figure 6A). Strikingly, we found that syncytia had restored the surface expression of both EWI-2 and CD81 to nearly the same level as uninfected T cells found within the same wells (Figure 6B). Viruses 2019, 11, 1082 17 of 23 Figure 6. Syncytia have higher surface expression of EWI-2 and CD81 than mononucleated infected cells. (A) Primary CD4+ T cells were infected with NL-sfGI, surface-labeled for either EWI-2 or CD81 (both shown in magenta), fixed, stained with DAPI (cyan) and AlexaFluor 647-conjugated secondary antibody, and imaged. Infected cells were identified by GFP (yellow) and discriminated as mono- or multinucleated infected cells by DAPI. Representative cells are shown. Scale bars = 5 μm. (B) Cells were prepared as described in (A) and analyzed for EWI-2 or CD81 surface expression on uninfected cells, mononucleated infected cells (Mono) and syncytia (Syn) by manually selecting the plasma membrane at the midline of each cell and quantifying the mean EWI-2 or CD81-associated fluorescence intensity. 3.4. EWI-2 and CD81 Surface Expression is Restored on HIV-1-Induced Syncytia Raw fluorescence intensity values were background-subtracted using the fluorescence intensity of a cell-free area within the same image and subsequently normalized to the average intensity value of uninfected cells within the same imaging set. Data shown are the pooled normalized intensity values of two independent biological replicates, each with two technical replicates. Each data point represents the normalized surface MFI of an individual cell. Error bars = SD. p-values are the result of two-tailed non-parametric Mann-Whitney U tests. Figure 6. Syncytia have higher surface expression of EWI-2 and CD81 than mononucleated infected cells. (A) Primary CD4+ T cells were infected with NL-sfGI, surface-labeled for either EWI-2 or CD81 (both shown in magenta), ﬁxed, stained with DAPI (cyan) and AlexaFluor 647-conjugated secondary antibody, and imaged. Infected cells were identiﬁed by GFP (yellow) and discriminated as mono- or multinucleated infected cells by DAPI. Representative cells are shown. Scale bars = 5 µm. (B) Cells were prepared as described in (A) and analyzed for EWI-2 or CD81 surface expression on uninfected cells, mononucleated infected cells (Mono) and syncytia (Syn) by manually selecting the plasma membrane at the midline of each cell and quantifying the mean EWI-2 or CD81-associated ﬂuorescence intensity. Raw ﬂuorescence intensity values were background-subtracted using the ﬂuorescence intensity of a cell-free area within the same image and subsequently normalized to the average intensity value of uninfected cells within the same imaging set. Data shown are the pooled normalized intensity values of two independent biological replicates, each with two technical replicates. Each data point represents the normalized surface MFI of an individual cell. Error bars = SD. p-values are the result of Kruskal-Wallis tests. Figure 6. Syncytia have higher surface expression of EWI-2 and CD81 than mononucleated infected cells. (A) Primary CD4+ T cells were infected with NL-sfGI, surface-labeled for either EWI-2 or CD81 (both shown in magenta), fixed, stained with DAPI (cyan) and AlexaFluor 647-conjugated secondary antibody, and imaged. Infected cells were identified by GFP (yellow) and discriminated as mono- or multinucleated infected cells by DAPI. Representative cells are shown. Scale bars = 5 μm. (B) Cells were prepared as described in (A) and analyzed for EWI-2 or CD81 surface expression on uninfected cells, mononucleated infected cells (Mono) and syncytia (Syn) by manually selecting the plasma membrane at the midline of each cell and quantifying the mean EWI-2 or CD81-associated fluorescence intensity. 4. Discussion The transient alignment of infected (producer) and uninfected (target) cells allows for eﬃcient transmission of virus particles. However, because of the presence of viral Env and CD4/co-receptor at the surface of producer and target cell, respectively, rather than separating after particle transfer, these cells could also easily fuse with each other, thus forming a syncytium. This study now identiﬁes EWI-2 as a host protein that contributes to the maintenance of viral homeostasis through fusion inhibition. Our investigations were partially prompted by two recent reports. In one of those studies, Rubinstein and colleagues documented a role for EWI-F, a close relative of EWI-2, in myoblast fusion regulation [26]. EWI-F was shown to act as fusion repressor in cooperation with the tetraspanins CD9 and CD81. With the other study, Yáñez-Mó and colleagues [32] showed the presence of EWI-2 at sites of contact between uninfected T cells and T cells stably expressing HIV-1 Env. In separate experiments, HIV-1-infected EWI-2 knockdown cells were also shown to have somewhat increased virus production and the authors mentioned (as data not shown) that this was accompanied by augmented syncytium formation, indicating that EWI-2 could be involved in the regulation of HIV-1-induced membrane fusion. Importantly, however, the study did not address the question of whether the reported increase in syncytium formation was (potentially) caused by the action of EWI-2 in producer or target cells, nor did it provide a dissection of where EWI-2 accumulates (producer and/or target cells). The authors did speculate that EWI-2, together with α-actinin, might be active in target cells, there possibly contributing to α-actinin’s actin bundling activity, thus ultimately inhibiting virus entry/fusion. They also explicitly stated, however, that even if their speculation about where α-actinin acts during virus replication should eventually be conﬁrmed (with subsequent studies), they cannot exclude an involvement of the partner protein EWI-2 in the “subsequent steps of the viral life cycle”. Our study now reveals that EWI-2 indeed acts during the late phase of the HIV-1 replication cycle: It accumulates on the producer cell side of the VS (Figures 1 and 2). Surprisingly, unlike tetraspanins, which have fusion-inhibitory roles at both sides of the VS (and thus are present at both the viral pre- and post-synapse [22,62]), EWI-2 accumulates (and inhibits fusion) only at the presynaptic terminal of the VS. 3.4. EWI-2 and CD81 Surface Expression is Restored on HIV-1-Induced Syncytia Raw fluorescence intensity values were background-subtracted using the fluorescence intensity of a cell-free area within the same image and subsequently normalized to the average intensity value of uninfected cells within the same imaging set. Data shown are the pooled normalized intensity values of two independent biological replicates, each with two technical replicates. Each data point represents the normalized surface MFI of an individual cell. Error bars = SD. p-values are the result of two-tailed non-parametric Mann-Whitney U tests. Figure 6. Syncytia have higher surface expression of EWI-2 and CD81 than mononucleated infected cells. (A) Primary CD4+ T cells were infected with NL-sfGI, surface-labeled for either EWI-2 or CD81 (both shown in magenta), ﬁxed, stained with DAPI (cyan) and AlexaFluor 647-conjugated secondary antibody, and imaged. Infected cells were identiﬁed by GFP (yellow) and discriminated as mono- or multinucleated infected cells by DAPI. Representative cells are shown. Scale bars = 5 µm. (B) Cells were prepared as described in (A) and analyzed for EWI-2 or CD81 surface expression on uninfected cells, mononucleated infected cells (Mono) and syncytia (Syn) by manually selecting the plasma membrane at the midline of each cell and quantifying the mean EWI-2 or CD81-associated ﬂuorescence intensity. Raw ﬂuorescence intensity values were background-subtracted using the ﬂuorescence intensity of a cell-free area within the same image and subsequently normalized to the average intensity value of uninfected cells within the same imaging set. Data shown are the pooled normalized intensity values of two independent biological replicates, each with two technical replicates. Each data point represents the normalized surface MFI of an individual cell. Error bars = SD. p-values are the result of Kruskal-Wallis tests. Viruses 2019, 11, 1082 18 of 23 18 of 23 4. Discussion This leads us to speculate whether EWI-2 accumulation at the presynaptic terminal might contribute to unique intracellular signaling events in HIV-1-infected cells [32,63], such as tuning T cell receptor function. Paralleling what we previously documented for tetraspanins [22], we found that fusion with uninfected target cells was inhibited by EWI-2, and we established that it does so in a dose-dependent manner (Figure 5). Also analogous to our ﬁndings about tetraspanins [54,56], we demonstrate that while EWI-2 accumulates at the virological presynapse, overall this protein is downregulated in infected cells (Figure 3). Our proteomic analysis (Figure 4) now shows that EWI-2 depletion from the infected cell surface, as is also the case for tetraspanins [56,57], is primarily mediated by Vpu (Figure 4). Since EWI-2 is a known interactor of tetraspanins CD81 and CD9, it is possible that EWI-2 downregulation by Vpu (with or without Nef) is “direct” (e.g., the canonical Vpu “targets” BST2 and CD4, as well as SNAT1 [53]) or “indirect,” possibly through its association with tetraspanins. Note, this is also true of CD81/other tetraspanins, which may likewise be “direct” or “indirect” targets (e.g., by their association with EWI-2). Our data do not distinguish these possibilities, and further mechanistic studies would be required to delineate the detailed mechanism of Vpu-mediated depletion. It should also be noted that in Table S1 of [64], EWI-2 depletion in CEM-T4 cells is (somewhat) dependent on the expression of Vpr. The eﬀect size is modest and likely “indirect,” and does not contradict the Vpu and Nef data shown here. It does, however, suggest that the mechanism of EWI-2 depletion in HIV-1 infected T cells may be complex. Overall, the combination of these two features (enrichment during assembly and transmission at the VS, and regulation by HIV-1 accessory proteins in infected cells), together with the fusion-preventing functions, strongly suggests that a particular host factor plays an important role in virus replication. We expect that EWI-2 also inhibits the fusion of virus particles to target cells, as tetraspanins do [54,56,59], and we are currently testing that hypothesis (within the context of an extensive follow-up analysis aimed at dissecting the molecular determinants responsible for EWI-2′s fusion-inhibitory 19 of 23 Viruses 2019, 11, 1082 functions). 4. Discussion It seems likely that tetraspanins and EWI-2 are not only tolerated but indeed enriched at virus budding sites because the beneﬁt of cell–cell fusion inhibition at the VS is balanced against any negative eﬀect of a reduction in virus infectivity. This is demonstrated by the fact that, in a native (unmanipulated) context, it is simultaneously true that (A) HIV-1-infected T cells routinely exhibit enrichment of these fusion inhibitors at virus release sites, (B) that cell–cell fusion is relatively infrequent, and (C) that HIV-1 spreads eﬃciently in those cell cultures. As mentioned, while fusion inhibition operates at many levels and is orchestrated by HIV-1 proteins during infection, syncytia do nevertheless form, including in vivo [7–9] and when using a transmitted/founder (T/F) R5-tropic Env or even full-length replication-competent T/F virus [10,12]. However, these syncytia seem to remain small, at 4 or fewer nuclei and the vast majority having only two nuclei [9]. Very large syncytia (dozens to thousands of nuclei) are only induced by HIV-1 infection of certain T cell lines, especially Sup-T1 cells [65], or in vivo but only with the involvement of macrophage or dendritic cells [66–68]. It is therefore possible that T cell–T cell fusion is inhibited not only when a mononucleated infected cell encounters a target cell, but also when a syncytium encounters a target cell. An alternative explanation is that syncytia may be less viable as they grow larger, though some evidence contradicts that [69]. Here, we present evidence that host fusion-inhibitory proteins EWI-2 and CD81 are present at higher levels on the surface of small T cell syncytia when compared to mononucleated infected cells in the same culture. Because we ﬁnd that the fusion-inhibitory capacity of EWI-2 and CD81 is also dose-dependent, it would therefore be expected that a higher “dose” of EWI-2 and/or CD81 in syncytia would make them less likely to undergo cell–cell fusion a second (or third) time. We are currently formally testing this hypothesis, and also investigating the surface levels on syncytia of other host proteins normally downregulated upon HIV-1 infection. Without implicating any particular fusion-inhibitory protein, we have in the past found evidence that indeed fusion-inhibitory factors may also be acting at syncytium-target cell VSs [9]: in Movie S7 of that report, we showed an example of a small syncytium containing two nuclei undergoing cell–cell fusion and acquiring a third nucleus. Author Contributions: E.E.W., N.J.M., M.S., and M.T. conceived and designed the experiments. E.E.W. performed the experiments and analyzed the results, with contributions by M.S. and P.B.M. in Figure 1. N.J.M. performed the proteomics and analyzed the results in Figures 3A and 4. S.P. performed the FG12 vector modiﬁcation to remove the GFP reporter cassette. E.E.W., N.J.M., and M.S. prepared the ﬁgures. E.E.W., N.J.M., M.S., and M.T. wrote and edited the manuscript. References 1. Jolly, C.; Kasheﬁ, K.; Hollinshead, M.; Sattentau, Q.J. HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse. J. Exp. Med. 2004, 199, 283–293. [CrossRef] [PubMed] 1. Jolly, C.; Kasheﬁ, K.; Hollinshead, M.; Sattentau, Q.J. HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse. J. Exp. Med. 2004, 199, 283–293. [CrossRef] [PubMed] 2. Hubner, W.; McNerney, G.P.; Chen, P.; Dale, B.M.; Gordon, R.E.; Chuang, F.Y.; Li, X.D.; Asmuth, D.M.; Huser, T.; Chen, B.K. Quantitative 3D video microscopy of HIV transfer across T cell virological synapses. Science 2009, 323, 1743–1747. [CrossRef] [PubMed] 3. Ladinsky, M.S.; Kieﬀer, C.; Olson, G.; Deruaz, M.; Vrbanac, V.; Tager, A.M.; Kwon, D.S.; Bjorkman, P.J. Electron tomography of HIV-1 infection in gut-associated lymphoid tissue. PLoS Pathog. 2014, 10, e1003899. [CrossRef] [PubMed] 3. Ladinsky, M.S.; Kieﬀer, C.; Olson, G.; Deruaz, M.; Vrbanac, V.; Tager, A.M.; Kwon, D.S.; Bjorkman, P.J. Electron tomography of HIV-1 infection in gut-associated lymphoid tissue. PLoS Pathog. 2014, 10, e1003899. [CrossRef] [PubMed] 4. Reh, L.; Magnus, C.; Schanz, M.; Weber, J.; Uhr, T.; Rusert, P.; Trkola, A. Capacity of Broadly Neutralizing Antibodies to Inhibit HIV-1 Cell-Cell Transmission Is Strain- and Epitope-Dependent. PLoS Pathog. 2015, 11, e1004966. [CrossRef] [PubMed] 4. Reh, L.; Magnus, C.; Schanz, M.; Weber, J.; Uhr, T.; Rusert, P.; Trkola, A. Capacity of Broadly Neutralizing Antibodies to Inhibit HIV-1 Cell-Cell Transmission Is Strain- and Epitope-Dependent. PLoS Pathog. 2015, 11, e1004966. [CrossRef] [PubMed] 5. Bracq, L.; Xie, M.; Benichou, S.; Bouchet, J. Mechanisms for Cell-to-Cell Transmission of HIV-1. Front. I 2018, 9, 260. [CrossRef] [PubMed] 6. Imle, A.; Kumberger, P.; Schnellbacher, N.D.; Fehr, J.; Carrillo-Bustamante, P.; Ales, J.; Schmidt, P.; Ritter, C.; Godinez, W.J.; Muller, B.; et al. Experimental and computational analyses reveal that environmental restrictions shape HIV-1 spread in 3D cultures. Nat. Commun. 2019, 10, 2144. [CrossRef] 7. Orenstein, J.M. In vivo cytolysis and fusion of human immunodeﬁciency virus type 1-infected lymphocytes in lymphoid tissue. J. Infect. Dis. 2000, 182, 338–342. [CrossRef] 8. Murooka, T.T.; Deruaz, M.; Marangoni, F.; Vrbanac, V.D.; Seung, E.; von Andrian, U.H.; Tager, A.M.; Luster, A.D.; Mempel, T.R. HIV-infected T cells are migratory vehicles for viral dissemination. Nature 2012, 490, 283–287. [CrossRef] 9. Symeonides, M.; Murooka, T.T.; Bellfy, L.N.; Roy, N.H.; Mempel, T.R.; Thali, M. HIV-1-Induced Small T Cell Syncytia Can Transfer Virus Particles to Target Cells through Transient Contacts. Viruses 2015, 7, 6590–6603. [CrossRef] 10. Viruses 2019, 11, 1082 Viruses 2019, 11, 1082 Viruses 2019, 11, 1082 Acknowledgments: The ﬂow cytometry data we presented were obtained at the Harry Hood Bassett Flow Cytometry and Cell Sorting Facility, Larner College of Medicine, University of Vermont. The imaging shown in Figure 1A was performed at the Imaging/Physiology Core Facility, Neuroscience Center of Biomedical Research Excellence, Larner College of Medicine, University of Vermont. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Discussion Subsequently, that syncytium encountered uninfected target cells and transferred virus particles to them through close contact but did not undergo further cell–cell fusion and instead fully separated from them despite exhibiting the ability to fuse only hours earlier. We can now speculate that, as a result of the cell–cell fusion event we captured at the beginning of that sequence, this syncytium likely acquired a dose of EWI-2 and/or CD81, which subsequently allowed the syncytium to mediate cell-to-cell virus transfer at the VS without further cell–cell fusion. Finally, repressing HIV-1 Env-induced cell–cell fusion not only allows for a continued increase in the number of infected cells (as that number doubles each time producer and target cells separate after virus transmission), but keeping Env’s fusion activity at bay may also be beneﬁcial for the virus for other reasons. For instance, we and others have recently shown that lowering Env’s fusion activity also allows HIV-1 to overcome a restriction factor (APOBEC3G; [60]), and even antiviral drugs [70]. Further, large syncytia, that could form if Env-induced cell–cell fusion is uncontrolled, are likely prone to be attacked by innate immune cells. It is therefore critical that HIV-1 recruits fusion-inhibitory host factors such as EWI-2 to the VS to prevent excess cell–cell fusion and keep T cell syncytia small when they do form. Author Contributions: E.E.W., N.J.M., M.S., and M.T. conceived and designed the experiments. E.E.W. performed the experiments and analyzed the results, with contributions by M.S. and P.B.M. in Figure 1. N.J.M. performed the proteomics and analyzed the results in Figures 3A and 4. S.P. performed the FG12 vector modiﬁcation to remove the GFP reporter cassette. E.E.W., N.J.M., and M.S. prepared the ﬁgures. E.E.W., N.J.M., M.S., and M.T. wrote and edited the manuscript. Funding: The work was supported by the National Institutes of Health (R01-GM117839 to M.T., P30-RR032135 and P30-GM103498 for the Neuroscience COBRE Imaging Facility), the University of Vermont Larner College of Medicine (Bridge Support Grant to M.T.), the University of Vermont Department of Microbiology & Molecular Genetics (Nicole J. Ferland Award to S.P.), the Medical Research Council (CSF MR/P008801/1 to N.J.M.), NHS Blood and Transplant (WPA15-02 to N.J.M.), the NIHR Cambridge BRC, and a Wellcome Trust Strategic Award to CIMR. 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https://openalex.org/W4200277556	https://periodicos.ufsc.br/index.php/mundosdotrabalho/article/download/80841/47952	Portuguese	null	O “Perigo Vermelho” em Americana (SP): a trajetória de Romeu Sturari, por uma história de lutas dos trabalhadores de Vila Operária de Carioba (1947-1967)	Revista Mundos do Trabalho	2,021	cc-by	12,421	O “Perigo Vermelho” em Americana (SP): a trajetória de Romeu Sturari, por uma história de lutas dos trabalhadores de Vila Operária de Carioba (1947-1967) O “Perigo Vermelho” em Americana (SP): a trajetória de Romeu Sturari, por uma história de lutas dos trabalhadores de Vila Operária de Carioba (1947-1967) The “Red Danger” in Americana (SP): the trajectory of Romeu Sturari, for a history of struggles of the workers of Vila Operária de Carioba (1947-1967) ∗ Mestrando em História Social do Trabalho pela Universidade Estadual de Campinas (Unicamp). Bolsista do Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). E-mail: luiscarboni.2@gmail.com. ORCID: https://orcid.org/0000-0002-2373-5312. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 municipality and, finally, the situation experienced by workers during the Carioba’s Great Strike in the mid-1960s. Keywords: Labor Movement (Americana); Worker Village of Fabrics Factory of Carioba; Romeu Sturari (1918-1978). Keywords: Labor Movement (Americana); Worker Village of Fabrics Factory of Carioba; Romeu Sturari (1918-1978). rds: Labor Movement (Americana); Worker Village of Fabrics Factory of Carioba; Sturari (1918-1978). Luís Henrique Carboni Junior∗ Resumo: Este artigo investiga a trajetória de Romeu Sturari, tecelão da Fábrica de Tecidos Carioba e morador em Vila Operária de Carioba, como meio de acesso ao conhecimento histórico sobre a organização do movimento operário em Vila Operária e em Americana (SP). Através de sua trajetória, pretende-se aferir: a influência do Partido Comunista Brasileiro (PCB) na cidade, a ação do sindicato têxtil, os modos de organização e de luta adotados pelos trabalhadores nas diferentes conjunturas, os efeitos do golpe militar de 1964 no município e, finalmente, a situação experienciada pelos trabalhadores durante a Grande Greve de Carioba em meados da década de 1960. Palavras-chave: Movimento operário (Americana); Vila Operária da Fábrica de Tecidos Carioba; Romeu Sturari (1918-1978). Abstract: This article investigates the trajectory of Romeu Sturari, weaver in Fabrics Factory of Carioba and residente in Worker Village of Carioba, as a means of accessing historical knowledge about the organization of the Labor Movement in the Worker Village and Americana (SP). With this trajectory we intend to measure: the influence of the Brazilian Communist Party (PCB) in the city, the action of Textile Syndicate, the ways of organization and struggle adopted by workers in diferents circunstances, the affects of the Military Coup of 1964 in the Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 1 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 1 Todas as citações diretas sem referência são retiradas dos documentos produzidos pelas autoridades policiais da Delegacia de Ordem Política e Social (São Paulo) em conjunto com a Delegacia de Americana. 2 P t A i A i Públi d E t d d Sã P l F d DEOPS/SP g ( ) j g asta Americana. Arquivo Público do Estado de São Paulo, Fundos: DEOPS/SP. p 5 Agradecimentos especiais a Vanessa Parazzi, neta de Romeu Sturari, que gentilmente cedeu fotografias e documentos pessoais do acervo particular da família. 1 Todas as citações diretas sem referência são retiradas dos documentos produzidos pelas autoridades policiais da Delegacia de Ordem Política e Social (São Paulo) em conjunto com a Delegacia de Americana. 2 Pasta Americana. Arquivo Público do Estado de São Paulo, Fundos: DEOPS/SP. 3 No sentido de que Romeu Sturari é um indivíduo capaz de representar uma coletividade em diferentes conjunturas históricas. 4 Um levantamento prévio dessa documentação foi apresentado em Simpósio Temático do IV Encontro Estadual da ANPUH/Amapá, 2020. 5 Agradecimentos especiais a Vanessa Parazzi, neta de Romeu Sturari, que gentilmente cedeu fotografias e documentos pessoais do acervo particular da família. 1 Todas as citações diretas sem referência são retiradas dos documentos produzidos pelas autoridades policiais da Delegacia de Ordem Política e Social (São Paulo) em conjunto com a Delegacia de Americana. 2 Pasta Americana. Arquivo Público do Estado de São Paulo, Fundos: DEOPS/SP. 3 No sentido de que Romeu Sturari é um indivíduo capaz de representar uma coletividade em diferentes j t hi tó i 4 Um levantamento prévio dessa documentação foi apresentado em Simpósio Temático do IV Encontro Estadual da ANPUH/Amapá, 2020. 5 Agradecimentos especiais a Vanessa Parazzi, neta de Romeu Sturari, que gentilmente cedeu fotografias e documentos pessoais do acervo particular da família. q , entido de que Romeu Sturari é um indivíduo capaz de representar uma coletividade em diferentes unturas históricas. evantamento prévio dessa documentação foi apresentado em Simpósio Temático do IV Encontro Estadual NPUH/Amapá, 2020. 1 Todas as citações diretas sem referência são retiradas dos documentos produzidos pelas autoridades O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) O artigo está dividido em sete principais seções, conforme critérios temáticos e temporais, mesclando momentos importantes da política nacional e da trajetória de vida do tecelão junto aos companheiros de trabalho: a primeira parte pretende esclarecer aspectos gerais da vila operária e expor a metodologia e objetivos de se investigar Romeu Sturari como meio de compreender tanto o regime patronal existente em Carioba quanto as relações dos trabalhadores com a política local e nacional. Posteriormente, é investigada a ação de Sturari em Carioba, tendo como base a recente legalização do PCB, as possibilidades eleitorais e os meios de propaganda e luta adotados pelo militante. O terceiro tópico aborda o período de clandestinidade de Sturari, condenado, pela primeira vez, na Lei de Segurança Nacional de 1953. O quarto tópico trata do período pós-anistia, quando Sturari retoma sua militância, priorizando o viés sindical. A quinta sessão investiga os impactos do golpe militar de 1964 e da caça aos comunistas para o movimento operário e para política local, processo que acarretou uma segunda condenação na Lei de Segurança Nacional. O penúltimo tópico aborda a conjuntura da Grande Greve de Carioba, privilegiando reconhecer os motivos do embate entre as classes, bem como os recursos de negociação adotados pelos operários. Por fim, a conclusão pretende retomar as constatações que a trajetória de Romeu Sturari nos permitiu aferir sobre as movimentações trabalhistas em Vila Operária de Carioba. Introdução D etido quando iria visitar sua família em Americana, no dia 25 de fevereiro de 1965, encontrava-se na Delegacia de Ordem Política e Social de São Paulo (DOPS), o até então foragido, Romeu Sturari. Tecelão de instrução primária, descendente de italianos, 1,75 m, contando 46 anos, casado com Genny Polido Sturari, era pai de 6 filhos e avô de 2 netos, mas que “desde jovem atuava como agitador”.1 Sua última prisão, decretada dois meses após o golpe militar, tinha como base a Lei de Segurança Nacional nº 1.802/53, seu crime consistia na “implantação do comunismo peleguista nos meios sindicais, forjando greves, forjando a luta de classes, e incutindo no trabalhador americanense as ideias esquerdistas”. Lembrava-se de ter sido detido por “quatro ou cinco vezes” pela polícia política e social, sendo identificado pelas autoridades como aquele que “participou e provou todas as greves de Americana”, era o “chefia dos comunistas americanenses”. D e e Menos de uma semana após o golpe militar, Sturari fugiu para São Paulo temendo ser preso, pois era “velho conhecido” das autoridades e de toda a população de Americana por atividades comunistas “em praça pública”. Quando detido, ocupava o cargo de presidente do Sindicato dos Trabalhadores na Indústria de Fiação e Tecelagem de Americana, desde dezembro de 1958; era juiz vogal dos trabalhadores na Junta de Conciliação e Julgamento da Justiça do Trabalho desde 1962; e, por fim, suplente no conselho da Federação dos Trabalhadores nas Indústrias de Fiação e Tecelagem do Estado de São Paulo. Mas sua trajetória junto aos trabalhadores americanenses nos arquivos do DOPS2 remonta a meados da década de 1940, podendo ser descrita literariamente como uma epopeia.3 Seu nome é citado uma centena de vezes na Pasta Americana, de 1947 a 1965. Há, também, menções de suas atividades no Dossiê Comunismo – PCB, bem como na imprensa, seja ela local ou operária. Sendo assim, são essas as principais fontes documentais4 que permitem a investigação de sua trajetória e a escrita do artigo.5 p , 5 Agradecimentos especiais a Vanessa Parazzi, neta de Romeu Sturari, que gentilmente cedeu fotografias e documentos pessoais do acervo particular da família. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Luís Henrique Carboni Junior Nas primeiras décadas do século XX, Carioba consolidou-se como independente polo urbano-industrial, concentrando a vida operária de Americana, reconhecida como “verdadeira cidadezinha”. A infraestrutura era ampla: havia a Sociedade de Mútuo Socorro; o Clube Recreativo e Sportivo de Carioba, que além do time de futebol contava com biblioteca, eventos de jogos e dança, banda e desfiles de carnaval; bem como o Clube de Regatas e Natação Carioba. No âmbito educacional e cultural havia o Grupo Escolar Carioba e o Cine Carioba. Assim, com a expansão fabril e oferta de empregos, Carioba tornou-se receptora de trabalhadores, ocupando, por mais de meio século, o posto de maior indústria da cidade, influenciando o curso e o ritmo do desenvolvimento de Americana. Na década de 1940, a cidade viveu um processo de multiplicação de centenas de pequenas fabriquetas têxteis, as chamadas facções, destinadas à produção de rayon, setor que, apesar das sucessivas crises, era a manufatura dominada pelos tecelões, dada a implementação pela Fábrica de Tecidos Carioba. Outro modo de influência deu-se nos cargos dos poderes Executivo e Legislativo da Câmara Municipal; todas as diferentes administrações da Carioba ocuparam cargos públicos. A popularidade política de seus administradores tem explicações na relação de trabalho estabelecida em Carioba, definida como paternalismo industrial, uma vez que os administradores residiam próximos à vila e transitavam pelos mesmos locais que os trabalhadores, permitindo interação e convívio entre as classes, o que contribuía para a construção de uma falsa concepção de harmonia entre patrão e empregado. Essa acessibilidade ao patrão fazia com que muitos trabalhadores os vissem como “compadres”. Outro fator importante era o “peculiar regime de política social [...] Geopoliticamente formando quase uma cidade com vida independente, têm os operários casa, luz e água fornecidos pelo patrão”. Havia, desse modo, uma concepção entre os trabalhadores de dívida de lealdade com os empregadores, provedores do bem-estar e dos meios de subsistência. A ampla variedade de atividades recreativas, a modernização, o sentimento de pertencimento e a falsa sensação de igualdade classista possibilitaram aos trabalhadores urdirem uma memória idílica sobre Vila Carioba. O sentimento de orgulho em se denominar cariobense6 revela, por meio de memorialistas e relatos orais, o imaginário de um lugar alheio aos conflitos de classe. Romeu Sturari e Vila Operária de Carioba: metodologia e objetivos Nascido no dia 12 de novembro de 1918, em Americana, Romeu Sturari iniciou sua vida operária em 1932, quando mudou-se com sua família para a Vila Operária de Carioba, propriedade da Fábrica de Tecidos Carioba. Lá, aos 14 anos, adquiriu saberes que o permitiram ascender ao cargo de tecelão. Sua trajetória profissional era semelhante à de muitos outros trabalhadores da tecelagem, “vindos de fazendas e sítios e que aqui [...] aprenderam a se tornar tecelões”. Localizada entre as margens do rio Piracicaba e do ribeirão Quilombo, no interior de São Paulo, a Fábrica de Tecidos Carioba foi um dos primeiros grandes empreendimentos industriais do ramo têxtil no Brasil. Fundada em 1875 por iniciativa dos irmãos Antônio e Augusto Souza e Queiroz, em sociedade com William Pultney Talston, a tecelagem passou por diversas administrações. Em 1882, a fábrica foi vendida para os irmãos ingleses Clement e Jorge Wilmont, os quais iniciaram a construção da Vila Operária de Carioba. Entretanto, de 1886 a 1901, a fábrica não operou até ser arrematada pela Rawlinson Müller e Companhia, principais responsáveis pela expansão e consolidação da vila operária. Em 1918, a vila concentrava mais de 220 casas operárias, sendo habitada, em sua maioria, por imigrantes italianos ou descendentes. Vale ressaltar que o recorte temporal abarcado pela pesquisa se concentra na administração do Grupo J. J. Abdalla, que se estendeu de 1944 até 1976. 3 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 7 NEGRO, Antonio Luigi; SILVA, Fernando Teixeira da. Trabalhadores, sindicatos e política (1945-1964). In: FERREIRA, Jorge; DELGADO, Lucilia de Almeira (org.). O Brasil Republicano: o tempo da experiência democrática. Rio de Janeiro: Civilização Brasileira, 2003. p. 89. 6 Os próprios americanenses se diferenciam, destacando seu local de origem, denominando-se cariobense aquele que nasceu ou por muito tempo viveu em Vila Operária de Carioba. 6 Os próprios americanenses se diferenciam, destacando seu local de origem, denominando-se cariobense aquele que nasceu ou por muito tempo viveu em Vila Operária de Carioba. 7 NEGRO, Antonio Luigi; SILVA, Fernando Teixeira da. Trabalhadores, sindicatos e política (1945-1964). In: FERREIRA Jorge; DELGADO Lucilia de Almeira (org ) O Brasil Republicano: o tempo da experiência 6 Os próprios americanenses se diferenciam, destacando seu local de origem, denominando-se cariobense aquele que nasceu ou por muito tempo viveu em Vila Operária de Carioba. 7 NEGRO, Antonio Luigi; SILVA, Fernando Teixeira da. Trabalhadores, sindicatos e política (1945-1964). In: FERREIRA, Jorge; DELGADO, Lucilia de Almeira (org.). O Brasil Republicano: o tempo da experiência democrática. Rio de Janeiro: Civilização Brasileira, 2003. p. 89. 8 GINZBURG, Carlo; PONI, Carlo. O nome e o como: troca desigual e mercado historiográfico. In: GINZBURG, Carlo; CASTELNUOVO, Enrico; PONI, Carlo. A micro-história e outros ensaios. Lisboa: Difel, 1989. p. 171. 9 Ibidem, p. 174-175. 10 SCHMIDT, Benito Bisso. História e biografia. In: CARDOSO, Ciro; VAINFAS, Ronaldo (org.). Novos domínios da história. Rio de Janeiro: Elsevier, 2012. p. 195. 11 Ibidem, p. 193-194. 12 SCHMIDT, Benito Bisso. Trajetórias e vivências: as biografias na historiografia do movimento operário brasileiro. Projeto História, São Paulo, v. 16, 1998, p. 238. 13 LORIGA, Sabina. A biografia como problema. In: REVEL, Jacques (org). Jogos de escalas: a experiência da microanálise. Rio de Janeiro: Editora FGV, 1998. p. 249. 14 LEVI Giovanni. Un problema de escala. Relaciones: Estudios de historia y sociedade, Mexico, v. 24, n. 95, ver. 2003, p. 282-284. 8 GINZBURG, Carlo; PONI, Carlo. O nome e o como: troca desigual e mercado historiográfico. In: GINZBURG, Carlo; CASTELNUOVO, Enrico; PONI, Carlo. A micro-história e outros ensaios. Lisboa: Difel, 1989. p. 171. 9 Ibidem, p. 174-175. 8 GINZBURG, Carlo; PONI, Carlo. O nome e o como: troca desigual e mercado historiográfico. In: GINZBURG, Carlo; CASTELNUOVO, Enrico; PONI, Carlo. A micro-história e outros ensaios. Lisboa: Difel, 1989. p. 171. 9 Ibidem, p. 174-175. 10 SCHMIDT, Benito Bisso. História e biografia. In: CARDOSO, Ciro; VAINFAS, Ronaldo (org.). Novos domínios da história. Rio de Janeiro: Elsevier, 2012. p. 195. 11 Ibidem, p. 193-194. 12 SCHMIDT Benito Bisso Trajetórias e vivências: as biografias na historiografia do movimento operário Luís Henrique Carboni Junior Contudo, a ação de Romeu Sturari junto aos trabalhadores, presente na documentação policial, revela a luta classista, com seus influxos e refluxos, de reivindicações dos trabalhadores, entre 1947 e 1967, empregando diferentes estratégias político-organizacionais e modos de luta por seus direitos, sendo Carioba o principal foco de organização operária. Como veremos, o meio sindical, mas não somente, consolidou-se como importante via para o questionamento do comando patronal e ruptura com a suposta dívida de lealdade.7 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) Foi após a aquisição da Fábrica de Tecidos Carioba pelo Grupo J. J. Abdalla que as greves e a perseguição a Sturari se tornaram constantes. A trajetória do comunista e sindicalista juntamente com a organização dos trabalhadores de Vila Carioba e de Americana aparecem entrelaçadas nos arquivos da repressão. Investigar o tecelão que dá título ao artigo não se justifica como um fim em si mesmo, mas pretende-se adotar o método onomástico como bússola para guiar a pesquisa nos arquivos, possibilitando a reunião de documentos e eventos aparentemente desconectados.8 Não obstante, optar por circunscrever a investigação a um determinado local (Vila Carioba), a um determinado indivíduo (Romeu Sturari) e durante um curto espaço temporal (1947-1967) são elementos que possibilitam sobrepor a série documental reunida e encontrar o mesmo indivíduo em diferentes contextos sociais, agindo a partir de diferentes estratégias.9 Escolher Romeu Sturari justifica-se, ainda, na suposição de que seja capaz de representar vários outros trabalhadores americanenses derrotados em seus projetos políticos e sociais.10 Ao resgatar o papel da ação individual na construção dos processos históricos, o historiador E. P. 8 GINZBURG, Carlo; PONI, Carlo. O nome e o como: troca desigual e mercado historiográfico. In: GINZBURG, Carlo; CASTELNUOVO, Enrico; PONI, Carlo. A micro-história e outros ensaios. Lisboa: Difel, 1989. p. 171. 9 Ibidem, p. 174-175. 10 SCHMIDT, Benito Bisso. História e biografia. In: CARDOSO, Ciro; VAINFAS, Ronaldo (org.). Novos domínios , p 14 LEVI Giovanni. Un problema de escala. Relaciones: Estudios de historia y sociedade, Mexico, v. 24, n. 95, ver. 2003, p. 282-284. Luís Henrique Carboni Junior Thompson tinha como objetivo recuperar tradições e projetos contestatórios derrotados, estabelecendo uma relação dialética entre a ação individual e as implicações sociais decorrentes, de acolhimento ou repressão.11 Enquanto Eric Hobsbawm, por sua vez, utilizava seu agente histórico como microcosmos para a compreensão de problemas mais amplos da historiografia do movimento operário e do tempo em que o indivíduo viveu.12 Nas palavras de Sabina Loriga, investigar movimentos individuais nos permite: [...] romper as homogeneidades aparentes (por exemplo, a instituição, a comunidade ou o grupo social) e revelar os conflitos que presidiram a formação e a edificação das práticas culturais: penso na inércia e na ineficácia normativas, mas também nas incoerências que existem entre as diferentes normas, e na maneira pela qual os indivíduos, ‘façam’ eles ou não a história, moldam e modificam as relações de poder.13 [...] romper as homogeneidades aparentes (por exemplo, a instituição, a comunidade ou o grupo social) e revelar os conflitos que presidiram a formação e a edificação das práticas culturais: penso na inércia e na ineficácia normativas, mas também nas incoerências que existem entre as diferentes normas, e na maneira pela qual os indivíduos, ‘façam’ eles ou não a história, moldam e modificam as relações de poder.13 Diante de tais premissas metodológicas, as ações humanas passam a ter peso decisivo na investigação de grupos bem delimitados no tempo e no espaço, devendo-se recompor as redes de interação entre os indivíduos e o contexto que os cerca para compreender as escolhas de estratégias de ação. Os modos de luta de que lançam mão não possuem dinâmica autônoma, pois, além do jogo relacional social, sofrem interferência das estruturas normativas. Trata-se de analisar como os trabalhadores lutavam dentro dos interstícios de liberdade existentes nas incoerências das normas.14 p 14 LEVI Giovanni. Un problema de escala. Relaciones: Estudios de historia y sociedade, Mexico, v. 24, n. 95, ver. 2003, p. 282-284. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Luís Henrique Carboni Junior Desse modo, ao darmos a devida atenção à Vila Carioba, temos maiores chances de desvendar os sistemas e os modos de exercício de poder entre as classes diante de um quadro empírico, privilegiando a trajetória como via de acesso para recomposição das tensões existentes entre o individual e o social em Carioba.15 Em suma, pretende-se investigar a agência de Romeu Sturari como prisma de observação para lançar novas perspectivas sobre a formação da classe operária de Americana. Portanto, o objetivo do artigo é evidenciar a existência, em Vila Carioba, de disputas classistas e projetos políticos dos trabalhadores, que sucumbiram durante o processo histórico, propondo um revisionismo, na memória e na historiografia sobre o município, sobre a agência dos trabalhadores. Para tanto, iremos recuperar agentes sociais e eventos históricos até então excluídos e silenciados. Além disso, investigar essa conjuntura nos permite aferir sobre a atuação do Partido Comunista Brasileiro (PCB) em Americana, a repressão política e ideológica durante o período de redemocratização, os efeitos do golpe militar na cidade e, por fim, analisar brevemente a “Grande Greve” de Carioba. , p , , p 16 NEGRO; SILVA, op. cit., p. 49-50. 15 SCHMIDT, op. cit., 1998, p. 139-140. 17 Ibidem, p. 51. 17 Ibidem, p. 51. 18 Hoje, São Paulo, 19 out. 1947. j , , 19 NEGRO; SILVA, op. cit., p. 56. “Perigo Vermelho”: o PCB em Americana (1947-1953) O período de redemocratização foi marcado pela industrialização rápida e migração de trabalhadores do campo para a indústria. Ao passo que, para os governantes e industriais, a figura do “trabalhador cidadão” não deveria ser almejada nesse perfil do migrante, pois era tido como servo do atraso, de ação histórica inepta, incapaz de inventar suas tradições sociais, culturais ou políticas de forma independente. Assim, o jovem operariado urbano, imaturo e sem tradição trabalhista, deveria ser assistido e vigiado pelo poder público, para preservar seu eminente caráter dócil, cordial e ordeiro. Portanto, o trabalhador tinha de ser protegido pelo Estado, via Ministério do Trabalho, que estaria incumbido de o resguardar tanto da luta classista quanto da exploração capitalista.16 Todavia, os trabalhadores de Americana, em especial Romeu Sturari, não se comportaram de modo inerte, tomando iniciativas na organização política e reivindicativa dos direitos da classe trabalhadora. A redemocratização que marcava o fim do sufocamento político “se abria para os trabalhadores com esperança de justiça e liberdade [...] A volta à democracia implicava invenção de direitos, que por sua vez implicava exigência de seu cumprimento, que dependia da organização e participação dos trabalhadores”.17 É nesse contexto que trabalhadores lançaram empreitadas eleitorais, envolvendo-se com projetos políticos até então ilegais, como o do PCB. Diante das novas possibilidades, Romeu Sturari concorreu a deputado estadual pelo PCB em janeiro de 1947. A partir de então, o anonimato do tecelão nos arquivos do DOPS Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 O “Perigo Vermelho” em Americana (SP) 20 Ibidem, p. 59. 21 Ibidem, p. 60 22 Hoje, São Paulo, 19 out. 1947. 23 Jornal do Commercio, Rio de Janeiro, 30 mar. 1950. (Fábrica de Tecidos Carioba recorre às decisões do TRT, pedido negado). 24 Correio Paulistano, São Paulo, 3 dez. 1951. (Romeu Sturari apela contra Fábrica de Tecidos Carioba, pedido negado). O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) terminou, entrando definitivamente na mira das autoridades. Contudo, ser anônimo para as autoridades não significava estar alheio ao movimento dos trabalhadores, pois, conforme o jornal Hoje, quando se candidatou, Sturari era “destacado dirigente sindical [...] tendo obtido expressiva votação”,18 apesar de não ter sido eleito. Com a legalização, o PCB buscou consolidar-se como um partido ordeiro, respeitando a via eleitora, todavia, associavam a luta pela liberdade democrática com os direitos da classe trabalhadora.19 Entretanto, antes das eleições municipais ocorrerem em novembro, o então presidente Dutra viu-se obrigado a colocar o Brasil na dinâmica da Guerra Fria para manter boas relações internacionais com os Estados Unidos, decretando, novamente, a ilegalidade do PCB e intervenções do Ministério do Trabalho nos sindicatos, sob o pretexto de que essas instituições não deveriam interferir nos movimentos sociais. Com a crescente pressão, o governador de São Paulo Ademar de Barros aceitou a caça aos comunistas, tornando-se traidor aos olhos dos trabalhadores que outrora lhe apoiaram e o elegeram.20 Em decorrência da ilegalidade, sedes do PCB foram invadidas e fechadas pela polícia, como ocorrido com o Comitê Municipal do Partido Comunista de Americana, do qual Sturari declarou-se secretário político. Durante a ação policial, foram apreendidas 34 fichas de filiação de americanenses, incluindo nove mulheres e oito residentes de Vila Carioba. Imagem I: Ficha de filiação ao PCB de Rosa Sturari, moradora de Vila Carioba Fonte: Dossiê Comunismo - PCB, Delegacia de Ordem Política e Social. Imagem I: Ficha de filiação ao PCB de Rosa Sturari, moradora de Vila Carioba Fonte: Dossiê Comunismo - PCB, Delegacia de Ordem Política e Social. 20 Ibidem, p. 59. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Luís Henrique Carboni Junior Órfãos de partido, muitos dos então filiados ao PCB migraram para o Partido Social Trabalhista (PST) como meio de viabilizar as candidaturas municipais.21 Graças ao acordo político entre “comunistas e democratas de Americana”,22 os comunistas candidatos a vereador foram lançados sob a legenda do PST, sendo recebidos com “caloroso apoio popular”. Conforme o DOPS, Sturari, o “candidato de Prestes” em ambas as eleições, tinha como pretensão angariar votos da imagem de “líder dos operários têxteis [...] na luta pelas reivindicações da classe, que lhe valeu perseguições dos empregadores”. Passadas as eleições, em dezembro de 1947, Sturari foi afastado de sua função de tecelão para responder ao processo administrativo instaurado pelos Abdalla. A justificativa era de que o empregado atuava “contra os interesses da empresa”, pois era “elemento agitador e incitador de greve”. O processo estendeu-se até 1951, conforme o Jornal do Commercio23 e o Correio Paulistano,24 visto que ambas as partes tentaram recorrer às decisões promulgadas pelo Tribunal Regional do Trabalho. Por fim, foi demitido, mas recebeu todos os valores que lhes eram legalmente cabíveis. Segundo as autoridades, dois anos após seu afastamento não exerceu nenhuma outra atividade profissional, dedicando-se somente a “agitar os operários, sendo [...] sustentado com sua família por elementos do PCB”. Outro “candidato de Prestes” nas eleições municipais, sob a legenda do PST, era Álvaro Cecchino, industriário do ramo têxtil que atuava conjuntamente com Sturari. Cecchino era fundador da Cooperativa Industrial de Tecidos de Rayon (CITRA), primeira experiência cooperativa no ramo têxtil; posteriormente, vendeu suas ações e fundou a Distribuidora de Tecidos de Rayon de Americana (DISTRAL). No final dos anos de 1940, Sturari e Cecchino eram os principais comunistas da cidade investigados pelo DOPS. Cecchino era presidente do Comitê Municipal do PCB, classificado como o “cérebro e o mentor do Partido”, mas “devido ao cargo que ocupa não aparece em público como executante”. Por outro lado, não era capaz de “despertar simpatia na massa operária”, pois, como patrão, “dispensava e entrava em constantes choques com seus operários”. Romeu Sturari, que aguardava a “saída dos operários, às 23 horas, para doutriná-los”, era o grande temor das autoridades: auxiliado por outros membros do Partido, está fazendo uma grande e eficiente propaganda, que trará consequências gravíssimas e prejudiciais caso esse indivíduo não seja detido o mais breve possível. Tem muita influência entre o operariado e não perde ocasião de usá-la. Luís Henrique Carboni Junior Seu palavreado tem o dom de convencer a massa inculta [...] está tentando por todos os meios levar os operários da firma Carioba S/A à greve. É tão audacioso que para conseguir seus fins não hesita em afirmar aos operários que dentro de poucos dias os comunistas tomarão as rédeas do governo brasileiro. Sua demagogia tem influído na mente dos operários e é bem provável que, caso Sturari não seja afastado, dentro de dias as fábricas de Carioba estarão em greve. auxiliado por outros membros do Partido, está fazendo uma grande e eficiente propaganda, que trará consequências gravíssimas e prejudiciais caso esse indivíduo não seja detido o mais breve possível. Tem muita influência entre o operariado e não perde ocasião de usá-la. Seu palavreado tem o dom de convencer a massa inculta [...] está tentando por todos os meios levar os operários da firma Carioba S/A à greve. É tão audacioso que para conseguir seus fins não hesita em afirmar aos operários que dentro de poucos dias os comunistas tomarão as rédeas do governo brasileiro. Sua demagogia tem influído na mente dos operários e é bem provável que, caso Sturari não seja afastado, dentro de dias as fábricas de Carioba estarão em greve. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) Em fevereiro de 1948, Sturari foi investigado por coordenar a distribuição de larga tiragem do Manifesto de Janeiro de 1948. A impressão teria sido financiada por Cecchino e o material distribuído em Vila Carioba e outros pontos de Americana, bem como em municípios vizinhos: Santa Bárbara d’Oeste e Piracicaba. Após a cassação do mandato dos parlamentares do PCB, o manifesto demarcava o término da política de união nacional e início da luta contra o governo de traição de Dutra e Barros. Período marcado pelo enfrentamento direto dos membros do partido com as autoridades policiais, igualmente pelo incentivo, conforme orientações do partido, ao abandono do corporativismo e dos sindicatos oficiais.25 Entre março e junho, Sturari foi duas vezes apresentado ao DOPS para prestar esclarecimento sobre a “distribuição de boletins nas fábricas de tecelagem”. Entretanto, devido à insuficiência de provas “não houve relatório” e foi colocado em liberdade. p 26 A Crítica, 26 ago. 1948. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 25 NEGRO; SILVA, op. cit., p. 60. Luís Henrique Carboni Junior Por estarem articulando greve prevista para final de outubro, as autoridades do DOPS cumpriram um mandado de prisão contra Cecchino e Sturari como meio de silenciar o movimento dos trabalhadores. Cecchino não foi localizado por estar em viagem, enquanto Sturari fugiu dos agentes que tentaram, à meia-noite, capturá-lo em sua residência em Vila Carioba, onde foi encontrado “farto material subversivo”. Contudo, nos últimos dias de novembro, Sturari foi apreendido por um praça que o localizou distribuindo jornais27 pela cidade, mas, lavrado o ato de apreensão, foi liberado. Com as novas diretrizes do PCB incentivando a criação de outras instituições, os comunistas americanenses fundaram, no início de 1949, a Comissão Municipal de Estudos e Defesa do Petróleo. Durante a cerimônia de posse, ocorrida no salão de festas do Rio Branco Futebol Clube, houve a formalização escrita de um protesto, encaminhado ao deputado Euzébio da Rocha, contra a Lei de Segurança. Alegavam que a lei consistia em um “castigo para o povo, impedindo-o de exercer o seu direito sagrado, que é defender o petróleo e as demais riquezas de nosso país”.28 O manifesto era assinado por Sturari, Cecchino e outros. As pautas das reuniões da comissão concentravam-se em atacar o capitalismo e o imperialismo norte-americano, criticando o acordo militar estabelecido entre Brasil e Estados Unidos. Apelavam aos operários para que, “em seus locais de trabalho, cooperassem com a Comissão, carreando adesão”. Para o DOPS, estava claro que a comissão era “a nova modalidade de atividades dos elementos extremistas, para reerguimento do extinto Partido Comunista”. Nesse período, Americana era uma cidade majoritariamente industrial e urbana, com 190 fábricas,29 em sua maioria do ramo têxtil. A população urbana do município, de aproximadamente 20 mil habitantes, era constituída em quase sua totalidade por operários. A Fábrica de Tecidos Carioba somava dois mil funcionários, sendo um local “isolado” que abrigava “cerca de 700 adeptos do credo vermelho”, constituindo “um ótimo ponto para sabotarem a indústria nacional”. As benfeitorias e o maquinário estavam avaliados em “mais de 100 milhões de cruzeiros”. Em contrapartida, a Delegacia de Polícia de Americana era uma delegacia de 5ª classe, tendo à disposição somente quatro praças. Temendo as manifestações dos trabalhadores, o investigador do DOPS sentia-se “forçado a admitir que esta Delegacia não possui elementos [...] suficientes para uma repressão à altura do que vem acontecendo”. Luís Henrique Carboni Junior Mas, o tecelão prosseguia sua militância, escrevendo artigos publicados em diversos jornais comunistas, como A Crítica.26 Além disso, recebia e distribuía, na Estação Ferroviária de Americana, materiais como Zé Brasil, Karl Marx, Luís Carlos Prestes e exemplares do Hoje, realizando campanha para angariar fundos para o jornal. Era, também, “encarregado da Campanha de Ajuda à Imprensa Popular. Cuidando exclusivamente dos interesses do Partido”. Imagem II: Carteira Profissional de Romeu Sturari (1937) Fonte: Acervo particular da família Sturari. Imagem II: Carteira Profissional de Romeu Sturari (1937) Fonte: Acervo particular da família Sturari. 28 Notícias de Hoje, São Paulo, 17 fev. 1949. 29 Relatório DOPS/SP, 21 nov. 1949. Em outro relatório, de 15 maio 1951, consta o número de 200 fábricas, sendo a grande maioria do ramo têxtil do tipo fação (tecelagens de pequeno porte). 27 No dia seguinte, exemplares de A Classe Operária chegavam aos Correios endereçados a Sturari. 28 N tí i d H j Sã P l 17 f 1949 g , p p g ç 28 Notícias de Hoje, São Paulo, 17 fev. 1949. 27 No dia seguinte, exemplares de A Classe Operária chegavam aos Correios endereçados a Sturari. 28 Notícias de Hoje, São Paulo, 17 fev. 1949. 29 Relatório DOPS/SP, 21 nov. 1949. Em outro relatório, de 15 maio 1951, consta o número de 200 fábricas, sendo a grande maioria do ramo têxtil do tipo fação (tecelagens de pequeno porte). ia seguinte, exemplares de A Classe Operária chegavam aos Correios endereçados a Sturari. cias de Hoje São Paulo 17 fev 1949 30 Manifesto ao Povo de Americana, 26 fev. 1950. Pasta Americana. Arquivo Público do Estado de São Paulo, Fundos DEOPS/SP. 31 NEGRO; SILVA, op. cit. p, 60. Luís Henrique Carboni Junior Os operários pichavam muros com os dizeres: “Queremos 40%”, “P.C.B.”, “Abaixo Ademar e a Lei de Segurança”, “O povo repudia Ademar” e “Viva Prestes”; por outro lado, o delegado de Americana, defensor dos interesses da elite industrial, estava certo de que, com a ajuda da polícia especializada, a cidade iria “continuar a passos largos para o progresso sem temer” os elementos comunistas. 10 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 O “Perigo Vermelho” em Americana (SP) Manifesto ao Povo de Americana e a Tribuna do Povo: o jornal do proletariado americanense Mesmo com a repressão policial, a célula comunista não cessava suas ações, concentrando seus esforços em Carioba. No dia 26 de fevereiro de 1950, foi distribuído o manuscrito intitulado Manifesto ao Povo de Americana, assinado pelo Comitê Municipal do Partido Comunista, tendo como foco central a luta organizada dos trabalhadores contra o imposto e o divisionismo sindical: O Comitê Municipal do P.C.B. vem a público conclamar o povo a se organizar e lutar contra [...] o tirano Ademar de Barros e Dutra, governo este que fez correr por muitas vezes o sangue de comunistas [...] mandam assassinar operários e camponeses partidários da paz e patriotas [...] não vacilam em aumentar cada vez mais os lucros dos latifundiários e grandes industriais à custa do esfomeamento dos trabalhadores. No imposto sindical foram gastos 5.156.227 milhões de cruzeiros no divisionismo da classe operária, imposto ilegal e inconstitucional [...] Conclamamos os trabalhadores a apoiar moral e financeiramente a Conferência Sindical de Montevidéu, que se realiza em março sob patrocínio da C.T.H.L. e da Federação Sindical Mundial, com representante na ONU. Conclamamos os trabalhadores a lutar organizadamente contra o imposto sindical, é por meio deste imposto que os pelegos se mantêm no sindicato, infestando e traindo miseravelmente a classe. [...] Por eleições livres no sindicato Contra o imposto sindical Contra o imperialismo e a guerra [...] Comitê Municipal do Partido Comunista Leia e divulgue Tribuna do Povo, jornal do proletariado de Americana.30 [...] Por eleições livres no sindicato Contra o imposto sindical Contra o imperialismo e a guerra [...] Comitê Municipal do Partido Comunista Leia e divulgue Tribuna do Povo, jornal do proletariado de Americana.30 As suspeitas de autoria e distribuição recaíram, nas palavras das autoridades, “inevitavelmente sobre o indivíduo Romeu Sturari”, mesmo com a “falta de elementos probatórios”. Não obstante, os agentes especulavam que o tipo de letra e os dizeres empregados no manifesto “coincidem perfeitamente” com as pichações encontradas nos muros. Apesar da diretriz do PCB aconselhar o abandono dos sindicatos, alguns militantes, como é o caso de Sturari, exerceram militância dupla, ou seja, contrariavam as ordens do partido, optavam por realizar campanhas para expulsar as juntas interventoras dos sindicatos, não abrindo mão dessas instituições.31 11 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 30 Manifesto ao Povo de Americana, 26 fev. 1950. Pasta Americana. Arquivo Público do Estado de São Paulo, Fundos DEOPS/SP. 31 NEGRO; SILVA, op. cit. p, 60. Fundos DEOPS/SP. Luís Henrique Carboni Junior A respeito da primeira iniciativa de um jornal operário de Americana, Tribuna do Povo,32 foi possível localizar apenas o primeiro número, distribuído por Romeu Sturari no dia 7 de setembro de 1949, em Carioba, juntamente com exemplares de A Luta.33 O material, uma folha tipografada frente e verso, chegou às mãos dos proprietários, os quais formalizaram a denúncia. A data escolhida para estreia do jornal não ocorreu ao acaso, seus idealizadores interpretavam o dia 7 de setembro como um marco de liberdade. O compromisso do jornal era estar “sempre alerta, guiando e orientando todo o proletariado no caminho da revolução”, que só poderia ser realizada “sob a direção do proletariado, dirigido pelo seu partido de vanguarda – o Partido Comunista”. Dentre as pautas estavam: 40% de aumento de salários, “assistência e direitos aos homens [...] igualdade em tudo para as mulheres”, além de questões do Matadouro Municipal e do Sindicato de Fiação e Tecelagem de Americana. Sobre o matadouro, o jornal denunciava o aumento no valor do abate, que de 12 cruzeiros passou para 25 por cabeça, acarretando a carestia da carne, enquanto os trabalhadores continuariam a receber o mesmo “salário de fome” para um turno das 3h às 17h. Incentivavam que uma comissão de reivindicação fosse instaurada pelos empregados para solicitarem 50% de aumento, devendo “paralisar os serviços”, pois a situação era “insalubre”, dada a “imundície” do local de trabalho. Por fim, voltavam-se contra os “pelegos” que atuavam no sindicato têxtil, exigiam a saída dos interventores, que eram “traidores a serviço dos patrões”, fazendo do sindicato um antro de “divisionismo, de traição da classe operária”. Como meio de encorajar os trabalhadores na luta, asseguravam que “essa junta de policiais tem medo de operários dispostos a lutar por seus direitos”. Reforçando que a ação dos operários deveria ser “ou os 40% ou greve, garantida pela Constituição Federal, art. 158”. Era o modo de evitar o intermédio das instituições governamentais, uma vez que a Justiça do Trabalho “só dá causa ganha para os operários em último caso”. Para as autoridades, apesar de o “ex-PCB estar articulado naquela cidade” e exercer forte propaganda, não havia ameaças de greve; mesmo assim, deter o responsável pelo manifesto e pelo jornal era uma questão de ordem. Manifesto ao Povo de Americana e a Tribuna do Povo: o jornal do proletariado americanense 31 NEGRO; SILVA, op. cit. p, 60. 30 Manifesto ao Povo de Americana, 26 fev. 1950. Pasta Americana. Arquivo Público do Estado de São Paulo, Fundos DEOPS/SP. 31 NEGRO; SILVA, op. cit. p, 60. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 32 Tribuna do Povo: o jornal do proletariado americanense, 7 set. 1949. Dossiê Comunismo – PCB. Arquivo Público do Estado de São Paulo, Fundos DEOPS/SP. 33 A Luta: órgão de luta dos trabalhadores de Campinas, ago. 1949. , 33 A Luta: órgão de luta dos trabalhadores de Campinas, ago. 1949. 32 Tribuna do Povo: o jornal do proletariado americanense, 7 set. 1949. Dossiê Comunismo – PCB. Arquivo Público do Estado de São Paulo, Fundos DEOPS/SP. 33 A L t ó ã d l t d t b lh d d C i 1949 Luís Henrique Carboni Junior Poucos dias após a distribuição do manifesto, agentes do DOPS dirigem-se para Americana para deter Sturari que, mais uma vez, foi capaz de fugir quando desembarcava na estação ferroviária trazendo jornais de Campinas. As autoridades acreditavam ser necessária a prisão de Romeu Sturari para “desarticular provisoriamente a rede vermelha naquela cidade”. Outrossim, falava-se em uma detenção que equivaleria a um “descanso de 10 dias”, pois acreditavam que seria tempo suficiente para diminuir seu fervor. Nos anos seguintes, o tecelão exercia sua militância muito ligada 12 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) à imprensa, assinando manifestos e artigos comemorativos em grandes jornais comunistas como o Hoje34 e Imprensa Popular.35 A essa altura, Romeu Sturari era reconhecido pelas autoridades como o “elemento de ligação entre parte do operariado e dos sindicatos”, sendo detentor de um discurso eloquente, capaz de “citar leis e decretos”, além disso, era comum “fazer barulho quando detido, mas nunca resiste à prisão”, pois era liberado devido à falta de provas e artifícios legais capazes de imputar um maior período de detenção. 34 Hoje, São Paulo, 24 jan. 1952. (Romeu Sturari assina manifesto de solidariedade a jornalistas presos). 35 Imprensa Popular, 8 nov. 1953. (Romeu Sturari assina artigo de comemoração ao 36º aniversário da R l ã d O t b d 1917) j , , j ( j p ) Imprensa Popular, 8 nov. 1953. (Romeu Sturari assina artigo de comemoração ao 36º aniversár Revolução de Outubro de 1917). 34 Hoje, São Paulo, 24 jan. 1952. (Romeu Sturari assina manifesto de solidariedade a jornalistas presos). 35 Imprensa Popular, 8 nov. 1953. (Romeu Sturari assina artigo de comemoração ao 36º aniversário da Revolução de Outubro de 1917). 36 Diário de Notícias Rio de Janeiro 30 set 1954 ç ) 36 Diário de Notícias, Rio de Janeiro, 30 set. 1954 37 Notícias da Noite, Rio de Janeiro, 6 out. 1954. oje, São Paulo, 24 jan. 1952. (Romeu Sturari assina manifesto de solidariedade a jornalistas presos). ç ) 36 Diário de Notícias, Rio de Janeiro, 30 set. 1954. Revolução de Outubro de 1917). 36 Diário de Notícias, Rio de Janeiro, 30 set. 1954. 37 N tí i d N it Ri d J i 6 t 1954 Luís Henrique Carboni Junior A cidade de Americana, em 1957, era qualificada como uma cidade “muito movimentada, embora não muito grande [...] cidade industrial muito expressiva, pois conta com mais ou menos 470 indústrias de rayon [...] o maior centro produtor de rayon da América do Sul”. Não havia “favelas, mocambos e construções de madeira”, sendo este ambiente impróprio para “ideias esquerdistas [...] na mente dos operários”. No âmbito político, os partidos estavam “organizados e em plena calma”, não havia presença de comunistas em outros partidos, tampouco ocupando cargos na Câmara Municipal, nas escolas ou no funcionalismo público. No aspecto religioso, Americana era descrita como “verdadeiro reduto de católicos”, o que corroborava para a “obediência às leis [...] dado o espírito ordeiro e cristão das massas”. Complementando a projeção social, no aspecto rural, os três principais produtos eram: cana-de-açúcar, cereais e algodão. As pequenas propriedades eram predominantes, não havendo “propagandas extremistas entre as classes rurais” ou núcleo da União dos Lavradores e Trabalhadores Agrícolas do Brasil. Mesmo foragido, o nome de Romeu Sturari não foi extinto dos relatórios do DOPS. Com a distribuição de pouco mais de 20 boletins aos operários de Carioba, às 5h do dia 23 de abril de 1957, a polícia prendeu José Martins Moreira, empregado daquela indústria, mas que, de qualquer modo, estava atuando como “elemento de ligação do foragido”. Todavia, de modo geral, a ausência de Romeu Sturari mostra-se coincidente ao momento de retração da organização dos trabalhadores. Segundo as autoridades, no início de 1957, os comunistas não apresentavam “qualquer atividade aparente, e de certa forma, bastante desorganizados e desmoralizados perante a opinião pública”. Jornais “do PCB” deixaram de ser distribuídos, “os elementos reconhecidos como perturbadores da ordem social não encontram o menor apoio no seio das classes operárias”. Mesmo enfrentando forte recessão econômica e demissões em massa por meses consecutivos, com estimativas de 800 despedidos em abril, os 4,5 mil têxteis38 de Americana não organizaram qualquer ação reivindicativa. Lei de Segurança Nacional: tempos de clandestinidade (1954-1957) O impasse enfrentado pelas autoridades da ausência de dispositivos legais capazes de impor prisões definitivas contra Romeu Sturari foi sanado com a Lei de Segurança Nacional nº 1.802/1953, chamada Lei de Crimes Contra o Estado e a Ordem Política e Social. Em meados de março de 1954, a delegacia de Americana instaurou inquérito contra o comunista, imputando-o o artigo 9º e o parágrafo 3º do artigo 11º da referida lei. Desse modo, seus crimes eram: tentar reorganizar o partido político dissolvido por força legal e fazer publicamente propagandas que fomentavam, por meio de boletins e panfletos, o ódio de classes, visando à subversão da ordem política e social. Seu mandado de prisão foi expresso pelo juiz, que o sentenciou a dois anos de reclusão. Todavia, Romeu Sturari foi capaz de permanecer foragido por anos, mas, mesmo escondendo- se, contava com amparo judicial, conforme registros do pedido de habeas corpus negado pelo Supremo Tribunal Federal, em setembro de 1954.36 O jornal Notícias da Noite, ao noticiar a petição, qualificou o tecelão que lutava pelos direitos de sua classe como “comunista e vadio”, que vinha “tentando reorganizar o partido vermelho” em Americana.37 Concomitantemente, em Americana, havia uma campanha pela concessão de anistia aos presos e condenados por crimes políticos, “abrangendo o líder comunista Romeu Sturari”. Muros amanheciam pichados com dizeres: “Anistia aos presos políticos”, “Anistia a Luís Carlos Prestes”, “Anistia a Romeu” e “Grande comício na cidade de Americana no dia 24/03”. A divulgação do comício de anistia ficava a cargo da emissora de rádio e do jornal O Liberal. Com a necessidade de sufocar o movimento, os policiais de Americana colocaram-se em alerta, prendendo, no dia 22 de março, dois indivíduos enquanto inscreviam nos muros. Ambos alegaram ser pintores de ofício, que nada entendiam de comunismo, estavam apenas realizando serviços para Álvaro Cecchino, que pagava 50 cruzeiros para cada pintor por noite de serviço. Desse modo, mediante a repressão policial que ameaçava agir contra os trabalhadores “com todo rigor legal”, o comício não ocorreu e a campanha de anistia se esmoreceu. 13 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 j 40 Notícias de Hoje, São Paulo, 3 jun. 1958. 38 Relatório DOPS/SP, 10 maio 1957. , 39 Jornal do Commercio, Rio de Janeiro, 18 jul 38 Relatório DOPS/SP, 10 maio 1957. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 38 Relatório DOPS/SP, 10 maio 1957. 39 Jornal do Commercio, Rio de Janeiro, 18 jul. 1957. O retorno de Romeu Sturari (1958-1963) Após julgado um novo pedido de habeas corpus pelo STF, ainda no ano de 1957, Romeu Sturari teve sua sentença anulada.39 Mas, somente um ano depois, o jornal Notícias de Hoje40 estampou uma fotografia de Romeu Sturari, advertindo que o Juiz da Comarca havia revogado sua prisão. Desse modo, estava o tecelão livre de seus crimes da Lei de Segurança Nacional, podendo reaparecer na cena pública junto ao movimento dos trabalhadores. Em poucos meses Sturari foi eleito presidente do Sindicato dos Trabalhadores na Indústria de Fiação e Tecelagem de Americana. Nas eleições municipais do ano de 1959, pretendeu disputar o Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) cargo de vereador, candidatando-se pelo Partido Republicano Trabalhista (PRT), mas teve sua candidatura impugnada pelo Tribunal Regional Eleitoral devido seu histórico de crimes contra a ordem política e social. Na perspectiva do DOPS, o sindicato então presidido por Sturari, “na ocasião das eleições, exercia influência no seio de seus associados”. No segundo mês de 1959 eclodiu um movimento grevista do setor têxtil, reivindicando o cumprimento da nova lei salarial que estabeleceu o salário-mínimo de 5.100 cruzeiros. A culpa do não atendimento às normas previstas pela legislação recai sobre as facções, embora a greve tenha abarcado todas as tecelagens do município como solidariedade. Com o cumprimento da reinvindicação e o fim da greve, os agentes do DOPS constataram, depois de alguns meses, que as “pequenas indústrias de tecelagem continuam lutando com sérias dificuldades [...] em sua grande maioria, terão que encerrar suas atividades”. Mas não só as pequenas tecelagens estavam sendo afetadas, a situação geral dos têxteis era precária. A reputação do Grupo J. J. Abdalla não era nada positiva, sendo a lei reconhecida como “letra morta na fábrica do deputado J. J. , 42 CORRÊA, Larissa Rosa. Trabalhadores têxteis e metalúrgicos a caminho da Justiça do Trabalho: leis e direitos na cidade de São Paulo – 1953 a 1964. 2007. Dissertação (Mestrado em História) – Programa de Pós-Graduação em História, Universidade Estadual de Campinas, Campinas, 2007, p. 175. 43 Última Hora, Paraná, 28 fev. 1961. 41 O Trabalhador Têxtil, set. 1957. 42 CORRÊA, Larissa Rosa. Trabalhadores têxteis e metalúrgicos a caminho da Justiça do Trabalho: leis 41 O Trabalhador Têxtil, set. 1957. Ê 41 O Trabalhador Têxtil, set. 1957. 42 CORRÊA, Larissa Rosa. Trabalhadores têxteis e metalúrgicos a caminho da Justiça do Trabalho: leis e direitos na cidade de São Paulo – 1953 a 1964. 2007. Dissertação (Mestrado em História) – Programa de Pós Graduação em História Universidade Estadual de Campinas Campinas 2007 p 175 O retorno de Romeu Sturari (1958-1963) Abdalla na cidade de Americana”.41 As causas dos conflitos entre os trabalhadores e o empregador gravitavam em torno de salários atrasados, violação dos contratos de trabalho e aumento do uso de teares, sendo os tecelões obrigados a movimentar quatro teares, o dobro do previsto.42 O jornal Última Hora noticiou, em fevereiro de 1961, que os tecelões de Americana estavam desamparados pela lei; era a segunda vez que Sturari, enquanto presidente do sindicato, recorria à Delegacia Regional do Trabalho solicitando fiscalização do cumprimento da legislação trabalhista, “violentada e burlada quase que pela maioria das 300 empresas do ramo”. O sindicalista acreditava que, ao menos, 40% dos 8 mil trabalhadores têxteis do município não eram registrados, além disso, somente 10 empresas estavam pagando o aumento de 37% acertado no último acordo. O pagamento de férias estava sendo igualmente negligenciado, bem como o adicional noturno ou insalubridade. Ademais, os salários dos trabalhadores menores de idade não eram respeitados, apesar de fazerem “serviço idêntico” ao dos adultos, e além disso, era recorrente “mulheres e menores trabalharem até altas horas da madrugada”.43 Segundo Sturari, essa situação gerava problemas, desde dificuldade para “sindicalização dos operários” até a “ausência total de assistência médico-hospitalar e social”. As empresas deixavam, ainda, de fazer os descontos em folhas de associados, alegando “não constarem seus nomes entre os empregados”, já que não possuíam registro. Por fim, o tecelão reclamava o não pagamento de 100 mil cruzeiros pelos Abdalla ao sindicato, que “recolheu aquela importância” de seus empregados, mas não efetuou o repasse. 15 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 44 Relatório DOPS/SP, 27 out. 1961. 45 NEGRO; SILVA, op. cit., p. 79. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 O golpe militar em Americana (1964) Com o advento do golpe militar, a política e o movimento operário de Americana sofreram drásticas consequências. Ainda em janeiro de 1964, foi declarada greve em Carioba que perdurou 28 dias, sendo utilizada como uma das justificativas para o impeachment do prefeito Jairo de Azevedo, eleito pelo Partido Trabalhista Brasileiro (PTB). Diante da agitação política e social gerada pela tomada do poder pelos militares, as autoridades de Americana encaminharam ao DOPS informações sobre 14 pessoas “ligadas ao fato” do combate ao comunismo. O grupo era formado, sobretudo, por políticos e trabalhadores. Sobre o ex-prefeito Azevedo, impichado pela Câmara Municipal em junho de 1964, constavam as acusações de “colaboração com Romeu Sturari”, cedendo “caminhões e prestígio ao Sindicato”, participando de reuniões sindicais afirmando “todo apoio ao sindicato, atacando os patrões, especialmente os Abdalla, dizendo que deviam vários milhões à Prefeitura”. Em fevereiro, participou da inauguração do Sindicato Rural de Americana, filiado à Superintendência da Política Agrária (SUPRA). No dia seguinte ao golpe, reuniu-se com líderes sindicais para organizar uma passeata contra o assalto ao poder democrático. A campanha de Azevedo contou com apoio de Sturari, que com “seu trabalho junto às massas”, estava encarregado de angariar “base popular” ao candidato. Os slogans usados em comícios eram o do “Pobre Contra o Rico” e do “Tostão Contra o Milhão”, proferindo discursos de “hostilidade entre o operário e o patrão”. O prefeito discursava, também, em favor de alguns projetos da agenda do PCB, como direito de voto aos analfabetos e reforma agrária. Ao passo que o corporativismo sindical era usado como mecanismo para controlar o operariado, a aliança PTB-PCB, expressa em Azevedo-Sturari, servia-se dos sindicatos para organizar os trabalhadores em favor do direito à greve.46 Na visão das autoridades, com a vitória do PTB os comunistas “sentiram-se fortes”, organizando a greve de Carioba por contarem com apoio do prefeito e outros cinco vereadores do PTB. Por outro lado, ao analisarmos os processos do Tribunal Regional do Trabalho sobre essa paralisação, vislumbramos motivações geradas por problemas trabalhistas e não puramente políticos. Quando, no dia 6 de janeiro, declararam greve, os trabalhadores não tinham recebido os salários dos meses de novembro e dezembro, juntamente com o abono de Natal de 1963. Luís Henrique Carboni Junior Posteriormente, Sturari escreveu em nome do sindicato para O Liberal,44 em outubro de 1961, alertando aos trabalhadores para que se preparassem para a greve. Após reunir-se, em São Paulo, com outros líderes sindicais, realizou carreatas durante o início de dezembro convocando os trabalhadores para discutir a organização do movimento grevista caso o abono de Natal não fosse quitado. Nesse período, a pauta do 13º salário era uma das principais lutas do PCB.45 Porém, os patrões e as autoridades municipais rapidamente estabeleceram acordo, evitando a eclosão da greve. Assim, ficou acertado que os trabalhadores poderiam gozar de um abono de 120 horas a ser pago ainda naquele ano. Durante o segundo trimestre de 1963, Romeu Sturari atuou em favor da greve da Companhia Paulista de Estada de Ferro, fomentando, também, uma greve de solidariedade em Carioba para que os trabalhadores aderissem à Greve dos 700 mil, contudo, não obteve sucesso. No mesmo período, a convite de Álvaro Cecchino, “amigo pessoal de Luís Carlos Prestes”, o ícone comunista visitou Americana para realização de um comício, evento que evidencia a relevância e a amplitude das relações do movimento comunista organizado na cidade. Imagem III: (da esquerda para direita) Romeu Sturari, Álvaro Cecchino e Luís Carlos Prestes em Americana (1963) (da esquerda para direita) Romeu Sturari, Álvaro Cec Luís Carlos Prestes em Americana (1963) Fonte: Acervo particular da família Sturari. Fonte: Acervo particular da família Sturari. Fonte: Acervo particular da família Sturari. 45 NEGRO; SILVA, op. cit., p. 79. 16 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) ; , p , p 47 Processo TRT/SP 23/64-A. 46 NEGRO; SILVA, op. cit., p. 85. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Luís Henrique Carboni Junior a não demissão dos grevistas, o pagamento dos dias de paralisação e o recebimento dos salários na fábrica. Uma vez que, conforme Larissa Corrêa, era prática habitual dos Abdalla orientar os trabalhadores para que fossem receber no banco, ocasionando longas filas de espera. Não raro, as agências encerravam suas operações e muitos operários tinham de retornar no dia seguinte para resgatar seus rendimentos.48 Retomando os indivíduos listados pelo delegado, constava o vereador Willi Cordenonsi (PTB), encarregado de um dos institutos de previdência que, durante reunião na Câmara, proferiu verbalmente o pedido da legalização do PCB. Em contrapartida, o Círculo Operário de Americana organizou um grupo de trabalhadores para irem até a Câmara entregar um abaixo- -assinado com 4 mil assinaturas de “católicos que protestavam”49 contra a fala do vereador. Do âmbito rural, consta somente o nome de Francisco Prado, “presidente do recém- -instaurado Sindicato Rural de Americana, semianalfabeto, influenciado, porém, por Sturari”. A instituição em questão é a primeira iniciativa sindical dos trabalhadores rurais da cidade. As reuniões de articulação e criação do sindicato ocorreram, segundo Prado, sob incentivo e mediação de Sturari, o qual tinha contato com representantes da SUPRA. No segmento metalúrgico, Dario Pompeu e Osório Voletti eram apontados como principais agitadores das Indústrias Nardini,50 sendo representantes do Sindicato dos Metalúrgicos de Campinas e dirigentes da Delegacia dos Metalúrgicos de Americana. Entre os servidores públicos, o diretor do Grupo Escolar do Jardim São Paulo Hipólito Gonçalves era acusado de atividades comunistas. Gonçalves atuou como propagandista da campanha de Azevedo, além de prestar serviço voluntário ao sindicato e participar de assembleias. Em sua posse foram encontrados mimeógrafo e máquina de escrever patrimoniados do sindicato, material cedido por Sturari. Tendo tomado parte na greve de Carioba, era o principal suspeito de ter redigido os boletins circulados durante o movimento. Dentre os trabalhadores têxteis, a atenção recaía sobre os dirigentes do sindicato: Romeu Sturari, presidente; Nelson Zanetti, tesoureiro; e, por fim, Dirceu Leão, secretário, que ocupava, também, o cargo de vereador suplente pelo PTB. Como garantia da ordem, a polícia de Americana invadiu, menos de uma semana após o golpe, a sede do sindicato à rua Vieira Bueno, nº 48. A essa altura, Sturari já se encontrava foragido, mas devido ao material encontrado na instituição, foi indiciado na Lei de Segurança Nacional nº 1.802/53, artigo 11º, alínea “a”, parágrafos 1º e 3º. O golpe militar em Americana (1964) Após tentativas frustradas de negociação entre o sindicato e o empregador, a Federação dos Trabalhadores Têxteis de Fiação e Tecelagem decidiu, no dia 20 de janeiro, instaurar processo na Justiça do Trabalho.47 No entendimento dos próprios trabalhadores, os Abdalla protelavam os pagamentos como meio de incentivar a rescisão de contrato para que os empregados abdicassem de seus direitos e de sua morada. Os grevistas exigiam, ainda, 17 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 17 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 49 O Liberal, Americana, 19 mar. 1964. 50 As Indústrias Nardini somavam 300 trabalhadores em junho de 1957, conforme relatório do DOPS/S , p , p 9 O Liberal, Americana, 19 mar. 1964. , p , p 49 O Liberal, Americana, 19 mar. 1964. 49 O Liberal, Americana, 19 mar. 1964. 50 As Indústrias Nardini somavam 300 trabalhadores em junho de 1957, conforme relatório do DOPS/SP. 49 O Liberal, Americana, 19 mar. 1964. 50 As Indústrias Nardini somavam 300 trabalhadores em junho de 1957 conforme relatório do DOPS/SP 48 CORRÊA, op. cit., p. 167-168. 48 CORRÊA, op. cit., p. 167-168. Luís Henrique Carboni Junior Seu crime consistia em fazer propaganda e distribuição de material textual para subversão da ordem política e social, havendo agravante por distribuir em fábricas. Para as autoridades, estava claro que o sindicato atuava como “verdadeira célula comunista”, sendo medida cabível a prisão de seus dirigentes. Quando interrogados sobre suas relações com Romeu Sturari, todos os investigados negaram ter ligação de caráter político com ele ou partilhar do ideário comunista. Os membros 18 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) da diretoria do sindicato diziam reprovar a conduta extremista do presidente, advertindo-o que essa “não era a finalidade do sindicato”. Outros alegavam partilhar dos “princípios católicos e democráticos”, afirmando ter participado da Marcha da Família com Deus pela Liberdade e da Campanha Ouro para o Bem do Brasil. O vereador Willi Cordenonsi (PTB), além de apresentar os argumentos citados acima, alegou ter votado favorável tanto ao impeachment de Jairo Azevedo (PTB) quanto à moção de louvor da Câmara Municipal aos militares, mantendo-se “firme ao lado dos verdadeiros democratas”. Por fim, afirmou ter se desligado do PTB, assim como os demais vereadores do partido. Álvaro Cecchino não foi poupado. As autoridades exigiam sua prisão preventiva devido à “célula comunista que criou dentro de sua indústria”, colaborando para a formação de “vários líderes esquerdistas”. Diferentemente de todos os outros depoentes, Cecchino disse abertamente partilhar da teoria marxista, sendo favorável à política de João Goulart. Questionado sobre Sturari, disse que apesar da relação de amizade, o tecelão passou a hostilizá-lo e chamá-lo de reacionário após assumir a presidência do sindicato. Como álibi para evidenciar que não era mais um comunista em atividade, Cecchino afirmou ter instaurado ações na Justiça do Trabalho contra alguns de seus empregados que vinham participando dos movimentos grevistas organizados por Sturari. Prestadas as declarações e por “estar já velho, doente, e não mais em atividade”, foi liberado. Após quase um ano foragido, Romeu Sturari foi detido, informando não mais residir em Vila Carioba, mas em Vila Manoel, no mesmo município. Não havia espaço para sua conduta comunista em Carioba. Em seu ponto de vista, toda a perseguição e prisões foram “ocasionadas por atritos entre seu ex-patrão J. J. Abdalla”, que o denunciava “visando a expulsá-lo da casa onde residia”. 51 NEGRO; SILVA, op. cit., p. 89. Luís Henrique Carboni Junior A prática de denunciar e promover a espionagem dos próprios empregados era recorrente, já que os industriais vislumbravam na polícia um costumeiro aliado na conservação de seus domínios privados sobre os mundos do trabalho.51 Perguntado sobre Cecchino, Sturari afirmou ser um companheiro que durante muitos anos atuou em conjunto, tendo também trabalhado na tecelagem do industriário durante o ano de 1953, mas suas relações foram abaladas, em 1962, após ser nomeado juiz vogal e atuar “em defesa dos empregados de Álvaro” na Justiça do Trabalho. Admitiu, ainda, ser comunista e ter participado, bem como organizado, diversos movimentos grevistas, os quais eram determinados sem, necessariamente, perpassar pelo sindicato. As reuniões e decisões eram tomadas em “assembleias organizadas por ele mesmo”. Em contrapartida, reafirmou seu caráter “de família católica”, salientando as boas relações que tinha com monsenhor Maggi, pois que “as festas do sindicato eram sempre feitas em salões de propriedade da Igreja”. Dadas as declarações, foi recolhido à prisão, momento em que é encerrada a trajetória de Romeu Sturari nos arquivos do DOPS. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 19 52 Arquivo Nacional, Fundo: Serviço Nacional de Informações (SNI). Luís Henrique Carboni Junior Por meio dos registros de sua carteira profissional, é possível dizer que o tecelão trabalhou, em meados de 1958, na Têxtil Carauma, ascendendo ao cargo de sócio dessa indústria no final de 1959, permanecendo nesse posto até sua prisão ser decretada em 1964. Após ser colocado em liberdade, Sturari não exerceria mais, pelo menos formalmente, a função de tecelão. O único registro posterior à prisão data de maio de 1967, na função de servente na construção civil na cidade de Campinas, contrato que seria encerrado três meses depois. Pode-se supor, assim, que tanto no ramo têxtil quanto na cidade de Americana, eram escassas as possibilidades de emprego para Romeu Sturari que, apesar de fiel servidor da causa operária, passou a ser evitado socialmente, inclusive, por seus vizinhos. Faleceu no dia 14 de dezembro de 1978, aos 60 anos de idade, sendo sepultado no Cemitério da Saudade, em Americana. Entretanto, três anos antes de sua morte, continuava a ser citado pelas autoridades, dessa vez, na documentação do Serviço Nacional de Informação (SNI), como “militante do PCB”. 52 Imagem IV: Romeu Sturari (1973) Fonte: Acervo particular da família Sturari. Imagem IV: Romeu Sturari (1973) Fonte: Acervo particular da família Sturari. Fonte: Acervo particular da família Sturari. O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) apresentou, na Câmara Federal, seu telegrama enviado ao ministro do Trabalho sobre a tecelagem. Seu discurso, noticiado pelos jornais Folha de São Paulo53 e O Liberal,54 afirmava ser “verdadeiramente dramática” a situação, pois os proprietários agiam de forma “desumana e antissocial”, que, além do atraso salarial “há meses”, realizavam demissões “sem pagamento de indenização”. Na última dispensa, somavam-se 200 trabalhadores sem ressarcimento. Diante desse contexto, Levy solicitava a “intervenção direta do governo”, uma vez que a “ação morosa da Justiça” não era suficiente para garantir os direitos dos trabalhadores. Contudo, conforme as forças policiais, apesar da situação de penúria, não era cogitada “paralisação do trabalho por greve”, não havendo “nenhum elemento incitador de subversão”. Mas quanto aos salários, além de estarem em atraso, os empregadores ofereciam a opção de pagamento através de produtos produzidos pelo Grupo J. J. Abdalla, como “cimento, materiais de construção, mantimentos” e até mesmo tecido. Em uma situação calamitosa, os trabalhadores tinham de vender a própria produção para garantirem sua subsistência. Com o “expurgo” de “agitadores profissionais e políticos inescrupulosos [...] extirpados do meio trabalhador [...] Romeu Sturari e Jairo Azevedo [...] a insatisfação não contou com o fermento prodígio para transformar-se em agitação”. Outro fator que contribuiu para a manutenção da ordem era o atendimento das necessidades básicas do operariado: casa, água e luz. Itens, como já dito, fornecidos sem custos pelo patrão. Entretanto, com a persistência da situação por um ano, os trabalhadores decidiram, no início de 1966, apelar para a Justiça do Trabalho, tendo em vista que em março daquele ano muitos trabalhadores ainda não tinham recebido o salário de dezembro. Diante dessa situação, inevitavelmente o empregador era condenado pela JT e propunha um acordo, o qual era aceito e acarretava o encerramento do processo. Nas palavras do delegado, “tudo voltava à estaca zero”, pois mesmo quando os acordos eram cumpridos, os atrasos voltavam a ocorrer, acarretando a sobrecarga da JT. O representante dos empregadores era Nicolau Abdalla, vereador eleito que “gozava de grande simpatia entre os trabalhadores por seu trato humano e bondade de coração”. Por residir em Carioba, era “acessível, amigo dos trabalhadores e ‘compadre’ de muitos deles”. Mas seu status de benquisto sofria constante depreciação entre os empregados, dadas as dificuldades de gestão. Para quantificarmos em dados aproximados, em 1964, a empresa possuía 1.100 funcionários, enquanto no início de 1966 contava com somente 650. 54 O Liberal, Americana, 24 ago. 1965. 53 Folha de São Paulo, São Paulo, 19 ago. 1965. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 21 A Grande Greve de Carioba (1966-1967) A Grande Greve de Carioba (1966-1967) No segundo semestre de 1965, Carioba apresentava sinais de graves problemas financeiros. A situação preocupava os parlamentares, sobretudo o deputado federal Herbert Levy que 52 Arquivo Nacional Fundo: Serviço Nacional de Informações (SNI) No segundo semestre de 1965, Carioba apresentava sinais de graves problemas financeiros. A situação preocupava os parlamentares, sobretudo o deputado federal Herbert Levy que 52 Arquivo Nacional, Fundo: Serviço Nacional de Informações (SNI). 20 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Luís Henrique Carboni Junior firmado um acordo. Ficou acertado que a empresa pagaria parcelas proporcionais ao número de operários que cada família possuísse. Para realização do acerto, cinco agentes do DOPS estavam em Carioba desde as primeiras horas do dia, igualmente outros 12 soldados da delegacia de Americana estavam de “sobreaviso para qualquer emergência”. Apesar de preparadas, as autoridades não tiveram de agir. Insatisfeitos, os trabalhadores reuniram-se, em meados de julho de 1966, em Assembleia Geral Extraordinária, organizada pelo Sindicato dos Trabalhadores na Indústria de Fiação e Tecelagem, em conjunto com o Sindicato de Mestres e Contramestres nas Indústrias de Fiação e Tecelagem. Assim, os sindicatos que deveriam atuar como mecanismo de controle estatal sobre os operários possibilitaram, mais uma vez, romper com a dívida de lealdade para com o patrão, protagonizando o conflito de classe.55 Conforme ata da reunião, dos 681 trabalhadores empregados em Carioba, 542 compareceram à reunião para discutirem: 1) complementação do pagamento relativo ao 13º salário (Abono de Natal correspondente ao ano de 1965); 2) pagamento das importâncias correspondentes às férias vencidas e gozadas por trabalhadores; 3) pagamento dos salários dos meses de abril, maio e junho de 1966 [...] deliberar sobre a decretação do movimento grevista.56 1) complementação do pagamento relativo ao 13º salário (Abono de Natal correspondente ao ano de 1965); 2) pagamento das importâncias correspondentes às férias vencidas e gozadas por trabalhadores; 3) pagamento dos salários dos meses de abril, maio e junho de 1966 [...] deliberar sobre a decretação do movimento grevista.56 No total, 523 trabalhadores computaram seus votos, sendo seis votos em branco e apenas um não favorável à paralização. Assim, ficou decidido que “absterão pacificamente do trabalho” a partir do dia primeiro do mês de agosto, “tudo em obediência às normas legais vigentes e conforme aceitação” dos empregados. Sendo a ata lavrada e uma cópia encaminhada à Justiça do Trabalho. Desde o primeiro dia de agosto de 1966 até o dia 15 de abril de 1967, Carioba não funcionou, os únicos serviços mantidos na fábrica foram os de portaria e vigilância noturna. A greve foi considerada legal pela Justiça do Trabalho, sendo determinado o pagamento dos dias de paralisação, o 13º de 1966 e os meses de salários atrasados. Contudo, a decisão considerou os atrasos desde agosto, causando descontentamento entre os empregados, que argumentaram haver atrasos anteriores que motivaram a deflagração da greve no mês respectivo. O “Perigo Vermelho” em Americana (SP) Era comum a aglomeração de grupos de trabalhadores nos escritórios da fábrica na expectativa de receber. Em março houve “ameaças de depredação e até de esforço físico”. A situação preocupava as autoridades do DOPS, assim, um agente da instituição solicitou e acompanhou Nicolau Abdalla e o presidente do sindicato até São Paulo para que fosse Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 21 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 ; , p , 56 Pasta Americana. Arquivo Público do Estado de São Paulo, Fundos DEOPS/SP. 55 NEGRO; SILVA, op. cit., 88-89. 56 Pasta Americana. Arquivo Público do Estado de São Paulo, Fundos DEOPS/SP. 55 NEGRO; SILVA, op. cit., 88-89. 55 NEGRO; SILVA, op. cit., 88-89. O “Perigo Vermelho” em Americana (SP) O “Perigo Vermelho” em Americana (SP) em aberto. Era uma “situação de verdadeira calamidade”, conforme analisava o delegado de Americana. A autoridade policial acreditava “não haver condições de trabalho após a longa paralisação, eis que a maquinaria é obsoleta. Grande parte da mesma foi vendida e outra encontra-se penhorada para o pagamento de executivos fiscais. Praticamente não há matéria-prima”. Como previsto, a situação de Carioba era irremediável, mesmo sob intervenção direta do governo federal e com a prisão e confisco de bens de José João Abdalla, a Fábrica de Tecidos Carioba agonizou até o final de 1976, encerrando suas atividades. A Vila Operária de Carioba foi tema de disputas judiciais entre os moradores e os herdeiros da família Abdalla; por fim, foi desocupada e demolida em meados de 1980. 57 Parte dos trabalhadores, após longa disputa jurídica, foram indenizados com terrenos na região correspondente ao bairro Cariobinha em Americana. 57 A análise do processo de disputa e demolição da Vila Operária de Carioba foge do escopo do presente artigo, entretanto, é reconhecida a importância da análise desse processo que carece de produções historiográficas a seu respeito. Luís Henrique Carboni Junior Outro problema que afligia os operários era a obrigatoriedade de retorno, mesmo sem haver data para os pagamentos dos valores, que apesar de estarem garantidos judicialmente, poderiam sofrer longas protelações até serem quitados. Entretanto, não podendo descumprir a decisão, decidiram retornar às atividades e interpelar através do sindicato. Quando as atividades foram retomadas, apenas 600 funcionários permaneciam empregados na tecelagem. Os salários em atraso estavam sendo quitados parceladamente, mas o processo sobre o pagamento dos dias de greve e valores rescisórios permaneciam 22 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 Conclusão Conclui-se, assim, que Romeu Sturari foi um grande representante dos trabalhadores de Carioba, os quais envolveram-se em lutas por seus direitos, sobretudo após 1966. Mas não somente, visto que sua trajetória nos permitiu analisar ações do Comitê Municipal do Partido Comunista Brasileiro em conjunto com os trabalhadores de Americana e de Vila Carioba, evidenciando a existência de projetos políticos da classe operária que, mesmo durante a redemocratização, sofreram severas perseguições das autoridades policiais. Além disso, vislumbramos as diferentes estratégias de organização e modos de luta adotados pelos trabalhadores nas variadas conjunturas. Não podemos deixar de levar em conta os objetivos da Delegacia de Ordem Política e Social na produção da documentação sobre Carioba. Ao supervalorizar a ação e o protagonismo de Romeu Sturari na mobilização operária, colaboravam com a cosmovisão dos agentes estatais que atribuíam a difusão do comunismo aos militantes de vanguarda, pois os trabalhadores, em sua maioria migrantes rurais do interior, eram compreendidos como politicamente ignorantes e facilmente manipuláveis. Assim, é necessário ponderar até que ponto os agentes legais desprezavam a politização das classes de base que, apesar de influenciados pelo paternalismo e pelas políticas assistencialistas existentes na tecelagem, foram capazes de se articular enquanto classe para reivindicações de direitos durante a Grande Greve de Carioba, mesmo sem a participação de Sturari. Assim, seja pelo viés partidário ou sindical, a luta classista se fazia presente e mobilizava os trabalhadores de Vila Operária de Carioba. Outrossim, a investigação permitiu aferir parte dos efeitos do golpe militar na política e no movimento operário do município. Por fim, apesar dos agentes do DOPS estarem frequentemente presentes em Carioba durante os 20 anos 57 A análise do processo de disputa e demolição da Vila Operária de Carioba foge do escopo do presente artigo, entretanto, é reconhecida a importância da análise desse processo que carece de produções historiográficas a seu respeito. Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841 23 24 Luís Henrique Carboni Junior analisados, seus relatórios reforçavam sempre os aspectos de combate ao comunismo e agitação operária, adotando silêncio sepulcral sobre aspectos como acidentes ou condições de trabalho na tecelagem. Recebido em 23/04/2021 Aprovado em 03/08/2021 24 Mundos do Trabalho, Florianópolis \| v. 13 \| p. 1-24 \| 2021 e-ISSN: 1984-9222 \| DOI: https://doi.org/10.5007/1984-9222.2021.e80841
https://openalex.org/W4303958728	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/77F6DAF48EA54EF008AC5B8BE19C60B1/S0008439522000601a.pdf/div-class-title-benford-behavior-and-distribution-in-residue-classes-of-large-prime-factors-div.pdf	English	null	Benford behavior and distribution in residue classes of large prime factors	Canadian mathematical bulletin	2,022	cc-by	10,094	Canad. Math. Bull. Vol. 66 (2), 2023, pp. 626–642 http://dx.doi.org/10.4153/S0008439522000601 © The Author(s), 2022. Published by Cambridge University Press on behalf of The Canadian Mathematical Society. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Canad. Math. Bull. Vol. 66 (2), 2023, pp. 626–642 http://dx.doi.org/10.4153/S0008439522000601 © The Author(s), 2022. Published by Cambridge University Press on behalf of The Canadian Mathematical Society. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Canad. Math. Bull. Vol. 66 (2), 2023, pp. 626–642 http://dx.doi.org/10.4153/S0008439522000601 © The Author(s), 2022. Published by Cambridge University Press on behalf of The Canadian Mathematical Society. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Canad. Math. Bull. Vol. 66 (2), 2023, pp. 626–642 http://dx.doi.org/10.4153/S0008439522000601 © The Author(s), 2022. Published by Cambridge University Press on behalf of The Canadian Mathematical Society. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Canad. Math. Bull. Vol. 66 (2), 2023, pp. 626–642 http://dx.doi.org/10.4153/S0008439522000601 © The Author(s), 2022. Published by Cambridge University Press on behalf of The Canadian Mathematical Society. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Received by the editors April 25, 2022; revised September 13, 2022; accepted October 4, 2022. Published online on Cambridge Core October 10, 2022. P.P. is supported by the National Science Foundation under award DMS-2001581. AMS subject classification: 11A63, 11N37, 11N64. Keywords: Benford’s law, smooth numbers, anatomy of integers. Benford behavior and distribution in residue classes of large prime factors Paul Pollack and Akash Singha Roy Abstract. We investigate the leading digit distribution of the kth largest prime factor of n (for each fixed k = 1, 2, 3, . . . ) as well as the sum of all prime factors of n. In each case, we find that the leading digits are distributed according to Benford’s law. Moreover, Benford behavior emerges simultaneously with equidistribution in arithmetic progressions uniformly to small moduli. 1 Introduction It should be noted that these examples obey https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press 627 Benford behavior of large prime factors Benford’s law in a weaker sense; namely, Benford’s law holds if asymptotic density is replaced with logarithmic density.) Benford’s law in a weaker sense; namely, Benford’s law holds if asymptotic density is replaced with logarithmic density.) When f is multiplicative, whether or not f is Benford in base b can be interpreted as a problem in the theory of mean values of multiplicative functions. Namely, f is Benford precisely when f (n)2πiℓ/ log b has mean value zero for each nonzero integer ℓ. This criterion was noted by Aursukaree and Chandee [3] and used by them to show that the divisor function d(n) is Benford in base 10. A more systematic study of the Benford behavior of multiplicative functions, leveraging Halász’s celebrated mean value theorem, was recently undertaken in [8]. For example, it is shown there that ϕ(n) is not Benford, but that ∣τ(n)∣is, where τ is Ramanujan’s τ-function.1 All of the work in [8] is carried out in base 10, but both of the quoted results hold, by simple modifications of the proofs, in each fixed base b ≥2. Our concern in the present paper is with certain nonmultiplicative functions. Roughly speaking, we show that (for each fixed k) the kth largest prime factor of n obeys Benford’s law, as does the sum of all of the prime factors of n. (Both results hold for each base b.) In fact, our results are somewhat stronger than this. We let Pk(n) denote the kth largest prime factor of n; when k = 1, we write P(n) in place of the more cumbersome P1(n). More precisely, if n = p1p2p3⋯pΩ(n), with p1 ≥p2 ≥p3 ≥⋅⋅⋅≥pΩ(n), we set Pk(n) = pk, with the convention that Pk(n) = 0 if k > Ω(n). Put Ψk(x, y) ∶= #{n ≤x ∶Pk(n) ≤y}. Ψk(x, y) ∶= #{n ≤x ∶Pk(n) ≤y}. (When k = 1, it is usual to write Ψ(x, y) in place of Ψ1(x, y).) Let a mod q be a coprime residue class. For real x, y ≥2, define (When k = 1, it is usual to write Ψ(x, y) in place of Ψ1(x, y).) Let a mod q be a coprime residue class. 1In this latter result, the notion of “asymptotic density” in the definition of a Benford function should be replaced with “asymptotic density relative to the set of n with τ(n) ≠0.” 1 Introduction Benford’s law, named for physicist Frank Benford (though discovered almost 60 years prior by Simon Newcomb), refers to the observation that in many naturally occurring datasets, the leading digits are far from uniformly distributed, with smaller digits more likely to occur. Let us make this precise. By the N leading digits of the positive real number x, we mean the N most significant digits. For example (working in base 10), 123.456 has the first 4 leading digits 1234, and this is the same for 0.00123456. Now, let D and b be integers with b ≥2. We say a positive real number “begins with D in base b” if its most significant digits in base b are those of the base b expansion of D. Then Benford’s law, in base b, predicts that the proportion of terms in the dataset beginning with D should be approximately log(1 + D−1)/log b. For example, since log 2 log 10 = 0.3010 . . . , we expect to see a leading digit 1 in base 10 about 30% of the time. log 10 For general background on Benford’s law, see [5, 22]. In this paper, we are interested in datasets arising from positive-valued arithmetic functions. Let f ∶N →R>0. We say f obeys Benford’s law in base b (or that f is Benford in base b) if, for each positive integer D, the asymptotic density of n for which f (n) begins with D in base b is log(1 + D−1)/log b. Results on the “Benfordity” of particular arithmetic functions are scattered throughout the literature. For example, f (n) = n! is Benford in every base b [11], as is the “primorial” f (n) = ∏n k=1 pk [21]. The classical partition function p(n) is also Benford in every base (see [2] or [21]). On the other hand, f (n) = n is not Benford; the asymptotic density in question does not exist. This same obstruction to Benford’s law persists if f (n) is any positive-valued polynomial function of n. (See, for instance, the final section of [21]. 1.1 Notation Most of our notation is standard. Of note, we allow constants in O-symbols to depend on any parameter that has been declared as “fixed.” When we refer to “large” x, the threshold for large enough may also depend on these parameters. We write A ≳B as an abbreviation for A ≥(1 + o(1))B. 1 Introduction Then Theorem 1.2 Fix an integer b ≥2, and a positive integer D. Fix real numbers U ≥1 and ε > 0. Then N(x, y; b, D, q, a) ∼1 q log(1 + D−1) log b Ψ(x, y), as x, y →∞, uniformly for y ≥x1/U and residue classes a mod q with q ≤(log x) 1 2 −ε. as x, y →∞, uniformly for y ≥x1/U and residue classes a mod q with q ≤(log x) 1 2 −ε. As before, taking y = x and q = 1 shows that A(n) satisfies Benford’s law. Again, the extra generality here seems interesting. For example, it is implicit in Theorem 1.2 that A(n) is equidistributed mod q, uniformly for q ≤(log x) 1 2 −ε, a result which we have not seen explicitly stated in the literature before. (See [12] for the case of fixed q.) The same range of uniformity may follow from the method of Hall in [15] (who considered the distribution mod q of ∑p∣n, p∤q p), but our proof exhibits the result as a simple consequence of quantitative mean value theorems. In addition to the already-mentioned references, the reader interested in number- theoretic investigations of Benford’s law might also consult [6, 7, 9, 18, 20, 24]. 1 Introduction For real x, y ≥2, define Ψk(x, y; b, D, q, a) ∶= #{n ≤x ∶Pk(n) ≤y, Pk(n) ≡a (mod q), Pk(n) begins with D in base b} Theorem 1.1 Fix positive integers k, b, and D, with b ≥2. Fix real numbers U ≥1 and ε > 0. Then Ψk(x, y; b, D, q, a) ∼ 1 ϕ(q) log(1 + D−1) log b Ψk(x, y), as x, y →∞, uniformly for y ≥x1/U and coprime residue classes a mod q with q ≤ log x (log log x)k−1+ε . In fact, if k = 1, we can take q ≤(log x)A for any fixed A. as x, y →∞, uniformly for y ≥x1/U and coprime residue classes a mod q with q ≤ log x (log log x)k−1+ε . In fact, if k = 1, we can take q ≤(log x)A for any fixed A. To deduce that Pk(n) is Benford, it suffices to take q = 1 and y = x. The additional generality of Theorem 1.1 seems of some interest. For example, Theorem 1.1 contains the result of Banks–Harman–Shparlinski [4] that P(n), on integers n ≤x, is uni- formly distributed in coprime residue classes mod q, for q up to an arbitrary fixed power of log x. Theorem 1.1 gives the corresponding result for Pk(n), when k > 1, in the more restricted range q ≤log x/(loglog x)k−1+ε. This appears to be new; more- https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press ll k d A S h 628 P. Pollack and A. Singha Roy over, this range of q is sharp up to the power of loglog x, since ≫x(loglog x)k−2/log x values of n ≤x have Pk(n) = 2.h Turning to the sum of the prime factors, we let A(n) = ∑pk∥n kp. That is, A(n) is the sum of the prime factors of n, counting multiplicity. (The sum of the distinct prime factors of n could be handled by similar arguments.) The function A(n) was introduced by Alladi and first investigated by Alladi and Erd˝os [1].i Define N(x, y; b, D, q, a) ∶= #{n ≤x ∶P(n) ≤y, A(n) ≡a (mod q), A(n) begins with D in base b}. N(x, y; b, D, q, a) ∶= #{n ≤x ∶P(n) ≤y, A(n) ≡a (mod q), Theorem 1.2 Fix an integer b ≥2, and a positive integer D. Fix real numbers U ≥1 and ε > 0. ρk(α) = 1 −∫ α 1 (ρk(t −1) −ρk−1(t −1))dt t , for α > 1 and k ≥1. (2.1) (2.2) (2.2) uniformly for y ≥x1/U, where u ∶= log x log y . In particular, Ψk(x, y) ∼ρk(u)x as x →∞, uniformly for y ≥x1/U. uniformly for y ≥x1/U, where u ∶= log x log y . In particular, Ψk(x, y) ∼ρk(u)x as x →∞, uniformly for y ≥x1/U. (In [19], it is assumed that the ratio log x log y is fixed, rather than merely bounded. However, the proof given actually establishes (2.2) in the full range of Proposition 2.1.) The next result is a variant of Theorem 1.1 where we require that Pk(n) be bounded below by a fixed power of x. (In [19], it is assumed that the ratio log x log y is fixed, rather than merely bounded. However, the proof given actually establishes (2.2) in the full range of Proposition 2.1.) The next result is a variant of Theorem 1 1 where we require that P (n) be bounded g y However, the proof given actually establishes (2.2) in the full range of Proposition 2.1.) The next result is a variant of Theorem 1.1 where we require that Pk(n) be bounded below by a fixed power of x. p g y g p The next result is a variant of Theorem 1.1 where we require that Pk(n) be bounded below by a fixed power of x. Proposition 2.2 Fix positive integers k, b, and D with b ≥2. Fix real numbers A ≥ 1, U ≥1, and fix a real number U′ > U. The number of n ≤x for which Pk(n) ≡a (mod q), Pk(n) begins with the digits of D in base b, and Pk(n) ∈(x1/U′, y] is 1 ϕ(q) log(1 + D−1) log b (ρk(u) −ρk(U′))x + o(x/ϕ(q)), where u ∶= log x log y , where x, y →∞with y ≥x1/U, and where a mod q is a coprime residue class with q ≤(log x)A. where u ∶= log x log y , where x, y →∞with y ≥x1/U, and where a mod q is a coprime residue class with q ≤(log x)A. The proof of Proposition 2.2 requires two classical results from the theory of primes in arithmetic progressions. Let π(x; q, a) denote the count of primes p ≤x with p ≡a (mod q). Proposition 2.3 (Brun–Titchmarsh) If a and q are coprime integers with 0 < 2q ≤x, then π(x; q, a) ≪ 1 ϕ(q) x log(x/q). Benford behav Much is know for instance, our purposes ρk (k = 1, 2, 3 limα→∞ρk(α The follow as equation (4 there). Proposition 2 (2.2) uniformly for uniformly for (In [19], it is However, the The next re below by a fix Proposition 2 1, U ≥1, and (mod q), Pk( where u ∶= lo lo residue class w The proof in arithmetic (mod q). Proposition 2 then Here, the imp Proposition 2 integers with 629 Benford behavior of large prime factors Much is known about the asymptotic behavior of ρk(α) as α →∞; for k = 1, see, for instance, [10], whereas for k ≥2, see equations (6.4) and (6.15) in [19]. For our purposes, much weaker information suffices. We assume as known that each ρk (k = 1, 2, 3, . . . ) is positive-valued and weakly decreasing on (0, ∞), and that limα→∞ρk(α) = 0.h The following result, which connects the ρk with the distribution of Pk(n), appears as equation (4.7) in [19] (and is a consequence of the stronger assertion (4.8) shown there). Proposition 2.1 Fix a positive integer k and a real number U ≥1. For all x, y ≥2, Proposition 2.1 Fix a positive integer k and a real number U ≥1. For all x, y ≥2, Ψk(x, y) = ρk(u)x + O(x/log x), (2.2) Ψk(x, y) = ρk(u)x + O(x/log x), 2 Benford’s law for Pk(n): proof of Theorem 1.1 We make crucial use of both the results and methods of Knuth and Trabb Pardo [19], who were the first to seriously investigate Pk(n) when k > 1. We define functions ρk(α), for integers k ≥0 and real α, as follows: ρk(α) = 0 if α ≤0 or k = 0, ρk(α) = 0 if α ≤0 or k = 0, ρk(α) = 1 for 0 < α ≤1 and k ≥1, ρk(α) = 1 for 0 < α ≤1 and k ≥1, ρk(α) = 1 −∫ α 1 (ρk(t −1) −ρk−1(t −1))dt t , for α > 1 and k ≥1. (2.1) (2.1) https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press (2.2) Here, the implied constant is absolute. Here, the implied constant is absolute. Proposition 2.4 (Siegel–Walfisz) Fix a real number A > 0. If a and q are coprime integers with 1 ≤q ≤(log x)A, and x ≥3, then π(x; q, a) = 1 ϕ(q) ∫ x 2 1 log t dt + OA(x exp(−C √ log x)). Here, C is a certain absolute constant. Here, C is a certain absolute constant. Here, C is a certain absolute constant. https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press 630 P. Pollack and A. Singha Roy For proofs of these results, see [23, Theorem 3.9, p. 90] and [23, Corollary 11.21, p. 382]. Proof of Proposition 2.2 First note that we can (and will) always assume that y ≤ x, since the cases when y > x are covered by the case y = x. By a standard compactness argument, when proving Proposition 2.2, we may assume that u = log x log y is fixed. To see this, suppose Proposition 2.2 holds when u is fixed but does not hold in general. Then, for some ε > 0, there are choices of x, y, a, and q with x arbitrarily large, x ≥y ≥x1/U, and q ≤(log x)A for which our count exceeds 1 ϕ(q) log(1 + D−1) log b (ρk(u) −ρk(U′) + ε)x, (2.3) (2.3) or there are such choices of x, y, a, and q for which our count falls below 1 ϕ(q) log(1 + D−1) log b (ρk(u) −ρk(U′) −ε)x. We will assume that we are in the former case; the latter can be handled analogously. By compactness, we may choose x, y, a, q so that u →u0, for some u0 ∈[1, U].i ( ) We will assume that we are in the former case; the latter can be handled analogously. h h f [ ] We first rule out u0 = 1. As y ≤x, the condition Pk(n) ≤y is always at least as strict as the condition Pk(n) ≤x (which holds vacuously, as we are counting numbers n ≤x). Moreover, the u = 1 case of Proposition 2.2 is true by hypothesis. Putting these observations together, we see that the count of n corresponding to x, y, a, q is at most 1 ϕ(q) log(1 + D−1) log b (ρk(1) −ρk(U′) + o(1))x. (2.2) by partial summation and the Brun–Titchmarsh theorem (Proposition 2.3). We proceed to estimate, for each j ∈J, the corresponding inner sums in (2.5) over p and n. If p is prime and Pk(n) = p, then n = mp where m ≤x/p, Pk(m) ≤p, and ( ) Th l h ld Th f J d I proceed to estimate, for each j ∈J, the corresponding inner sums in (2.5) over p and n. If p is prime and Pk(n) = p, then n = mp where m ≤x/p, Pk(m) ≤p, and Pk−1(m) ≥p. The converse also holds. Thus, if j ∈J and p ∈Ij, j g p If p is prime and Pk(n) = p, then n = mp where m ≤x/p, Pk(m) ≤p, and Pk−1(m) ≥p. The converse also holds. Thus, if j ∈J and p ∈Ij, ∑ n≤x Pk(n)=p 1 = Ψk(x/p, p) −Ψk−1(x/p, p −ε) for (say) ε = 1 2. Hence, for (say) ε = 1 2. Hence, 2 ∑ p∈Ij p≡a (mod q) ∑ n≤x Pk(n)=p 1 = ∑ p∈Ij p≡a (mod q) Ψk(x/p, p) − ∑ p∈Ij p≡a (mod q) Ψk−1(x/p, p −ε). To continue, observe that, for j ∈J, ∑ p∈Ij p≡a (mod q) Ψk(x/p, p) − 1 ϕ(q) ∫Ij Ψk(x/t, t) dt log t = ∑ u j≤p<v j p≡a (mod q) ∑ n≤x/p Pk(n)≤p 1 − 1 ϕ(q) ∫ v j u j ∑ n≤x/t Pk(n)≤t 1 log t dt = ∑ n≤x/u j ⎛ ⎜⎜⎜ ⎝ ∑ m<p≤M p≡a (mod q) 1 − 1 ϕ(q) ∫ M m dt log t + O(1) ⎞ ⎟⎟⎟ ⎠ , where m and M are defined by ∑ p∈Ij p≡a (mod q) ∑ n≤x Pk(n)=p 1 = ∑ p∈Ij p≡a (mod q) Ψk(x/p, p) − ∑ p∈Ij p≡a (mod q) Ψk−1(x/p, p −ε). (2.2) However, if u →1, then ρk(u) →ρk(1), and this estimate is eventually incompatible with (2.3).h Thus, it must be that u0 > 1. Here, we may obtain a contradiction by a slightly tweaked argument. For any fixed δ > 0, we eventually have u > u0 −δ. So the condi- tion Pk(n) ≤y is eventually stricter than the condition Pk(n) ≤x1/(u0−δ). If δ is fixed sufficiently small (in terms of ε), then the u = u0 −δ case of Proposition 2.2 gives an estimate contradicting (2.3).i We thus turn to proving the modified statement with the extra condition that u is fixed. i For each nonnegative integer j, let Ij denote the interval Ij ∶= [u j, v j), where u j ∶= Db j, v j ∶= (D + 1)b j. (2.4) (2.4) Then our count of n is given by Then our count of n is given by ∑ j≥0 ∑ p∈Ij∩(x1/U′ ,y] p≡a (mod q) ∑ n≤x Pk(n)=p 1. (2.5) ∑ j≥0 ∑ p∈Ij∩(x1/U′ ,y] p≡a (mod q) ∑ n≤x Pk(n)=p 1. (2.5) Let J be the collection of nonnegative integers j with Ij ⊂(x1/U′, y/exp( √ log x)). Then, at the cost of another error of size o(x/ϕ(q)), we can restrict the triple sum in (2.5) to j ∈J. Indeed, the n counted by the triple sum above that are 601 Published online by Cambridge University Press https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press Benford behavior of large prime factors 63 631 Benford behavior of large prime factors excluded by this restriction have either a prime divisor in P ∶= (x1/U′, bx1/U′] or in P′ ∶= [y/b exp( √ log x), y], and the number of such n ≤x is at most excluded by this restriction have either a prime divisor in P ∶= (x1/U′, bx1/U′] or in P′ ∶= [y/b exp( √ log x), y], and the number of such n ≤x is at most or x ∑ p∈P∪P′ p≡a (mod q) 1/p = o(x/ϕ(q)), x ∑ p∈P∪P′ p≡a (mod q) 1/p = o(x/ϕ(q)), by partial summation and the Brun–Titchmarsh theorem (Proposition 2.3). We proceed to estimate, for each j ∈J, the corresponding inner sums in (2.5) over p and n. by partial summation and the Brun–Titchmarsh theorem (Proposition 2.3). We proceed to estimate, for each j ∈J, the corresponding inner sums in (2.5) over p and n. (2.2) To continue, observe that, for j ∈J, ∑ p∈Ij p≡a (mod q) Ψk(x/p, p) − 1 ϕ(q) ∫Ij Ψk(x/t, t) dt log t = ∑ u j≤p<v j p≡a (mod q) ∑ n≤x/p Pk(n)≤p 1 − 1 ϕ(q) ∫ v j u j ∑ n≤x/t Pk(n)≤t 1 log t dt = ∑ n≤x/u j ⎛ ⎜⎜⎜ ⎝ ∑ m<p≤M p≡a (mod q) 1 − 1 ϕ(q) ∫ M m dt log t + O(1) ⎞ ⎟⎟⎟ ⎠ , where m and M are defined by where m and M are defined by where m and M are defined by m ∶= max{u j, Pk(n)}, M ∶= min{x/n, v j}, and where the last displayed sum on n is understood to be extended only over those n ≤x/u j for which m ≤M. By the Siegel–Walfisz theorem (Proposition 2.4), and where the last displayed sum on n is understood to be extended only over those n ≤x/u j for which m ≤M. By the Siegel–Walfisz theorem (Proposition 2.4), ∑ m<p≤M p≡a (mod q) 1 − 1 ϕ(q) ∫ M m dt log t ≪M exp(−C √ log M) ≪x n exp(−C′√ log x), where C is an absolute positive constant and C′ = C/ √ U′. (This use of the Siegel–Walfisz theorem explains the restriction q ≤(log x)A in the statement of where C is an absolute positive constant and C′ = C/ √ U′. (This use of the Siegel–Walfisz theorem explains the restriction q ≤(log x)A in the statement of https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press 632 P. Pollack and A. Singha Roy 632 P. Pollack and A. Singha Roy P. Pollack and A. Singha Roy Proposition 2.2.) Putting this back in the above and summing on n, we find that (for large x) Proposition 2.2.) Putting this back in the above and summing on n, we find that (for large x) (2.6) (2.6) ∑ p∈Ij p≡a (mod q) Ψk(x/p, p) − 1 ϕ(q) ∫Ij Ψk(x/t, t) dt log t ≪x log x ⋅exp(−C′√ log x) + x u j . (2.6) A nearly identical calculation gives the same bound for the difference ∑ p∈Ij p≡a (mod q) Ψk−1(x/p, p −ε) − 1 ϕ(q) ∫Ij Ψk−1(x/t, t) dt log t . (2.2) Since u j+1/u j ≥2 and the smallest j ∈J has u j ≥x1/U′, the expression on the right- hand side of (2.6), when summed on j ∈J, is ≪x(log x)2 exp(−C′√ log x) + x1−1/U′, and this is certainly o(x/ϕ(q)). As a consequence, instead of our original triple sum (2.5), it is enough to estimate x ϕ(q) ∑ j∈J 1 x ∫Ij (Ψk(x/t, t) −Ψk−1(x/t, t)) dt log t . (2.7) (2.7) We now apply Proposition 2.1, noting that for each t ∈Ij, we have log (x/t) log t = log x log t −1 ≤U′ −1 as well as log(x/t) ≥log(y/t) ≥ √ log x. We find that We now apply Proposition 2.1, noting that for each t ∈Ij, we have log (x/t) log t = log x log t −1 ≤U′ −1 as well as log(x/t) ≥log(y/t) ≥ √ log x. We find that 1 x ∫Ij (Ψk(x/t, t) −Ψk−1(x/t, t)) dt log t = ∫Ij 1 t (ρk (log x log t −1) −ρk−1 (log x log t −1)) dt log t + O ⎛ ⎝∫Ij 1 t √ log x dt log t ⎞ ⎠. 1 x ∫Ij (Ψk(x/t, t) −Ψk−1(x/t, t)) dt log t = ∫Ij 1 t (ρk (log x log t −1) −ρk−1 (log x log t −1)) dt log t + O ⎛ ⎝∫Ij 1 t √ log x dt log t ⎞ ⎠. The error term, when summed on j ∈J, is ≪ 1 √ log x ∫ x 2 dt t log t ≪loglog x/ √ log x, and so is o(1); inserted back into (2.7), we see that this gives rise to a final error of size o(x/ϕ(q)) in our count, which is acceptable. To deal with the remaining integrals, we write u j = x μ j and v j = xνj and make the change of variables α = log x log t . Then dα = − log x t(log t)2 dt, so that dt t log t = −dα α and ∑ j∈J∫Ij 1 t (ρk (log x log t −1) −ρk−1 (log x log t −1)) dt log t = ∑ j∈J∫ 1/νj 1/μ j −ρk(α −1) −ρk−1(α −1) α dα. (2.2) ∑ j∈J∫Ij 1 t (ρk (log x log t −1) −ρk−1 (log x log t −1)) dt log t α From (2.1), −ρk(α−1)−ρk−1(α−1) α = ρ′ k(α), so that this last sum on j simplifies to ∑j∈J(ρk(1/νj) −ρk(1/μ j)). Now, following [19], we introduce the function Fk(β) From (2.1), −ρk(α−1)−ρk−1(α−1) α = ρ′ k(α), so that this last sum on j simplifies to ∑j∈J(ρk(1/νj) −ρk(1/μ j)). Now, following [19], we introduce the function Fk(β) https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press behavior of large prime factors 633 Benford behavior of large prime factors Benford behavior of large prime factors 633 defined for β ∈(0, 1] by Fk(β) = ρk(1/β). By the mean value theorem, defined for β ∈(0, 1] by Fk(β) = ρk(1/β). By the mean value theorem, ρk(1/νj) −ρk(1/μ j) = Fk(νj) −Fk(μ j) defined for β ∈(0, 1] by Fk(β) = ρk(1/β). By the mean value theorem, ρk(1/νj) −ρk(1/μ j) = Fk(νj) −Fk(μ j) defined for β ∈(0, 1] by Fk(β) = ρk(1/β). By the mean value theorem, ρk(1/νj) −ρk(1/μ j) = Fk(νj) −Fk(μ j) = (νj −μ j)F′ k(t j) = log(1 + D−1) log x F′ k(t j) ) = Fk(νj) −Fk(μ j) = (νj −μ j)F′ k(t j) = log(1 + D−1) log x F′ k(t j) for some t j ∈(μ j, νj). Thus, for some t j ∈(μ j, νj). Thus, ∑ j∈J (ρk(1/νj) −ρk(1/μ j)) = log(1 + D−1) log b ∑ j∈J F′ k(t j) ⋅log b log x = log(1 + D−1) log b ∑ j∈J F′ k(t j) ⋅(μ j+1 −μ j). Since each t j ∈(μ j, νj) ⊂(μ j, μ j+1), the final sum on j is essentially a Riemann sum. To make this precise, let j0 = minJ and j1 = max J. Then F′ k(1/U′)(μ j0 −1 U′ ) + ∑ j∈J F′ k(t j)(μ j+1 −μ j) + F′ k(1/u)( 1 u −μ j1+1) is a genuine Riemann sum for ∫ 1/u 1/U′ F′ k(t)dt, whose mesh size goes to 0 as x →∞. However, the terms we have added to the sum on j ∈J contribute o(1), as x →∞. It follows that ∑j∈J F′ k(t j)(μ j+1 −μ j) →∫ 1/u 1/U′ F′ k(t)dt = Fk(1/u) −Fk(1/U′) = ρk(u) −ρk(U′). Collecting estimates completes the proof of the proposition in the case when u is fixed. (2.2) ∎ To deduce Theorem 1.1, it remains to handle the contribution from n with Pk(n) ≤x1/U′.h The following lemma bounds the number of integers with a large smooth divisor. A proof is sketched in Exercise 293 on page 554 of [26], with a solution in [25, pp. 305–306]. By the y-smooth part of a number n, we mean ∏pe∥n p≤y pe. Lemma 2.5 For all x ≥z ≥y ≥2, the number of n ≤x whose y-smooth part exceeds z is O (x exp(−1 2 log z log y )). Lemma 2.5 For all x ≥z ≥y ≥2, the number of n ≤x whose y-smooth part exceeds z is O (x exp(−1 2 log z log y )). Lemma 2.5 For all x ≥z ≥y ≥2, the number of n ≤x whose y-smooth part exceeds z is O (x exp(−1 2 log z log y )). Lemma 2.6 Fix a positive integer k and a real number B ≥1. Lemma 2.6 Fix a positive integer k and a real number B ≥1. • When k = 1, the number of n ≤x with Pk(n) ≤y and Pk(n) ≡a (mod q) is ≪ x ϕ(q) exp(−1 8u) + x (log(3q) log x ) B ⋅exp(−1 8u) , uniformly for x ≥y ≥3 with y ≤x1/4, and a mod q any coprime residue class with q ≤x1/8. As usual, u = log x log y . g y • When k ≥2, the number of n ≤x with Pk(n) ≤y and Pk(n) ≡a (mod q) is ≪ x log x (loglog x)k−2 log(3q) + x ϕ(q) (logu)k−2 u , uniformly in the same range of x, y, and q. 634 P. Pollack and A. Singha Roy P. Pollack and A. Singha Roy Proof We will restrict attention to n > x3/4; this is permissible, since x3/4 is dwarfed by either of our target upper bounds. We let p = Pk(n) and write n = p1⋯pk−1ps, where p1 ≥p2 ≥⋅⋅⋅≥pk−1 ≥p and P(s) ≤p.i / /h p p p p ( ) p We first show that we can assume s ≤x1/2. Indeed, suppose s > x1/2. Then, with m = n/p, we have that m ≤x/p and that the p-smooth part of m exceeds x1/2. (2.2) https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press 000601 Published online by Cambridge University Press Benford behavior of large prime factors Benford behavior of large prime factors Benford behavior of large prime factors 635 Hence, the number of possibilities for n given p is Hence, the number of possibilities for n given p is Hence, the number of possibilities for n given p is ≪ x log x (log log x log p) k−2 log p p . ≪ x log x (log log x log p) k−2 log p p . We now sum on p ≤y with p ≡a (mod q). Estimating crudely, we see that the p ≤3q2 contribute We now sum on p ≤y with p ≡a (mod q). Estimating crudely, we see that the p ≤3q2 contribute ≪ x log x (loglog x)k−2 log(3q). ≪ x log x (loglog x)k−2 log(3q). To handle the remaining contribution in the case when y > 3q2, we apply partial summation; by Brun–Titchmarsh, ∑ 3q2<p≤y p≡a (mod q) (log log x log p) k−2 log p p ≪ 1 ϕ(q)(logu)k−2 −∫ y 3q2 π(t; q, a)d⎛ ⎝(log log x log t ) k−2 log t t ⎞ ⎠. Since (log log x log t ) k−2 log t t is a decreasing function of t on [3q2, y], the bound π(t; q, a) ≪t/ϕ(q)log t implies that Since (log log x log t ) k−2 log t t is a decreasing function of t on [3q2, y], the bound π(t; q, a) ≪t/ϕ(q)log t implies that −∫ y 3q2 π(t; q, a)d⎛ ⎝(log log x log t ) k−2 log t t ⎞ ⎠ ≪− 1 ϕ(q) ∫ y 3q2 t log t d⎛ ⎝(log log x log t ) k−2 log t t ⎞ ⎠. (2.2) Applying Lemma 2.5, we see that for every p ≤y, the number of corresponding m is ≪x p exp(−1 4 log x log p) ≪x p exp(−1 8 log x log p) ⋅exp(−1 8 log x log p) ≪ x (log x)B (log p)B p exp(−1 8 log x log p) ≪ x (log x)B (log p)B p exp(−1 8u). Now, we sum on p ≤y with p ≡a (mod q). We split the sum at 3q2, using Mertens’ theorem to bound the first half and the Brun–Titchmarsh theorem (with partial summation) for the second; this gives ∑ p≤y p≡a (mod q) (log p)B p ≤∑ p≤3q2 (log p)B p + ∑ 3q2<p≤y p≡a (mod q) (log p)B p ≪(log(3q))B−1 ∑ p≤3q2 log p p + 1 ϕ(q)(log y)B ≪(log(3q))B + (log y)B ϕ(q) . Substituting this estimate into the previous display, we conclude that the n with s > x1/2 contribute ≪ x uBϕ(q) exp(−1 8u) + x (log(3q) log x ) B ⋅exp(−1 8u) ≪ x ϕ(q) exp(−1 8u) + x (log(3q) log x ) B ⋅exp(−1 8u) . 8) (2.8) This is already enough to settle the k = 1 case of Lemma 2.6. Indeed, in that case, n = ps, where p = P(n), and s = n/P(n) ≥n/y > x3/4/y ≥x1/2. 1/2h This is already enough to settle the k = 1 case of Lemma 2.6. Indeed, in that case, n = ps, where p = P(n), and s = n/P(n) ≥n/y > x3/4/y ≥x1/2. /h Now, suppose that k ≥2 and that s ≤x1/2. Then pk 1 ≥p1⋯pk−1p = n/s > x3/4/x1/2 = x1/4, so that p1 ≥x1/4k. Hence, given p2, . . . , pk−1, p, and s, the number of possibilities for p1 (and thus also for n) is ≪π(x/p2⋯pk−1ps) ≪x/p2⋯pk−1ps log x. Observe that s is p-smooth, while each pi ∈[p, x]. We have that ∑s p-smooth 1/s = ∏prime ℓ≤p(1 −1/ℓ)−1 ≪log p. Moreover (when p ≤y), ∑p≤pi≤x 1/pi ≪log log x log p. (2.2) −∫ y 3q2 π(t; q, a)d⎛ ⎝(log log x log t ) k−2 log t t ⎞ ⎠ −∫ y 3q2 π(t; q, a)d⎛ ⎝(log log x log t ) k−2 log t t ⎞ ⎠ Integrating by parts again, Integrating by parts again, ∫ y 3q2 t log t d⎛ ⎝(log log x log t ) k−2 log t t ⎞ ⎠ ∫ y 3q2 t log t d⎛ ⎝(log log x log t ) k−2 log t t ⎞ ⎠ = −∫ y 3q2 (log log x log t ) k−2 log t t d( t log t ) + O((loglog x)k−2) ≪∫ y 3q2 (log log x log t ) k−2 dt t + O((loglog x)k−2). = −∫ y 3q2 (log log x log t ) k−2 log t t d( t log t ) + O((loglog x)k−2) ≪∫ y 3q2 (log log x log t ) k−2 dt t + O((loglog x)k−2). Making the change of variables α = log t log x , Making the change of variables α = log t log x , Making the change of variables α = log t log x , Making the change of variables α = log t log x , ∫ y 3q2 (log log x log t ) k−2 dt t ≤log x ∫ 1/u 0 (log(1/α))k−2 dα ≪log x ⋅1 u(logu)k−2. (In the last step, we use that ∫ z 0 (log(1/α))k−2 dα has the form z ⋅Q(log(1/z)), where Q is a monic polynomial with degree k −2.) Collecting estimates, we conclude that (In the last step, we use that ∫ z 0 (log(1/α))k−2 dα has the form z ⋅Q(log(1/z)), where Q is a monic polynomial with degree k −2.) Collecting estimates, we conclude that https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press 636 P. Pollack and A. Singha Roy P. Pollack and A. Singha Roy 636 when k ≥2, the n with s ≤x1/2 make a contribution when k ≥2, the n with s ≤x1/2 make a contribution ≪ x log x (loglog x)k−2 log(3q) + x ϕ(q) (logu)k−2 u . Since this upper bound dominates the contribution (2.8) from n with s > x1/2, the k ≥2 cases of Lemma 2.6 follow. ∎ Proof of Theorem 1.1 Fix η > 0. (2.2) We will show that the count of n in question is eventually2 larger than 1 ϕ(q) log(1+D−1) log b (ρk(u) −η) x and eventually smaller than 1 ϕ(q) log(1+D−1) log b (ρk(u) + η) x, and hence is ∼ 1 ϕ(q) log(1+D−1) log b ρk(u)x. Since Ψk(x, y) ∼ ρk(u)x, Theorem 1.1 then follows.hfi h The required lower bound is immediate from Proposition 2.2: it suffices to apply that proposition with U′ fixed large enough that ρk(U′) < η. We turn now to the upper bound. Apply Lemma 2.6, taking B = A + 1 in the case k = 1. That lemma implies the existence of a constant C, depending only on k (and on A, if k = 1) such that the following holds: for any fixed U′ ≥4, the number of n ≤x with Pk(n) ≡a (mod q) and Pk(n) ≤x1/U′ is eventually at most C x ϕ(q) (log U′)k−2 U′ . If we choose U′ > U so large that C (log U′)k−2 U′ < η log(1+D−1) log b , the desired upper bound then follows from Proposition 2.2. ∎ https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press 2Here and later in this proof, “eventually” refers to the limit as taken in Theorem 1.1. That is, a statement holds eventually if there is a real number T such that the statement is true whenever x, y ≥T, with y ≥ x1/U, and with a mod q a coprime residue class modulo q ≤ log x (log log x)k−1+ε or, when k = 1, modulo q ≤ (log x)A. 3 Benford’s law for the sum of the prime factors: proof of Theorem 1.2 For multiplicative functions F, G taking values on or inside the complex unit circle, we define (following [13]) the distance between F and G, up to x, by D(F, G; x) = & ' ' ' ∑ p≤x 1 −Re(F(p)G(p)) p . D(F, G; x) = & ' ' ' ∑ p≤x 1 −Re(F(p)G(p)) p . The following statement (Corollary 4.12 on page 494 of [26]), due to Montgomery and Tenenbaum, makes quantitatively precise a result of Halász [14] that F has mean value 0 unless F “pretends” to be nit for some t. Proposition 3.1 Let F be a multiplicative function with ∣F(n)∣≤1 for all n. For x ≥2 and T ≥2, let m(x, T) = min ∣t∣≤T D(F, nit; x)2. m(x, T) = min ∣t∣≤T D(F, nit; x)2. 2Here and later in this proof, “eventually” refers to the limit as taken in Theorem 1.1. That is, a statement holds eventually if there is a real number T such that the statement is true whenever x, y ≥T, with y ≥ x1/U, and with a mod q a coprime residue class modulo q ≤ log x (log log x)k−1+ε or, when k = 1, modulo q ≤ (log x)A. 637 Benford behavior of large prime factors Benford behavior of large prime factors 637 Then ∑ n≤x F(n) ≪x 1 + m(x, T) em(x,T) + x T . Here, the implied constant is absolute. When F is real-valued, the following (slightly weakened version of a) theorem of Hall and Tenenbaum [16] allows us to consider only D(F, 1; x). Proposition 3.2 Let F be a real-valued multiplicative function with ∣F(n)∣≤1 for all n. Then ∑ n≤x F(n) ≪x exp(−0.3 ⋅D(F, 1; x)2). Lemma 3.3 Fix δ > 0 and fix U ≥1. For all large x, the number of n ≤x with P(n) ≤y and A(n) ≡a (mod q) is Lemma 3.3 Fix δ > 0 and fix U ≥1. For all large x, the number of n ≤x with P(n) ≤y and A(n) ≡a (mod q) is Ψ(x, y) q + O(x/(log x) 1 2 −δ), for all x ≥y ≥x1/U and residue classes a mod q with q ≤log x. for all x ≥y ≥x1/U and residue classes a mod q with q ≤log x. Proof By the orthogonality relations for additive characters, Proof By the orthogonality relations for additive characters, Proof By the orthogonality relations for additive characters, 1 = ∑ n≤x 1P(n)≤y1A(n)≡a (mod q) = ∑ n≤x 1P(n)≤y ⎛ ⎝ 1 q ∑ r mod q e2πir(A(n)−a)/q⎞ ⎠ = Ψ(x, y) q + 1 q ∑ r mod q r/≡0 (mod q) e−2πiar/q ∑ n≤x 1P(n)≤ye2πirA(n)/q. Hence, it suffices to show that ∑ n≤x 1P(n)≤ye2πirA(n)/q = O(x/(log x)1/2−δ) (3.1) (3.1) for each nonzero residue class r mod q. Write r/q = r′/q′ in lowest terms, so that q′ > 1. If q′ = 2, then r′ = 1, and F(n) ∶= 1P(n)≤ye2πirA(n)/q = 1P(n)≤y(−1)A(n) is a real-valued multiplicative function of mod- ulus at most 1. Moreover, D(F, 1; x)2 ≥∑2<p≤y 2/p = 2loglog x + O(1). By Proposi- tion 3.2, the left-hand side of (3.1) is O(x/(log x)0.6), which is more than we need. So we may assume q′ > 2. y q When q′ > 2, we apply Proposition 3.1 taking T = log x. Let t be any real number with ∣t∣≤T. We set z = exp((log x)δ) and start from the lower bound 01 Published online by Cambridge University Press https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press 638 P. Pollack and A. Singha Roy P. Pollack and A. Singha Roy D(F, nit; x)2 ≥∑ z<p≤y 1 −Re(e2πir′ p/q′ p−it) p . (3.2) To estimate the right-hand sum, we split the range of summation into blocks on which p−it is essentially constant. Cover (z, y] with intervals I = (u, u(1 + 1/(log x)2)], allowing the rightmost inter- val to jut out slightly past y but no further than y + y/(log x)2. On each interval I, every p ∈I satisfies ∣t log p −t logu∣≤∣t∣/(log x)2 ≤1/log x, so that ∣p−it −u−it∣= ∣∫ t log p t log u exp(−iθ) dθ∣≤1/log x, and and ∑ p∈I 1 −Re(e2πir′ p/q′ p−it) p = ∑ p∈I 1 −Re(e2πir′ p/q′u−it) p + O ⎛ ⎝ 1 log x ∑ p∈I 1 p ⎞ ⎠. (3.3) (3.3) The error term when summed over all intervals I will be O(loglog x/log x), which is negligible for us. So we focus on the main term. Observe that p = (1 + o(1))u for every p ∈I. Proof By the orthogonality relations for additive characters, (Here and below, asymptotic notation refers to the behavior as x →∞.) Thus, ∑ p∈I 1 −Re(e2πir′ p/q′u−it) p ≳1 u ∑ p∈I (1 −Re(e2πir′ p/q′u−it)) ≳1 u ∑ a′ mod q′ gcd(a′,q′)=1 (1 −Re(e2πir′a′/q′u−it))π(I; q′, a′), where π(I; q′, a′) denotes the number of primes p ∈I with p ≡a′ (mod q′). By the Siegel–Walfisz theorem (Proposition 2.4), π(I; q′, a′) ∼ 1 ϕ(q′)π(I), where π(I) is the total count of primes belonging to I. Thus, the above right-hand side is ≳ π(I) ϕ(q′)u ∑ a′ mod q′ gcd(a′,q′)=1 (1 −Re(e2πir′a′/q′u−it)) = π(I) ϕ(q′)u(ϕ(q′) −Re(μ(q′)u−it)) ≥1 2π(I)/u ≳1 2 ∑1 p; (3.4) ≳ π(I) ϕ(q′)u ∑ a′ mod q′ gcd(a′,q′)=1 (1 −Re(e2πir′a′/q′u−it)) = π(I) ϕ(q′)u(ϕ(q′) −Re(μ(q′)u−it)) ≥1 2π(I)/u ≳1 2 ∑ p∈I 1 p; ≥1 2π(I)/u ≳1 2 ∑ p∈I 1 p; (3.4) (3.4) here, we use that ∑a′ (mod q′), gcd(a′,q′)=1 e2πia′r′/q′ = μ(q′) (see, for example, [17, Theorem 272, p. 309]) and that ϕ(q′) −Re(μ(q′)u−it) ≥ϕ(q′) −1 ≥1 2 ϕ(q′), as q′ > 2. Combining the last two displays and summing on I, ∑ I ∑ p∈I 1 −Re(e2πir′ p/q′u−it) p ≳1 2 ∑ I ∑ p∈I 1 p ≥1 2 ∑ z<p≤y 1 p ≳1 2(1 −δ)loglog x. From (3.3) (and the immediately following remark about the error term), the same lower bound holds for ∑I ∑p∈I 1−Re(e2πir′ p/q′ p−it) p . This double sum essentially coincides with the right-hand side of (3.2), except for possibly including contributions 601 Published online by Cambridge University Press oi.org/10.4153/S0008439522000601 Published online by Cambridge University Press Benford behavior of large prime factors 639 from a few values of p > y. However, those contributions are O(1), in fact ≪∑y<p<y+y/(log x)2 1/p ≪1/(log x)2. Thus, D(F, nit; x)2 ≳1 2(1 −δ)loglog x. In particular, D(F, nit; x)2 ≥( 1 2 −9 10 δ)loglog x once x is sufficiently large (in terms of δ and U). Since this lower bound holds uniformly in t with ∣t∣≤T, the desired inequality (3.1) follows from Proposition 3.1. ∎ Using Lemma 3.3, we can establish the following A(n)-analogue of Proposition 2.2. Proposition 3.4 Fix positive integers k, D, and b with b ≥2. Fix real numbers U′ > U ≥1, and fix ε > 0. Proof By the orthogonality relations for additive characters, The number of n ≤x for which A(n) ≡a (mod q), P(n) begins with the digits of D in base b, and P(n) ∈(x1/U′, y] is 1 q log(1 + D−1) log b (ρ(u) −ρ(U′)) x + o(x/q), where u ∶= log x log y , where x, y →∞with y ≥x1/U, and where a mod q is any residue class with q ≤(log x) 1 2 −ε. Proof (sketch) The proof is similar to the case k = 1 of Proposition 2.2, with the needed input on Ψ(x, y) replaced by appeals to Lemma 3.3. We may assume y = x1/u where u ≥1 is fixed. With the intervals Ij defined as in (2.4), the desired count of n is given by the triple sum ∑ j≥0 ∑ p∈Ij∩(x1/U′ ,y] ∑ n≤x P(n)=p A(n)≡a (mod q) 1. (3.5) (3.5) At the cost of a negligible error, we may restrict the outer sum to j ∈J, where J is the collection of nonnegative integers j with Ij ⊂(x1/U′, y/exp( √ log x)); indeed, defining (as before) P ∶= (x1/U′, bx1/U′] and P′ ∶= [y/b exp( √ log x), y], the incurred error is of size At the cost of a negligible error, we may restrict the outer sum to j ∈J, where J is the collection of nonnegative integers j with Ij ⊂(x1/U′, y/exp( √ log x)); indeed, defining (as before) P ∶= (x1/U′, bx1/U′] and P′ ∶= [y/b exp( √ log x), y], the incurred error is of size ≪x ∑ p∈P∪P′ 1/p ≪x/(log x)1/2, which is o(x/q). Now, suppose j ∈J and p ∈Ij; then, by Lemma 3.3, which is o(x/q). Now, suppose j ∈J and p ∈Ij; then, by Lemma 3.3, ∑ n≤x P(n)=p A(n)≡a (mod q) 1 = ∑ m≤x/p P(m)≤p A(m)≡a−p (mod q) 1 = 1 q Ψ(x/p, p) + O ( x p(log(x/p)) 1 2 (1−ε) ) . Summing on all j ∈J and all p ∈Ij, the contribution from O-terms is Summing on all j ∈J and all p ∈Ij, the contribution from O-terms is ≪x ∑ x1/U′<p≤x/2 1 p(log(x/p)) 1 2 (1−ε) ≪ x (log x) 1 2 (1−ε) , which is o(x/q). Proof By the orthogonality relations for additive characters, (Perhaps the simplest way to estimate this last sum on p is to consider, for each j, the contribution from p with x/p ∈(e j, e j+1].) On the other hand, the https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press 640 P. Pollack and A. Singha Roy calculations from the proof of Proposition 2.2 (with k = 1, q = 1) already show that calculations from the proof of Proposition 2.2 (with k = 1, q = 1) already show that ∑ j∈J ∑ p∈Ij Ψ(x/p, p) = log(1 + D−1) log b (ρ(u) −ρ(U′) + o(1))x. Collecting estimates, we deduce that (3.5) is 1 q log(1+D−1) log b (ρ(u) −ρ(U′)) x + o(x/q), as desired. ∎ Collecting estimates, we deduce that (3.5) is 1 q log(1+D−1) log b (ρ(u) −ρ(U′)) x + o(x/q), as desired. ∎ Proposition 3.4 implies the following variant of Theorem 1.2, with the leading digits of P(n) prescribed (instead of those of A(n)). Proposition 3.5 Fix positive integers k, D, and b with b ≥2. Fix a real number U ≥1, and fix ε > 0. The number of n ≤x for which A(n) ≡a (mod q), P(n) begins with the digits of D in base b, and P(n) ≤y is ∼1 q log(1 + D−1) log b Ψ(x, y), where x, y →∞with y ≥x1/U, and where a mod q is any residue class with q ≤ (log x) 1 2 −ε. Proof The proof parallels that of Theorem 1.1. It suffices to show that the count of n in question is eventually larger than 1 q log(1+D−1) log b (ρ(u) −η) x and eventually smaller than 1 q log(1+D−1) log b (ρ(u) + η) x. The lower bound follows from Proposition 3.4, fixing U′ large enough that ρ(U′) < η. For the upper bound, we fix U′ large enough that ρ(U′) < η log(1+D−1) log b ; the upper bound inequality then follows from Lemma 3.3 and Proposition 3.4. ∎ Proof The proof parallels that of Theorem 1.1. It suffices to show that the count of n in question is eventually larger than 1 q log(1+D−1) log b (ρ(u) −η) x and eventually smaller To finish the proof of Theorem 1.2, we show that P(n) and A(n) usually have the same leading digits. We begin by observing that P(n) and A(n) are usually close. Lemma 3.6 Fix δ > 0. Proof By the orthogonality relations for additive characters, For large x, the number of n ≤x for which A(n) > (1 + δ)P(n) is O(x(loglog x)2/log x). Proof Put y ∶= x1/2 log log x. We may suppose that P(n) > y, since by standard results on the distribution of smooth numbers (e.g., Theorem 5.1 on page 512 of [26]) this condition excludes only O(x/log x) integers n ≤x. If A(n) > (1 + δ)P(n) for one of these remaining n, then δP(n) < ∑k>1 Pk(n) ≤Ω(n)P2(n) ≤2P2(n)log x. Hence, n is divisible by pp′ for primes p, p′ with p > y and p′ ∈( δ 2 p/log x, p]. The number of such n ≤x is x ∑ y<p≤x ∑ δ 2 p log x <p′≤p 1 pp′ ≪x ∑ y<p≤x 1 p loglog x log p ≪x loglog x log y ≪x (loglog x)2 log x . Here, the sum on p′ has been estimated using Mertens’ theorem with the usual 1/log error term [26, Theorem 1.10, p. 18]. ∎ 1 Published online by Cambridge University Press .4153/S0008439522000601 Published online by Cambridge University Press 641 Benford behavior of large prime factors Lemma 3.7 Fix positive integers N and b, with b ≥2, and fix a real number ε > 0. Among all n ≤x with A(n) ≡a (mod q), the number of n for which the N leading base b digits of P(n) do not coincide with those of A(n) is o(x/q), as x →∞, uniformly in residue classes a mod q with q ≤(log x) 1 2 −ε. Lemma 3.7 Fix positive integers N and b, with b ≥2, and fix a real number ε > 0. Among all n ≤x with A(n) ≡a (mod q), the number of n for which the N leading base b digits of P(n) do not coincide with those of A(n) is o(x/q), as x →∞, uniformly in residue classes a mod q with q ≤(log x) 1 2 −ε. Theorem 1.2 follows from combining Proposition 3.5 with Lemma 3.7. Remark The range of uniformity in q can be widened under the assumption that q is supported on sufficiently large primes. More precisely, for any fixed Q ≥2, the result of Theorem 1.2 holds uniformly for q ≤(log x)1−1/Q−ε, provided the least prime P−(q) dividing q is at least Q + 1. The key observation is that, in the notation of Lemma 3.3, such q have ϕ(q′) ≥P−(q) −1 ≥Q, which shows that π(I) ϕ(q′)u(ϕ(q′) −Re(μ(q′)u−it)) ≥(1 −1 Q ) π(I) u in the display (3.4). The remainder of the proof requires only minor modifications. Acknowledgment We thank the referees for their careful reading of the manuscript. Acknowledgment We thank the referees for their careful reading of the manuscript. Acknowledgment We thank the referees for their careful reading of the manuscript. References [1] K. Alladi and P. Erd˝os, On an additive arithmetic function. Pacific J. Math. 71(1977), no. 2, 275–294.fi Proof By the orthogonality relations for additive characters, Proof Since b and N are fixed, it is enough to prove the estimate of the lemma under the assumption that the N leading digits in the base b expansion of P(n) are fixed, say as the digits of the positive integer D.ii i For M a (fixed) positive integer to be specified momentarily, we let D′ be the integer obtained by tacking M copies of the digit “b −1” on to the end of the b-ary expansion of D. Thus, D′ = bMD + (bM −1). Suppose P(n) begins with D in base b, but A(n) does not. We take two cases. First, it may be that P(n) begins with D but not D′; in that case, for A(n) to not begin with D, we must have A(n)/P(n) > 1 + 1/D′. By Lemma 3.6, the number of such n ≤x is O(x(loglog x)2/log x), which is o(x/q). On the other hand, if P(n) begins with D′, we apply Proposition 3.5. Taking y = x there, we see that the number of n ≤x for which P(n) begins with D′ and A(n) ≡a (mod q) is ∼log(1+1/D′) log b x q . Since the coefficient log(1+1/D′) log b of x q in this estimate can be made as small as we like by fixing M large enough, we obtain the lemma. ∎ [1] K. Alladi and P. Erd˝os, On an additive arithmetic function. Pacific J. Math. 71(1977), no. 2, 275–294. [2] T. C. Anderson, L. Rolen, and R. Stoehr, Benford’s law for coefficients of modular forms and partition functions. Proc. Amer. Math. Soc. 139(2011), 1533–1541. [3] S. Aursukaree and V. Chandee, Equidistribution of log (d(n)). In: Proceedings of annual pure and applied mathematics conference, Chulalongkorn University, Thailand, 2016, pp. 399–410. [4] W. D. Banks, G. Harman, and I. E. Shparlinski, Distributional properties of the largest prime factor. Michigan Math. J. 53(2005), 665–681. [5] A. Berger and T. P. Hill, An introduction to Benford’s law, Princeton University Press, Princeton, NJ, 2015. f [3] S. Aursukaree and V. Chandee, Equidistribution of log (d(n)). In: Proceedings of annual pure and applied mathematics conference, Chulalongkorn University, Thailand, 2016, pp. 399–410. [4] W. D. Banks, G. Harman, and I. E. Shparlinski, Distributional properties of the largest prime factor. Michigan Math. J. 53(2005), 665–681. f g J ( ), [5] A. Berger and T. P. Hill, An introduction to Benford’s law, Princeton University Press, Princeton, NJ, 2015. References [1] K. Alladi and P. Erd˝os, On an additive arithmetic function. Pacific J. Math. 71(1977), no. 2, 275–294. [2] T. C. Anderson, L. Rolen, and R. Stoehr, Benford’s law for coefficients of modular forms and partition functions. Proc. Amer. Math. Soc. 139(2011), 1533–1541. [3] S. Aursukaree and V. Chandee, Equidistribution of log (d(n)). In: Proceedings of annual pure and applied mathematics conference, Chulalongkorn University, Thailand, 2016, pp. 399–410. [4] W. D. Banks, G. Harman, and I. E. Shparlinski, Distributional properties of the largest prime factor. Michigan Math. J. 53(2005), 665–681. [5] A. Berger and T. P. Hill, An introduction to Benford’s law, Princeton University Press, Princeton, NJ, 2015. p f [3] S. Aursukaree and V. Chandee, Equidistribution of log (d(n)). In: Proceedings of annual pure and applied mathematics conference, Chulalongkorn University, Thailand, 2016, pp. 399–410. [4] W. D. Banks, G. Harman, and I. E. Shparlinski, Distributional properties of the largest prime factor. Michigan Math. J. 53(2005), 665–681. f g J ( ), [5] A. Berger and T. P. Hill, An introduction to Benford’s law, Princeton University Press, Princeton, NJ, 2015. https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press 642 P. Pollack and A. Singha Roy P. Pollack and A. Singha Roy [6] [6] A. Best, P. Dynes, X. Edelsbrunner, B. McDonald, S. J. Miller, K. Tor, C. Turnage-Butterbaugh, and M. Weinstein, Benford behavior of Zeckendorf decompositions. Fibonacci Quart. 52(2014), no. 5, 35–46. [7] [7] A. Best, P. Dynes, X. Edelsbrunner, B. McDonald, S. J. Miller, K. Tor, C. Turnage-Butterbaugh, and M. Weinstein, Benford behavior of generalized Zeckendorf decompositions. In: Combinatorial and additive number theory. II, Springer Proceedings in Mathematics & Statistics, 220, Springer, Cham, 2017, pp. 25–37. [8] pp [8] V. Chandee, X. Li, P. Pollack, and A. Singha Roy, Benford’s law for multiplicative functions. Preprint, 2022. arXiv:2203.13117 p [9] E. Chen, P. S. Park, and A. A. Swaminathan, On logarithmically Benford sequences. Proc. Amer. Math. Soc. 144(2016), 4599–4608.h [10] N. G. de Bruijn, The asymptotic behaviour of a function occurring in the theory of primes. J. Indian Math. Soc. (N.S.) 15(1951), 25–32.h [11] P. Diaconis, The distribution of leading digits and uniform distribution mod 1, Ann. Probab. 5(1977), 72–81. [12] D. Goldfeld, On an additive prime divisor function of Alladi and Erd˝os. In: Analytic number theory, modular forms and q-hypergeometric series, Springer Proceedings in Mathematics & Statistics, 221, Springer, Cham, 2017, pp. 297–309. p g pp [13] A. e-mail: pollack@uga.edu akash01s.roy@gmail.com References Granville and K. Soundararajan, Pretentious multiplicative functions and an inequality for the zeta-function. In: Anatomy of integers, CRM Proceedings & Lecture Notes, 46, American Mathematical Society, Providence, RI, 2008, pp. 191–197. y pp [14] G. Halász, Über die Mittelwerte multiplikativer zahlentheoretischer Funktionen. Acta Math. Acad. Sci. Hungar. 19(1968), 365–403. g [15] R. R. Hall, On the probability that n and f (n) are relatively prime. III. Acta Arith. 20(1972), 267–289.f [16] R. R. Hall and G. Tenenbaum, Effective mean value estimates for complex multiplicative functions. Math. Proc. Cambridge Philos. Soc. 110(1991), 337–351. g [17] G. H. Hardy and E. M. Wright, An introduction to the theory of numbers, 6th ed., Oxford University Press, Oxford, 2008.hfi y [18] M. Jameson, J. Thorner, and L. Ye, Benford’s law for coefficients of newforms. Int. J. Number Theory 12(2016), 483–494.h h y [19] D. E. Knuth and L. Trabb Pardo, Analysis of a simple factorization algorithm. Theor. Comput. Sci. 3(1976/77), 321–348. ( ) [20] A. V. Kontorovich and S. J. Miller, Benford’s law, values of L-functions and the 3x + 1 problem. Acta Arith. 120(2005), 269–297.i [21] B. Massé and D. Schneider, Fast growing sequences of numbers and the first digit phenomenon. Int. J. Number Theory. 11(2015), 705–719. h y [22] S. J. Miller (ed.). Benford’s law: theory and applications, Princeton University Press, Princeton, NJ, 2015. [23] H. L. Montgomery and R. C. Vaughan, Multiplicative number theory. I. Classical theory, Cambridge Studies in Advanced Mathematics, 97, Cambridge University Press, Cambridge, 200 [23] H. L. Montgomery and R. C. Vaughan, Multiplicative number theory. I. Classical theory, Cambridge Studies in Advanced Mathematics, 97, Cambridge University Press, Cambridge, 2007. [24] P. Pollack and A. Singha Roy, Dirichlet, Sierpi´nski, and Benford. J. Number Theory 239(2022), 352–364.h É [24] P. Pollack and A. Singha Roy, Dirichlet, Sierpi´nski, and Benford. J. Number Theory 239(2022), 352–364.h [25] G. Tenenbaum, Théorie analytique et probabiliste des nombres: 307 exercices corrigés, Échelles collection, Belin, 2014, prepared with the collaboration of Jie Wu. [26] G. Tenenbaum, Introduction to analytic and probabilistic number theory, 3rd ed., Graduate Studies in Mathematics, 163, American Mathematical Society, Providence, RI, 2015. Department of Mathematics, Boyd Research and Education Center, University of Georgia, Athens, GA 30602, USA https://doi.org/10.4153/S0008439522000601 Published online by Cambridge University Press
https://openalex.org/W2906064573	https://europepmc.org/articles/pmc6331028?pdf=render	English	null	Targeting melanoma residual disease by USP7	Oncotarget	2,018	cc-by	1,153	Monika Vishnoi and Dario Marchetti proteasome targets (e.g., tumor suppressors), along with abilities for disrupting protein-protein interactions in ligases overseeing their degradation [7, 8]. We evaluated USP7/HAUSP, a deubiquitinase which we found associated with worst overall survival of melanoma patients. USP7 is involved in multiple functions of cell maintenance by regulating post-translational modifications of key tumor suppressors such as p53 and PTEN [9, 10]. Pharmacological inhibition of USP7 in melanoma CDXs eliminated minimal residual disease at metastatic sites and arrested BMRTCs at BM locales [6]. USP7 expression correlated with PTEN expression, and inhibition of USP7 restored nuclear localization of PTEN which is essential for normal hematopoietic stem-cell functions. PTEN plays critical roles switching hematopoietic stem cells quiescence to a metastatic state by the regulation of G1 to S cell-cycle transition. USP7 inhibition arrested BMRTCs residence at BM, conceivably mediating G1/S phase of cell-cycle progression. Targeting residual melanoma cells by USP7 inhibition induced p53/p21-mediated senescent phenotype which may lead to activation of apoptotic pathways [6, 10]. We demonstrated that USP7 modulated the senescent phenotype of BMRTCs while reducing melanoma micro-metastasis. This suggests that different microenvironment cues regulate USP7 in melanoma cells and will determine the fate and function of BMRTCs. Melanoma metastasis is an aggressive disease which exhibit a latency period between the onset of primary and metastatic tumors. Overall patient survival is reduced from 98% to 19%, respectively [1]. While pre-existing adjuvant and neo-adjuvant immunotherapies improve clinical outcome, they are inefficient to provide cure in metastatic melanoma settings [2]. Accordingly, there is the urgent need to develop novel therapeutic regimens which can prevent the colonization of tumor cells at distant sites [3]. Non-invasive liquid biopsy procedures represent an effective approach to monitor clinical progression in real- time and to identify candidate biomarkers in early vs late disease stages [4, 5]. They will provide precision oncology therapy to inhibit disease recurrence, ultimately reducing cancer mortality. During metastasis latency period, disseminated circulating tumor cells (CTCs), known “seeds” of metastasis, home to and reside at distant organs, undergoing temporal-spatial selection to acquire metastatic traits which lead to disease recurrence [2, 3]. In the clinically asymptomatic phase, CTCs follow a stochastic route to reside for prolonged periods in bone marrow (BM), a reservoir of CTCs. These BM-resident CTCs (BMRTCs) interact with the surrounding environment to maintain balance between stem cell renewal and metastatic abilities required for their survival and homing [4, 5]. Oncotarget, 2018, Vol. 9, (No. 101), pp: 37464-37465 Oncotarget, 2018, Vol. 9, (No. 101), pp: 37464-37465 www.oncotarget.com Editorial Correspondence to: Dario Marchetti, email dmarchetti@houstonmethodist.org Correspondence to: Dario Marchetti, email dmarchetti@houstonmethodist.org Keywords: USP7; bone marrow resident cells; circulating tu mor cells; melanoma; liquid biopsy Received: December 10, 2018 Published: December 25, 2018 Published: December 25, 2018 Published: December 25, 2018 Monika Vishnoi and Dario Marchetti Targeting BMRTC stem-cell properties to eliminate residual cells or delaying their ability to acquire metastatic traits can therefore be a promising therapeutic strategy to delay or overcome metastasis [3]. i Figure 1: Roles of USP7 regulating CTC properties We have provided first-time evidence recapitulating the biology of clinically undetectable melanoma metastasis by injection of Lin-neg/CTC-enriched population (DAPI-/ CD45-/CD34-/CD73-/CD90-/CD105- cells) sorted from blood of melanoma patients in CTC-derived xenografts (CDXs) [6]. We discovered high heterogeneity in these “biomarker-agnostic” CTCs which also beared distinct transcriptional signatures of BMRTCs vs CTCs isolated in parallel from CDXs. Further, we assessed the prognostic relevance of low-proliferating, however metastasis- competent BMRTCs, and identified protein ubiquitination as the most significantly altered canonical pathway (BMRTCs vs CTCs) [6]. The ubiquitin proteasome system is a complex network of proteins responsible for intracellular protein degradation and turnover. Recent advances have resulted in the development of a new generation of deubiquitinating enzymes and proteasome inhibitors targeting previously “undruggable” ubiquitin Figure 1: Roles of USP7 regulating CTC properties www.oncotarget.com www.oncotarget.com Oncotarget 37464 Dario Marchetti: Biomarker Research Program Center, Houston Methodist Research Institute, Houston, TX, USA USP7 maintains regulatory T-cell (Treg) function by deubiquitination of transcription factors FOXP3, Notch1, etc. [10]. USP7-mediated direct or indirect NF- κB deubiquitination asserts its role in inflammation and immune signaling pathways to regulate various cellular functions such as differentiation, survival and proliferation [10](see also Figure 1). Interactions between USP7 and immune cells might facilitate the stem-cell phenotype of BMRTCs and provide a pre-metastatic niche for effective organ colonization. Further USP7 mechanistic insights will provide effective strategies to target residual cells. For example, USP7 may regulate the extrinsic and intrinsic behavior of BMRTCs, either by acquiring genetic traits (e.g., APC/SMAD acquired mutations) during disease progression or by interacting with the surrounding microenvironment. Cross-talks of tumor cells with osteoblasts, myeloid cells, natural-killer cells, T-cells, etc., may discriminate the fate of disseminated cells to remain into a quiescent state or switch to a fully competent metastatic cell. Transcriptional profiling revealed that USP7 affected multiple BMRTC functions such as migration, invasion, and reseeding in the circulation, possibly altering cell growth and proliferative capacities during the metastatic cascade [6]. Dario Marchetti: Biomarker Research Program Center, Houston Methodist Research Institute, Houston, TX, USA REFERENCES 1. Howlader N, et al. SEER cancer statistics review. 1975- 2014. Bethesda, MD: NCI. 1. Howlader N, et al. SEER cancer statistics review. 1975- 2014. Bethesda, MD: NCI. 2. Luke JJ, et al. Nat Rev Clin Oncol. 2017; 14:463–82. 2. Luke JJ, et al. Nat Rev Clin Oncol. 2017; 14:463–82. 3. Anderson RL, et al. Nat Rev Clin Oncol. 2018 Dec 4. [Epub ahead of print]. https://doi.org/10.1038/s41571-018-0134-8. 4. Müller V, et al. Eur J Cancer. 2010; 46:1189-97. 5. Joose SA, et al. Cancer Metastasis Rev. 2016; 35:41-8. 6. Vishnoi M, et al. Cancer Res. 2018; 78:5349-5362. 7. Turnbull AP, et al. Nature. 2017; 550:481-486. 8. Desroses M, et al. Cell Chemical Biology. 2017; 24:1429- 1431. 9. Zhou J, et al. Med. Chem. 2018; 14:3–18. USP7 is an ideal anticancer target molecule which modulate BMRTCs at several metastatic steps [6]. Therefore, its pro-oncogenic and immune-modulatory roles to regulate survival, metastatic initiation and colonization of residual cell population during cancer asymptomatic phase warrants further investigation (Figure 1). 10. Wu J, et al. J Med. Chem. 2018; 61:422-443. Copyright: Vishnoi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and re production in any medium, provided the original author and source are credited. Concluding, liquid biopsy-based screening is poised to provide new surrogate biomarkers to reduce the risk of metastatic recurrence. Pre-clinical studies to assess the therapeutic relevance of potent, substrate- specific, druggable, small-molecule USP7 inhibitors in large patient cohort need to be considered as a valuable option [7, 8]. Additional investigations on USP7 roles in cancer progression employing more selective/drug- like inhibitors than ones used in our study will have important implications to develop precise therapy. Potential synergistic combinations of standard cancer therapeutic drugs with USP7 inhibitors in clinical settings will ultimately impact cancer patient survival in general, melanoma in particular. www.oncotarget.com www.oncotarget.com Oncotarget 37465
https://openalex.org/W4387066415	https://www.degruyter.com/document/doi/10.1515/nleng-2022-0310/pdf	English	null	Target recognition and detection system based on sensor and nonlinear machine vision fusion	Nonlinear engineering	2,023	cc-by	7,816	Hongbin Jia, Fanwen Yang, Tao Li, and R. Suresh Kumar Target recognition and detection system based on sensor and nonlinear machine vision fusion Hongbin Jia, Fanwen Yang, Tao Li, and R. Suresh Kumar https://doi.org/10.1515/nleng-2022-0310 received March 27, 2023; accepted July 28, 2023 for electrical sensor welds. The object detection and recogni- tion method proposed in this article can be applied to dynamic changes and complex scenes in various complex backgrounds and has a good application prospect. The system proposed in this article has some limitations, such as the algorithm in the calculation accuracy, real-time, and other aspects that have room for improvement. Abstract: In order to realize the automatic detection system of electric sensor, a method based on sensor and nonlinear machine vision is proposed. Aiming at complex scenes and dynamic changes in target recognition and detection in large- scale industrial ﬁeld, a target recognition and detection system based on the fusion of vision sensor and nonlinear machine vision is proposed. The system introduces nonlinear features and uses deep neural network to realize multi-scale analysis and recognition of image data on the basis of tradi- tional machine vision. The system uses C++ language devel- opment and has a good user interface. The photoelectric sensor weld image is collected by machine vision technology, the target area of the image is detected by Gaussian model, the feature points of the target area are extracted by com- bining Hessian matrix, the extracted feature points are input into the quantum gate neural network model, and the recog- nition results are obtained. The simulation results show that the author’s method has the highest value among the three test indicators, with the highest accuracy rate of 97%, the highest recall rate of 98%, and the highest F1 value of 94. The time consumed by the author’s method for automatic identiﬁcation of photoelectric sensor welding is within 6 s, the time spent on the ﬁlm wall recognition method for auto- matic identiﬁcation of photoelectric sensor welding is within 20 s, and the time spent by the feature extraction and identi- ﬁcation method for automatic identiﬁcation of photoelectric sensor weld is within 22 s. It has been proven that the method based on the fusion of sensors and nonlinear machine vision can achieve an automatic recognition and detection system Keywords: sensors, non linearity, machine vision, target recognition This work is licensed under the Creative Commons Attribution 4.0 International License. Nonlinear Engineering 2023; 12: 20220310 Open Access. © 2023 the author(s), published by De Gruyter. This hed by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 2 various ﬁelds because it contains a lot of information. Digital image processing refers to the conversion of image information into digital signals suitable for computer processing [1]. Digital image processing technology was developed in the early 1960s, and its early purpose was to simply improve the eﬀect and quality of images; in digital image processing systems, low- quality images that to be improved are the input and high- quality images or speciﬁc information are the output. Common digital image processing includes image denoising, image edge detection, and image segmentation; for the ﬁrst time, image processing was applied in the Jet Propulsion Laboratory of the United States, and the applied image processing methods included geometric correction, noise removal, and gray scale transformation. Image processing has made great progress in theoretical research. The research of image processing tech- nology includes the following aspects: Image digitization, image preprocessing, and image analysis. is the key to achieving robot welding. The development of robot technology is still in a stage where teaching program- ming is the main focus; oﬄine programming technology is becoming increasingly mature, and research on autonomous programming is just beginning. Therefore, developing oﬄine programming technology suitable for welding is currently the most feasible and eﬀective research direction. The foundation of digital image processing technology is solid mathematical knowledge, and the task of studying digital image processing is to design various processing algorithms and implement them in the form of programs. At present, digital image processing technology has been applied to all ﬁelds of production and life. It mainly includes remote sensing technology, medical image processing tech- nology, character recognition technology, and industrial applications and has made good achievements. According to diﬀerent application ﬁelds, it is mainly divided into image acquisition, image enhancement, image edge detec- tion, image transmission, etc. [2]. Welding is a very important processing technology, which is an essential part of modern mechanical manufac- turing and holds a signiﬁcant proportion. Due to the processing methods used in welding, the processing envir- onment is relatively harsh, and manual welding cannot ensure the consistency of processing under large-scale pro- duction conditions. In addition, the eﬃciency of manual welding is low, and it often requires a huge investment of human resources to ensure the requirements of the con- struction period. Modern labor costs and workers’ require- ments for working conditions are increasing, and manual welding is gradually unable to meet the requirements of modern processing. With the emergence and increasing maturity of robot technology, as well as the successful appli- cation of robots in other manufacturing ﬁelds, it is particu- larly necessary and important to study specialized robot technology suitable for welding processing. In China, the processing and manufacturing industry plays an important role in the national economy; it includes many processes, and welding plays a huge role in the man- ufacturing industry. In essence, welding is a comprehensive process of luminescence and heating, which involves com- plex physical and chemical changes. In the welding process, there is a lot of information that can be used; with the rapid development of computer vision technology and image pro- cessing technology, visual sensing technology is widely used in the automatic seam tracking system. According to statis- tics of eﬀective ﬁgures, in manual and semi-automatic pro- cess control systems, experienced workers can perceive the state by observing the shape and change of the molten pool and arc, and the welding position is controlled by adjusting various parameters in the welding process to obtain high- quality welding eﬀect. The rapid development of image pro- cessing technology has made visual sensing technology widely used in many ﬁelds; in the automatic seam recogni- tion and tracking system, the camera is equivalent to the human eye in the manual welding process. It acquires infor- mation from the collected images, extracts the information we want from the information to control the welding posi- tion, and the whole controller processes it by collecting the input information; then, the control signal that can be used is output to realize the automatic control of welding, and the whole control system is fully automated through closed-loop feedback, which ensures good welding quality and greatly improves the automation level of the production process and the ﬂexibility of welding [3]. With the emergence and development of visual sensing technology, real-time control of welding dynamic position and real-time monitoring of The core part of robot technology is the programming module that implements machining control. Based on this, the development of robot technology can be divided into three stages: teaching and playback stage, oﬄine program- ming stage, and autonomous programming stage. 1 Introduction The application of machine vision in the industrial ﬁeld mainly focuses on automatic detection, automatic assembly, automatic recognition, and classiﬁcation. Traditional machine vision mainly uses traditional image processing methods to extract target information. With the continuous develop- ment of artiﬁcial intelligence technology, more and more researchers pay attention to deep learning methods. Deep learning is a method of training a classiﬁer model using large-scale data sets to automatically identify patterns in images or make classiﬁcations. The combination of tradi- tional machine vision technology and deep neural network can not only better complete the target detection task, but also improve recognition eﬃciency and accuracy. In recent years, with the rapid development of mechanical automa- tion, welding seam technology has been widely applied in ﬁelds such as computer, automotive manufacturing, che- mical industry, and manufacturing. In order to improve the eﬃciency of welding seams and improve the quality of ﬁnished products, Chinese welding workers have been con- ducting research on photoelectric sensor welding seams, making eﬀorts to achieve eﬃcient welding process. The key is to innovate and transform the photoelectric sensor welding seam automatic recognition system. Based on experimental proof and theoretical basis, it can be seen that automatic recognition of welds using photoelectric sensors is of great signiﬁcance on the basis of machine vision. With the rapid development of computer, digital image processing tech- nology has developed rapidly, and image is widely used in * Corresponding author: Hongbin Jia, School of Information Engineering, Xinxiang vocational and Technical College, Xinxiang 456002, China, e-mail: HongbinJia7@163.com Fanwen Yang: School of Information Engineering, Xinxiang vocational and Technical College, Xinxiang 456002, China, e-mail: FanwenYang6@126.com Tao Li: School of Information Engineering, Xinxiang vocational and Technical College, Xinxiang 456002, China, e-mail: TaoLi17@163.com R. Suresh Kumar: Center for System Design, Chennai Institute of Technology, Chennai, India, e-mail: sureshkumarr@citchennai.net  Hongbin Jia et al. welding quality during the welding process have been achieved. Improved the processing quality of welding and solved the problem of poor weld consistency. Some researchers have used the diﬀerences in high- frequency and low-frequency weld paths for automatic identiﬁcation, completing the automatic identiﬁcation of photoelectric sensor welds, but this method has high com- putational complexity. The scattered data model is used to simulate the visual curve relationship between welds, and the whole region is recognized by the dual-laser texture sensing technology; the information extraction of the weld region is completed, and thus the automatic recognition of the photoelectric sensor weld is completed. However, this method takes a long time to identify welds. In order to solve the problems in the aforementioned methods, the author uses machine vision technology to study the auto- matic recognition method of photoelectric sensor weld seam [6]. In order to realize the automatic detection system of electric sensor, a method based on sensor and nonlinear machine vision is proposed. The photoelectric sensor weld image is collected by machine vision technology, the target area of the image is detected by Gaussian model, the fea- ture points of the target area are extracted by combining Hessian matrix, the extracted feature points are input into the quantum gate neural network model, and the recogni- tion results are obtained. 2 Literature review The visual calibration technology of the robot includes two parts: the internal and external parameter calibration of the camera and the hand–eye system parameter calibra- tion. Obviously, the development of visual calibration tech- nology stems from the application of visual sensors in robots. Its core is to establish the relationship between the camera, workpiece, and robot. Whether it is welding visual tracking or welding gun posture control, achieving high- quality visual calibration is an essential step. The appear- ance of weld seam tracking technology is mainly due to the existence of thermal deformation and groove consistency in the welding process, and the realization of real-time tracking of weld seam can eﬀectively avoid the adverse impact of the aforementioned problems on welding quality. The develop- ment of seam tracking technology is mainly reﬂected in the continuous progress of the sensor technology used; it has gone through the development process from contact sensor to non-contact sensor, especially the appearance of visual sensing technology, which has largely solved the problem of complex and changeable welding processing environ- ment, and made people realize that the welding operation under certain limit conditions in modern processing can be realized through the visual tracking system [4]. Target recognition and detection system  3 3 Teaching programming cannot meet the needs of small batch and multivariety ﬂexible processing, and large workpieces with complex structure have great teaching diﬃculties, which increase the time for processing preparation. In addition, teaching programming often requires the operator to com- plete teaching in a position close to the processing station, which increases the operational risk of the programmer under certain limiting conditions, especially welding proces- sing, whose working environment and processing workpiece types are complex and changeable. Therefore, developing a better programming method suitable for welding processing Target recognition and detection system  3.2 Target area detection based on Gaussian model One is the frequency domain processing method, which involves ﬁrst performing Fourier transform on image pixels, and then performing homomorphic ﬁltering, fre- quency domain high (low) pass ﬁltering, and other methods on the transformed complex pixel geometry. Another method is spatial domain processing, which generally uses methods such as neighborhood averaging, median ﬁltering, and histogram correction to directly process image pixels. The high computational complexity of fre- quency domain processing methods results in poor real- time performance, so they are rarely used in machine vision systems with real-time requirements. Due to the advantage of reducing noise while not blurring edges, the median ﬁltering method has been widely used among all existing spatial domain processing methods. Its prin- ciple is to replace the target pixel’s grayscale value with the median of neighboring grayscale values. In order to automatically identify the weld seam of photoelec- tric sensor, it is ﬁrst necessary to detect the weld seam image. The common detection method is the Gaussian model, which is generally composed of two to six single Gaussian models. If the number of models is L, the weight is y, and the distribution is h, the expression of Gaussian model is as follows: In order to automatically identify the weld seam of photoelec- tric sensor, it is ﬁrst necessary to detect the weld seam image. The common detection method is the Gaussian model, which is generally composed of two to six single Gaussian models. If the number of models is L, the weight is y, and the distribution is h, the expression of Gaussian model is as follows: ( ) ∑ ∑ ⎧ ⎨ ⎪ ⎪ ⎩ ⎪ ⎪ = = > − = = + Q y v ζ F y h y U v v ; , 1 , x L L L L L L L L L 2 1 1 1 (2) (2) where Q represents the target data density, V represents the average value of Gaussian model, U represents threshold, ζ represents the variance value of Gaussian model, and x represents the target data. According to the characteristics of Gaussian model, the target area detection of photoelectric sensor weld image is divided into the following steps: 1) Calculate the mean diﬀerence value of adjacent models and compare it with the threshold value. 3.2 Target area detection based on Gaussian model If the mean diﬀerence is less than the threshold value, use the fol- lowing formula (3) to update the model, where λ is the parameter learning rate. ( ) ( ) · ( ) · ⎪ ⎪ ⎧ ⎨ ⎩ = = − + = − + + + + y y y v λ v v λ ζ λ ζ ζ λ , max 1 1 . L L L L 1 1 2 1 2 (3) (3) (3) 2) The environmentforautomaticrecognitionofweldsbyphoto- electricsensorsiscomplex;inordertoimprovetherecognition eﬃciency, the noise recognition factor is introduced and expressed by formula (4), where O represents the noise recog- nition factor, m is a function, and g is a model function. 2) The environmentforautomaticrecognitionofweldsbyphoto- electricsensorsiscomplex;inordertoimprovetherecognition eﬃciency, the noise recognition factor is introduced and expressed by formula (4), where O represents the noise recog- nition factor, m is a function, and g is a model function. When collecting the weld image of the photoelectric sensor to be recognized by the camera charge coupled device (CCD), the light source should be considered to make the lighting eﬀect in the best state so as to obtain a clear image. Therefore, according to the size and working requirements of the photoelectric sensor weld, the author combined the camera CCD and selected the XF-5MDT05X65 lens to collect the photoelectric sensor weld image to be recognized, in order to ensure that the gray value of the photoelectric sensor weld image is uniform [8]. (( ) ) ∏ ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ = = − mg y m y L L , / . L L L L O 1 (4) (4) 3) After the noise recognition factor is added, use the fol- lowing formula (5) to update the algorithm, discard the model with weight less than zero, and repeat the operation to suppress the impact of noise on the recognition results. 3.1 Image acquisition based on machine vision The research on the application of visual sensing tech- nology to weld seam tracking started early abroad; it began in the early 1960s but was limited to the development level of visual sensing and computer technology at that time, especially the slow processing speed of the computer at that time, and so the developed visual tracking system needed to be taught in advance before use, which could not meet the requirements of actual welding. Therefore, satisfactory progress has not been achieved. This situation did not change until the emergence of the second genera- tion of robots in the 1980s. At this point, both the relevant technology of the robot body, as well as the corresponding visual sensing and computer technology, have reached a high level, which shows the possibility of applying visual sensors to welding robots. The research on weld seam tracking technology based on visual sensor in China started relatively late compared with foreign countries; in the 1970s, domestic universities and scientiﬁc research insti- tutes began to research in relevant ﬁelds, and the research focus is mostly on the control strategy of visual tracking [5]. Machine vision is widely used in diﬀerent ﬁelds of complex technologies. In simple applications (e.g., using a backlight to detect the proﬁle of opaque objects), the imaging system can operate reliably with loose system parameters. However, in more demanding applications (such as detecting mirror sur- face defects), the main parameters must be set within a smaller tolerance range or use more expensive high-preci- sion hardware for the imaging system to operate stably. The number of major parameters involved in the ima- ging system increases with the complexity of imaging applications. Edmund Optics Tokyo Imaging Laboratory conducted research on this and proposed up to 20 precise adjustable parameters. Image acquisition is the key link for automatic recog- nition of photoelectric sensor weld seam: only by acquiring a high-deﬁnition photoelectric sensor weld seam image can the target area of the image be detected more eﬀectively and accurately, so as to improve the recognition accuracy of the 4  Hongbin Jia et al. 4 3.2 Target area detection based on Gaussian model photoelectric sensor weld seam. Machine vision technology is an image acquisition technology, which is simple in opera- tion and of high quality. Therefore, the author uses machine vision technology to collect the weld image of photoelectric sensor, laying a foundation for the subsequent automatic recognition of photoelectric sensor weld [7]. The noise during the welding process, as well as the transmission and conversion of images, can aﬀect the quality of the image. Therefore, in general, the image is ﬁrst preprocessed by using certain ﬁltering and image restoration techniques to denoise and repair the obtained image, thereby reducing the impact of various interference signals and enhancing the strength of the required feature signals. The commonly used preprocessing methods can generally be divided into two types. One is the frequency domain processing method, which involves ﬁrst performing Fourier transform on image pixels, and then performing homomorphic ﬁltering, fre- quency domain high (low) pass ﬁltering, and other methods on the transformed complex pixel geometry. Another method is spatial domain processing, which generally uses methods such as neighborhood averaging, median ﬁltering, and histogram correction to directly process image pixels. The high computational complexity of fre- quency domain processing methods results in poor real- time performance, so they are rarely used in machine vision systems with real-time requirements. Due to the advantage of reducing noise while not blurring edges, the median ﬁltering method has been widely used among all existing spatial domain processing methods. Its prin- ciple is to replace the target pixel’s grayscale value with the median of neighboring grayscale values. photoelectric sensor weld seam. Machine vision technology is an image acquisition technology, which is simple in opera- tion and of high quality. Therefore, the author uses machine vision technology to collect the weld image of photoelectric sensor, laying a foundation for the subsequent automatic recognition of photoelectric sensor weld [7]. The noise during the welding process, as well as the transmission and conversion of images, can aﬀect the quality of the image. Therefore, in general, the image is ﬁrst preprocessed by using certain ﬁltering and image restoration techniques to denoise and repair the obtained image, thereby reducing the impact of various interference signals and enhancing the strength of the required feature signals. The commonly used preprocessing methods can generally be divided into two types. 3.2 Target area detection based on Gaussian model The gray pixel value of photoelectric sensor weld visual distribution is set to ( ) d x y , , and the constructed photoelectric sensor weld image acquisition model is as follows: ( ) ( ) ∏ ∏ ∑ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎧ ⎨ ⎪ ⎪⎪ ⎩ ⎪ ⎪ ⎪ ⎛ ⎝ ⎞ ⎠ + ⎛ ⎝ ⎞ ⎠ = ⎡ ⎣⎢ ⎛ ⎝ ⎞ ⎠ + ⎤ ⎦⎥ = = + ∂ − −∂ = − − = − mg y mg y m y L μ y m σ σy y y O y O O , / 0 , L L L O L L L O L L L L L L L L 1 1 1 1 ( ( )) ∑ = = F x E d x y , , i T i 1 (1) (1) (5) where E is the image fractal coeﬃcient and xi represents the pixel value. O 5 Target recognition and detection system  calculate the covariance of all sets of eigenvectors, and set D as the matrix model and U as the matrix dimension. calculate the covariance of all sets of eigenvectors, and set D as the matrix model and U as the matrix dimension. where μ represents the mean value of Gaussian model, σ represents the reference value, and ∂represents the number of samples. 4) With the target area as the reference, select the area with the average variance strength greater than the assumed threshold as the key area, and use the fol- lowing formula (6) to complete the detection of the target area of the weld image, where D is the target image in the key area and Q is the characteristic of the key area. ( ) ( ) ( )· ∑ ⎧ ⎨ ⎪ ⎩ ⎪ = − − = − < < = D Y y v y v y y v Y y v U 1 . L Y L U L U 1 (9) (9) 3.3 Feature extraction The photoelectric sensor weld seam has characteristics such as instability, which requires feature extraction to reduce the data density; the speciﬁc process is as follows: 1) Using the Hessian matrix model J for feature point detection, the Hessian model can be described by the following formula (7), where x and y represent any point in the matrix, ζ represents the matrix scale dis- tance, and M represents the Hessian derivative. 1) Using the Hessian matrix model J for feature point detection, the Hessian model can be described by the following formula (7), where x and y represent any point in the matrix, ζ represents the matrix scale dis- tance, and M represents the Hessian derivative. Start Data preprocessing Input data Computed network error Update matrix model Gradient calculation Optimal iteration Output result End Figure 1: Flow of quantum gate nonlinear neural network model. ( ) ( ) ( ) ( ) ( ) ⎡ ⎣⎢ ⎤ ⎦⎥= M x ζ M x ζ M x ζ M x ζ J x ζ , , , , , . xx xy xy xy (7) (7) 2) In order to ensure the stability of the photoelectric sensor weld feature, it is necessary to determine a fea- ture point that can be reused. First, take the previous feature point as the center of the circle, take ζ as the diameter, divide the feature area, calculate the conver- sion rate of x and y, and then predict the direction of the feature point [9,10]. 2) In order to ensure the stability of the photoelectric sensor weld feature, it is necessary to determine a fea- ture point that can be reused. First, take the previous feature point as the center of the circle, take ζ as the diameter, divide the feature area, calculate the conver- sion rate of x and y, and then predict the direction of the feature point [9,10]. 3) The gradient direction is corrected by the direction of the feature point, and the gradient direction is calculated according to the following formula (8), where ϑ repre- sents the direction of the feature point, Y stands for nor- malized feature vector, V represents vector dimension, and λ represents the ontology vector coeﬃcient. 3.4 Automatic recognition model of quantum gate nonlinear neural network · ∑ ⎧ ⎨ ⎪ ⎩⎪ = = + m yg m Q D D D 1 . g y g (6) · ∑ ⎧ ⎨ ⎪ ⎩⎪ = = + m yg m Q D D D 1 . g y g (6) (6) Quantum is a representative unit of calculation; there can be two quantum states at the same time, so the automatic recognition model of quantum gate nonlinear neural net- work has excellent distinguishing function, strong accu- racy, and obvious eﬀect on automatic recognition. When the quantum gate nonlinear neural network is used to automatically identify the photoelectric sensor weld, the ﬂow can be seen as shown in Figure 1, and the speciﬁc steps are as follows: 6  Hongbin Jia et al. 6  Hongbin Jia et al. ac- ork he xel 10) Table 1: Experimental parameter setting Sensor height 5 mm Voltage 21–22 V Welding current 140–160 A Welding wire φ1.2 Welding speed 35.6 cm/min e pretreated photoelectric sensor weld charac- data into the quantum gate neural network o calculate the quantum state S, where σ is the ity amplitude and i is the point where the pixel d, as shown in formula (10). ( ) \| \| ⎪ ⎪⎧ ⎨ ⎩ = ⎡ ⎣ − ⎤ ⎦ = + > S σ σ σ σ σ S σ σ cos sin sin cos sin 1 cos 0 i i (10) Table 1: Experimental parameter setting Sensor height 5 mm Voltage 21–22 V Welding current 140–160 A Welding wire φ1.2 Welding speed 35.6 cm/min 1) Input the pretreated photoelectric sensor weld charac- teristic data into the quantum gate neural network model to calculate the quantum state S, where σ is the probability amplitude and i is the point where the pixel is located, as shown in formula (10). 1) Input the pretreated photoelectric sensor weld charac- teristic data into the quantum gate neural network model to calculate the quantum state S, where σ is the probability amplitude and i is the point where the pixel is located, as shown in formula (10). ( ) \| \| ⎪ ⎪⎧ ⎨ ⎩ = ⎡ ⎣ − ⎤ ⎦ = + > S σ σ σ σ σ S σ σ cos sin sin cos sin 1 cos 0 . i i (10) ( ) \| \| ⎪ ⎪⎧ ⎨ ⎩ = ⎡ ⎣ − ⎤ ⎦ = + > S σ σ σ σ σ S σ σ cos sin sin cos sin 1 cos 0 . i i (10) (10) The experimental sample used by the author is a 350 mm × 80 mm × 4 mm Q235A steel test plate; use a white chalk to draw the butt weld on the sample [15]. 2) Calculate the network error, delete the quantum that produces the network error, recalculate, and get the back propagation error information [11]. Comparative experiments were conducted on machine vision based optoelectronic sensor welding seam auto- matic recognition methods, ﬁlm wall recognition methods, high and low frequency combination recognition methods, and feature extraction recognition methods using F1 value, ﬂoating-point operation times, and time as indicators. 4.1 F1 value (11) F1 value is an indicator used to measure the performance of the automatic recognition method, which is very repre- sentative in statistics; its calculation formula is as follows: 4) According to the optimal iterative method, output the data with the highest accuracy, and combine the gra- dient reduction algorithm to obtain the calculation results in the quantum neural network model, and then complete the automatic identiﬁcation of the photo- electric sensor weld. The gradient reduction formula is as follows (12): ( ) ( ) ( ) ⎪ ⎪ ⎧ ⎨ ⎩ = + + + = + = − + J A F A J A J TP/ TP TN FP FN TP/ TP FP 2 / , z c 1 c z c z (13) (13) where Jz represents the recall rate, Ac stands for accuracy, TP represents the correctly identiﬁed normal photoelectric sensor weld results, TN represents the correctly identiﬁed abnormal photoelectric sensor weld results, FP represents the incorrectly identiﬁed results, and FN represents the incorrectly identiﬁed abnormal results [16]. ( ) ( ) ( ) ∏ ∏ ∏ ∏ ⎧ ⎨ ⎪ ⎪⎪ ⎩ ⎪ ⎪ ⎪ = + = ⎛ ⎝ ⎜ ⎡ ⎣⎢ + ⎤ ⎦⎥ ⎞ ⎠ ⎟ = + = = = = i σ ϑ A γ σ B ϑ σ sin sin sinarc cos sin . i i i i ρ i n i i ρ i 1 1 1 1 ( ) ( ) ( ) ∏ ∏ ∏ ∏ ⎧ ⎨ ⎪ ⎪⎪ ⎩ ⎪ ⎪ ⎪ = + = ⎛ ⎝ ⎜ ⎡ ⎣⎢ + ⎤ ⎦⎥ ⎞ ⎠ ⎟ = + = = = = i σ ϑ A γ σ B ϑ σ sin sin sinarc cos sin . i i i i ρ i n i i ρ i 1 1 1 1 (12) The judgment of the advantages and disadvantages of the photoelectric sensor weld seam recognition mainly depends on the identiﬁcation accuracy, recall rate, and F1 value; the accuracy, recall rate, and F1 value of the proposed method, membrane wall recognition method, combined high- and low-frequency recognition method, and feature extrac- tion recognition method are calculated, respectively. The recognition rate of diﬀerent methods is shown in Table 2, the recall rate test result is shown in Figure 2, and the F1 value test result is shown in Figure 3 [17]. 6  Hongbin Jia et al. 3) According to the gradient reduction rule, use the fol- lowing formula (11) to update the values of ϑ and y. In formula (11), A represents the hidden range, B represents the probability amplitude of the model output, ϑ stands for hidden matrix, γ represents the model output matrix, and N represents the number of hidden matrices [12]. ( ) ( ) ∏ ∏ ⎧ ⎨ ⎪⎪ ⎩ ⎪ ⎪ ⎡ ⎣⎢ + ⎤ ⎦⎥= ⎡ ⎣⎢ + ⎤ ⎦⎥= = = σ ϑ A σ γ s B arcsin sin arcsin in . i n i i i n i i 1 1 (11) 3.3 Feature extraction ( ) ( ) ( )· · ∑ ⎧ ⎨ ⎪ ⎩ ⎪ − = = \| \| = − = ϑ x ϑ ϑ x Y Y Y y y v Y λ M / / . Y L L M L L L L 1 (8) (8) 4) In order to reduce the operation time, the principal com- ponent analysis method is used to reduce the dimension of the target vector; ﬁrst, use the following formula (9) to 4) In order to reduce the operation time, the principal com- ponent analysis method is used to reduce the dimension of the target vector; ﬁrst, use the following formula (9) to Figure 1: Flow of quantum gate nonlinear neural network model. 6  Hongbin Jia et al. 4.2 Floating-point operands Floating-point operands are used to measure the com- plexity of the weld recognition method, F is used to repre- sent. The smaller the F value is, the smaller the recognition complexity is. The larger the F value, the greater the recog- nition complexity. F can be calculated by the following formula (14): ( )· ( )· ⎪ ⎪ ⎧ ⎨ ⎩ = + = − ≥ F LX DL D F J P F J 2 1 2 1 . (14) (14) stable, without too much ﬂuctuation, whereas the recogni- tion rates of the membrane wall method and the feature extraction recognition method ﬂuctuate greatly. With the increase in the number of experiments, the experimental results show that the proposed method has a high recogni- tion rate for the automatic recognition of the photoelectric sensor weld [18]. stable, without too much ﬂuctuation, whereas the recogni- tion rates of the membrane wall method and the feature extraction recognition method ﬂuctuate greatly. With the increase in the number of experiments, the experimental results show that the proposed method has a high recogni- tion rate for the automatic recognition of the photoelectric sensor weld [18]. In formula (14), I represents the height of the input data matrix, X represents the width of the input data matrix, D represents the channel coeﬃcient of the data matrix, L represents matrix size, J represents the number of dimensions of the input model, and P represents the number of dimensions of the output matrix. The test results of ﬂoating-point operands of the pro- posed method, combined high-low frequency recognition method, and feature extraction recognition method are shown in Figure 4. According to Table 2, Figures 2 and 3, the author’s method has the highest value among the three test indica- tors, with the highest accuracy of 97%, the highest recall of 98%, and the highest F1 value of 94. The test index values of the author’s method are higher than those of the three methods. The reason is that the author uses Hessian matrix to extract the feature points of the photoelectric sensor weld target area, and then inputs the extracted feature The analysis of Figure 4 shows that the F values of the proposed method, combined high- and low-frequency recog- nition, and feature extraction recognition methods are dif- ferent. 7 Table 2: Recognition rate of diﬀerent methods Number of experiments/ time Recognition rate/% Proposed method Membrane wall identiﬁcation method Feature extraction and recognition method 10 97 30 70 20 73 70 57 30 84 54 62 40 86 68 57 50 90 54 71 60 89 24 63 70 79 39 25 80 93 42 46 90 87 39 70 10 85 51 61 Table 2: Recognition rate of diﬀerent methods points into the quantum gate neural network model to accurately obtain the automatic recognition results of the photoelectric sensor weld. 4 Experiment and analysis In order to verify the eﬀectiveness of the photoelectric sensor weld seam automatic recognition method based on machine vision, the simulation test is carried out by MATLAB simulation software. The parameters of the afore- mentioned experimental environment are set as shown in Table 1 [13,14]. According to the data in Table 2, with the increase of the number of experiments, the recognition rate of the pro- posed method is higher than that of the membrane wall method and the feature extraction recognition method; the recognition rate of the proposed method is relatively Target recognition and detection system  7 4.2 Floating-point operands No matter which data set, the F value of the proposed 10 20 30 40 50 60 70 80 90 20 40 60 80 100 Recall rate Number of tests Proposed method Membrane wall recognition Combined high and low frequency identification Figure 2: Recall rate test results. 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 100 F1 Number of tests Proposed method Membrane wall recognition Combined high and low frequency identification Figure 3: F1 value test results. 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 100 F1 Number of tests Proposed method Membrane wall recognition Combined high and low frequency identification 10 20 30 40 50 60 70 80 90 20 40 60 80 100 Recall rate Number of tests Proposed method Membrane wall recognition Combined high and low frequency identification Figure 2: Recall rate test results. Figure 3: F1 value test results. Figure 2: Recall rate test results. Figure 3: F1 value test results. 8  Hongbin Jia et al. 8 10 20 30 40 50 60 70 80 90 20 30 40 50 60 70 80 F Number of tests Proposed method Combined high and low frequency identification Feature extraction and recognition Figure 4: Floating-point operation values of diﬀerent methods. and analyze the automatic recognition time of photoelec- tric sensor welds; the comparison results are shown in Figure 5 [19,20]. According to Figure 5, the time consumed by the author’s method for automatic identiﬁcation of photoelec- tric sensor welds is within 6 s, and the time consumed by ﬁlm wall identiﬁcation method for automatic identiﬁcation of photoelectric sensor welds is within 20 s, and the time consumed by feature extraction and recognition method for automatic recognition of photoelectric sensor weld seam is within 22 s; the time consumed by the author’s method for automatic recognition of photoelectric sensor weld seam is the shortest and the recognition eﬃciency is the highest [21,22]. In traditional machine vision, vision sensors generally only collect and analyze two-dimen- sional images, cannot conduct multi-scale analysis of target objects, and are aﬀected by environmental factors such as lighting, which limits their application. 4.2 Floating-point operands Aiming at the short- comings of traditional machine vision, this article intro- duces nonlinear features and deep neural networks on the basis of traditional machine vision to realize multi- scale analysis and recognition of target objects. In order to extract the feature information of the target object eﬀec- tively, the original image is ﬁltered by Gaussian ﬁlter, and the edge information of the image is obtained by edge detec- tion. Then the histogram statistical method is used to ana- lyze the edge information. On this basis, nonlinear features are used for multi-scale analysis and identiﬁcation. Figure 4: Floating-point operation values of diﬀerent methods. method is higher than that of the joint high- and low-frequency recognition and feature extraction recognition methods. More- over, with the change of the quantity set, the F value of the proposed method does not change much, while the F value of the combined high- and low-frequency recognition and feature extraction recognition methods vary greatly, indicating that the recognition complexity of the proposed method is low. 4.3 Time side test The proposed method, membrane wall recognition method, and feature extraction recognition method are used to compare 9 Target recognition and detection system  9 wall recognition method for automatic identiﬁcation of photoelectric sensor welding is within 20 s, the time spent by the feature extraction and identiﬁcation method for auto- matic identiﬁcation of photoelectric sensor weld is within 22 s.This article mainly focuses on welding seam visual recog- nition, welding pose control, and visual system calibration and develops corresponding functional modules through a mixed programming method of VC and MATLAB. However, various obstacles that may exist during the welding process are not considered. In the future, obstacle avoidance tech- nology based on machine vision can be studied, and the development of visual obstacle avoidance function can be completed in the same way as this article. In this article, a target recognition and detection system based on the fusion of vision sensor and deep neural network is designed. The system mainly consists of two parts: target detection based on vision sensor and target recognition based on deep neural network. The object detection and recognition method pro- posed in this article can be applied to dynamic changes and complex scenes in various complex backgrounds and has a good application prospect. The system proposed in this article has some limitations, such as the algorithm in the calculation accuracy, real-time, and other aspects that have room for improvement. In addition, in the process of target recogni- tion, due to the problems existing in the algorithm itself, the accurate recognition of the target needs further research. [2] Fan XA, Gao X, Liu G, Ma N, Zhang Y. Research and prospect of welding monitoring technology based on machine vision. Int J Adv Manuf Technol. 2021;115:3365–91. [3] Chen S, Teng X, Sang X, Zhang H, Zhuang J. Automatic recognition of welding seam defects in tofd images based on tensorﬂow. Autom Control Comput Sci. 2022;56(1):58–66. [4] Wang Z, Li L, Chen H, Wu X, Dong Y, Tian J, et al. Penetration recognition based on machine learning in arc welding: a review. Int J Adv Manuf Technol. 2023;125:3899–923. [5] Tyystjärvi T, Virkkunen I, Fridolf P, Rosell A, Barsoum Z. Automated defect detection in digital radiography of aerospace welds using deep learning. Weld World. 2022;66(4):643–71. [6] Kumar S, Gaur V, Wu C. Machine learning for intelligent welding and manufacturing systems: research progress and perspective review. Int J Adv Manuf Technol. 2022;123:3737–65. [7] Zhou B, Pychynski T, Reischl M, Kharlamov E, Mikut R. Machine learning with domain knowledge for predictive quality monitoring in resistance spot welding. J Intell Manuf. 2022;33(4):1139–63. [8] Mishra A, Pathak T. Estimation of grain size distribution of friction stir welded joint by using machine learning approach. ADCAIJ: Adv Distrib Comput Artif Intell J. 2021;10(1):99–110. [9] Han SC, Park HM, Uhm SH, Choi DY, Jeong HC, Kim YJ, et al. Evaluation of liquid metal embrittlement crack in resistance spot welds under intensive welding condition using industrial X-ray computed tomography and machine learning. Weld World. 2021;65:1887–97. [10] Chen G, Sheng B, Luo R, Jia P. A parallel strategy for predicting the quality of welded joints in automotive bodies based on machine learning. J Manuf Syst. 2022;62:636–49. [11] Xiao M, Yang B, Wang S, Chang Y, Li S, Yi G. Research on recog- nition methods of spot-welding surface appearances based on transfer learning and a lightweight high-precision convolutional neural network. J Intell Manuf. 2023;34:2153–70. Funding information: This study did not receive any funding in any form. [12] Miao R, Jiang Z, Zhou Q, Wu Y, Gao Y, Zhang J, et al. Online inspection of narrow overlap weld quality using two-stage convo- lution neural network image recognition. Mach Vis Appl. 2021;32:27. Author contributions: Each author made signiﬁcant indi- vidual contributions to this manuscript. Hongbin Jia: writing and performing surgeries; Fanwen Yang: data analysis and performing surgeries; Tao Li and Dr. R. Suresh Kumar: article review and intellectual concept of the article. [13] Yemelyanova MG, Smailova SS, Baklanova OE. Detection of surface defects in welded joints during visual inspections using machine vision methods. Comput Opt. 2023;47(1):112–7. [14] Asif K, Zhang L, Derrible S, Indacochea JE, Ozevin D, Ziebart B. Machine learning model to predict welding quality using air- coupled acoustic emission and weld inputs. J Intell Manuf. 2022;33:881–95. Conﬂict of interest: The authors declare that they have no competing interests. [15] Tran TA, Lobov A, Kaasa TH, Bjelland M, Midling OT. CAD integrated automatic recognition of weld paths. Int J Adv Manuf Technol. 2021;115(7-8):2145–59. Data availability statement: The data used to support the ﬁndings of this study are available from the corresponding author upon request. [16] Zhang Z, Liu W, Sun X. Image recognition of limited and imbal- anced samples based on transfer learning methods for defects in welds. Proc Inst Mech Eng B: J Eng Manuf. 2022;236(12):1643–52. [17] Bologna F, Tannous M, Romano D, Stefanini C. Automatic welding imperfections detection in a smart factory via 2-D laser scanner. J Manuf Process. 2022;73:948–60. 5 Conclusion Through side test, it is found that the current photoelectric sensor weld automatic recognition technology has problems of low recognition rate and recall rate, and high recognition complexity and long recognition time; for this reason, an automatic recognition method of photoelectric sensor weld seam based on machine vision is proposed. This method ﬁrst collects the photoelectric sensor weld seam image through machine vision technology; then the Gaussian model is used to extract the target area, and the processed image is input into the quantum gate network recognition model to complete the recognition, which reduces the recog- nition complexity and improves the recognition rate, F1 value, and recognition eﬃciency. The author’s method has the highest value among the three test indicators, with the highest accuracy rate of 97%, the highest recall rate of 98%, and the highest F1 value of 94. The time consumed by the author’s method for automatic identiﬁcation of photoelec- tric sensor welding is within 6 s, the time spent on the ﬁlm 10 20 30 40 50 60 70 80 90 4 6 8 10 12 14 16 18 20 22 Time Number of tests Proposed method Membrane wall recognition Feature extraction and recognition 10 20 30 40 50 60 70 80 90 4 6 8 10 12 14 16 18 20 22 Time Number of tests Proposed method Membrane wall recognition Feature extraction and recognition Figure 5: Weld seam identiﬁcation time of photoelectric sensor in three methods. Time Figure 5: Weld seam identiﬁcation time of photoelectric sensor in three methods. 10  Hongbin Jia et al. 10 References [18] Yu R, Kershaw J, Wang P, Zhang Y. Real-time recognition of arc weld pool using image segmentation network. J Manuf Process. 2021;72:159–67. [18] Yu R, Kershaw J, Wang P, Zhang Y. Real-time recognition of arc weld pool using image segmentation network. J Manuf Process. 2021;72:159–67. 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W4389420261.txt	https://jurnal.pcr.ac.id/index.php/jakb/issue/view/79/Dewan%20Redaksi	null		null	Jurnal akuntansi keuangan dan bisnis	2,020	cc-by	349	Jurnal Akuntansi Keuangan dan Bisnis Vol.13 No. 1, Mei 2020 Dewan Redaksi Pelaksana : Unit Penelitian dan Pengabdian Masyarakat Politeknik Caltex Riau Pembina : Direktur Politeknik Caltex Riau Dewan Penyunting Ketua : Meliza Putriyanti Zifi, S.E., M.Acc Anggota : Atika Zarefar,S.E., M.Ak., Ak Adhitya Agri Putra, S.E., M.Acc., Ak. Hamdani Arifulsyah, S.E., M.Ak Vidiyanna Rizal Putri, S.E., M.Si Reviewer Arumega Zarefar,S.E., M.Ak., CA. Universitas Riau Prof. Dr. Rita Anugerah, MAFIS., Ak., CA. Universitas Riau Desi Handayani, S.E., M.Ak., CA. Politeknik Negeri Padang Heri Ribut Yuliantoro, S.E., M.Ak., CA. Politeknik Caltex Riau Yefni, S.E., M.Acc Politeknik Caltex Riau Associate Professor Dr. Razana Juhaida Johari C.A (M) Universiti Teknologi MARA Shah Alam Sekretariat UPPM Politeknik Caltex Riau Jl. Umbansari No 1 Rumbai Pekanbaru 28265 Telp (0761)53939, Fax (0761)554224 Email: jurnalakb@pcr.ac.id ii Jurnal Akuntansi Keuangan dan Bisnis Vol.13 No. 1, Mei 2020 Daftar Isi Halaman Judul Dewan Redaksi Daftar Isi i ii iii-iv Pengaruh Perencanaan Anggaran, Pelaksanaan Anggaran, Pencatatan Administrasi, dan Kompetensi Sumber Daya Manusia Terhadap Tingkat Penyerapan Anggaran (Studi Empiris pada OPD Kota Dumai) Sasmita Atika Sari Harahap, Taufeni Taufik, dan Nurazlina Pengaruh Good Corporate Governance Terhadap Profitabilitas Perusahaan Dengan Corporate Social Responsibility Sebagai Variabel Moderasi Pada Perusahaan Peserta Corporate Governance Perception Index Pada Tahun 2013-2017 Atika Rahmi Pratiwi dan Asniati Bahari Pengaruh Pendidikan Pemilik, Pengetahuan Akuntansi Pemilik, Budaya Perusahaan, Umur Usaha, Skala Usaha, dan Pelatihan Akuntansi Terhadap Penggunaan Informasi Akuntansi Pada UMKM Dengan Ketidakpastian Lingkungan Sebagai Variabel Moderasi (Studi Empiris Pada UMKM di Kabupaten Kuantan Singingi) Nurul Aini Yolanda, Raja Adri Satriawan Surya, dan Arumega Zarefar 1-10 11-20 21-30 Pengaruh Intelectual Capital Terhadap Kinerja Keuangan Perusahaan Perkebunan Hamdani Arifulsyah dan Suci Nurulita 31-40 Analisis Penerapan SAK ETAP pada Penyajian Laporan Keuangan PT. XYZ Susanto, Atika zarefar, dan Fifitri Ali 41-50 Analisis Pengaruh Financial Performance Terhadap Unemployment Rate Dengan Economic Growth Sebagai Variabel Intervening Pada Pemda Provinsi di Indonesia Suci Nurulita, Yefni, dan Hamdani Arifulsyah 51-60 Penggunaan Model Springate Untuk Mendeteksi Penurunan Kinerja Keuangan (Financial Distress) Sektor Pertambangan di Bursa Efek Indonesia Rangga Putra Ananto 61-70 Analisis Predeksi Kebangkrutan Dengan Menggunakan Metode Altman Z-Score Yeni Melia dan Rini Deswita iii 71-80
https://openalex.org/W4387043207	https://revistas.unal.edu.co/index.php/esrj/article/download/103801/89415	English	null	Vertical accuracy evaluation free access digital elevation models (DEMs): case Fergana Valley in Uzbekistan	Earth sciences research journal/Earth Sciences Research Journal	2,023	cc-by	5,914	GEOPHYSICS GEOPHYSICS GEOPHYSICS EARTH SCIENCES RESEARCH JOURNAL Earth Sci. Res. J. Vol. 27, No. 2 (June, 2023): 85 - 91 uation free access digital elevation models (DEMs): case Fergana Valley in Uzbekistan Dilbarkhon Fazilova1,2,3, Obidjon Arabov1 1National University of Uzbekistan named after Mirzo Ulugbek, Department of geodesy and geoinformatics, 1000174, 4, University str., Tashkent, Republic of Uzbekistan 2Astronomical Institute of Uzbek Academy of Sciences, Space researches laboratory, 100052, 33, Astronomical str., Tashkent, Republic of Uzbekistan 3Tashkent State Technical University named after Islam Karimov, Department of Mine Surveying and Geodesy, Republic of Uzbekistan Corresponding Author: dilbar@astrin.uz Keywords: open-source DEMs; vertical accuracy; SRTM; ASTER GDEM2; ALOS AWD3D30; GPS; geoid undulations; ABSTRACT In this study, the vertical accuracy of the Shuttle Radar Topography Mission Digital Elevation Model Version 2.0 (SRTM30), the Advanced Spaceborne Thermal Emission and Reflection Radiometer Global DEM Version 2.0 (ASTER GDEM2), and Advanced Land Observing Satellite World 3D Digital Surface Model Version 2.1 (ALOS AW3D30) was statistically assessed using GPS data. The Fergana Valley area was chosen as a study region, where the land surface can reflect tectonic processes. The values of ellipsoidal heights of 27 points of the regional GPS network were chosen as reference data. The geometric approach using GPS/leveling data and EGM96 global geopotential model-based geoid undulations was applied for geoid surface fitting. The geoid height corrections range ranged from –0.66 m to 0.87 m. Root-Mean-Square errors of ~10.0 m, ~16.4 m, and ~6.6 m was obtained for SRTM30, ASTER GDEM2, and ALOS AW3D30, respectively. It was found that compared with the reference model, all the global DEMs in mountainous areas generally overestimated elevation and the value of vertical ac- curacy at a 90% confidence level by 3-6 meters exceeded the declared by distributors. But ALOS AW3D30 proved to be the most accurate DEM that best represents the topography of the earth’s surface and could be used for some engineering applications in Fergana Valley. Evaluación de precisión vertical de los Modelos Digitales de Elevación de acceso abierto: caso del Valle de Fergana, en Uzbekistán RESUMEN Keywords: open-source DEMs; vertical accuracy; SRTM; ASTER GDEM2; ALOS AWD3D30; GPS; geoid undulations; How to cite this item: Fazilova, D., & Arabov, O. (2023). Vertical accuracy evaluation free access digital elevation models (DEMs): case Fergana Valley in Uzbekistan. Earth Sciences Research Journal, 27(2), 85-91. https://doi. org/10.15446/esrj.v27n2.103801 RESUMEN Palabras Clave: Modelos Digitales de Elevación de acceso abierto; precisión vertical; SRTM; ASTER GDEM2; ALOS AWD3D30; GPS; ondulaciones geoidales; Palabras Clave: Modelos Digitales de Elevación de acceso abierto; precisión vertical; SRTM; ASTER GDEM2; ALOS AWD3D30; GPS; ondulaciones geoidales; En este studio se evaluó estadísticamente la precisión vertical de los modelos digitales de elevación de la Misión Topográfica Shuttle Radar 2.0 (SRTM30), del sistema radiométrico de emisión y reflexión térmica espacial Terra Advanced 2.0 (ASTER GDEM2), y del Satélite de Observación Terrestre Mundial 3D 2.1 (ALOS AW3D30). El Valle de Fergana, en Asia Central, fue elegido como la región de estudio porque la superficie terrestre puede reflejar los procesos tectónicos. Para este trabajo se determinaron los valores de alturas elipsoidales de 27 puntos de la red regional GPS como valores de referencia. Para el ajuste geoidal de la superficie de estudio se aplicó la aproximación geométrica con los datos de nivelación GPS y el modelo geopotencial global EGM96, basado en ondulaciones geoidales. El rango de corrección de la altura geoidal se establece entre -0.66 m y 0.87 m. Con los modelos SRTM30, ASTER GDEM2 y ALOS AW3D30 se obtuvieron los errores de raíz cuadrada media de ~10.0 m, ~16.4 m, and ~6.6 m, respectivamente. Al compararse con el modelo de referencia se encontró que todos los modelos digitales de elevación en las áreas montañosas por lo general sobreestimaban la elevación y el valor de precisión vertical de un 90 % de exactitud excedía entre 3 y 6 metros el margen declarado por los distribuidores. Sin embargo, el modelo ALOS AW3D30 fue el modelo digital de elevación más preciso y que mejor representa la topografía de la superficie terrestre y que, por lo tanto, podría ser usado para algunas aplicaciones ingenieriles en el Valle de Fergana. Manuscript received: 19/07/2022 Accepted for publication: 17/05/2023 Manuscript received: 19/07/2022 Accepted for publication: 17/05/2023 How to cite this item: Fazilova, D., & Arabov, O. (2023). Vertical accuracy evaluation free access digital elevation models (DEMs): case Fergana Valley in Uzbekistan. Earth Sciences Research Journal, 27(2), 85-91. https://doi. org/10.15446/esrj.v27n2.103801 Vertical accuracy evaluation free access digital elevation models (DEMs): case Fergana Valley in Uzbekistan Dilbarkhon Fazilova1,2,3, Obidjon Arabov1 1National University of Uzbekistan named after Mirzo Ulugbek, Department of geodesy and geoinformatics, 1000174, 4, University str., Tashkent, Republic of Uzbekistan 2Astronomical Institute of Uzbek Academy of Sciences, Space researches laboratory, 100052, 33, Astronomical str., Tashkent, Republic of Uzbekistan 3Tashkent State Technical University named after Islam Karimov, Department of Mine Surveying and Geodesy, Republic of Uzbekistan Corresponding Author: dilbar@astrin.uz 1. Introduction Digital elevation models (DEMs) are necessary for the solution of many practical problems in the areas of the earth and environmental sciences (such as tectonics, the study of landslides, mineral exploration, water and land resources management, disaster prevention, and others) in Uzbekistan (Sayyidkosimov & Kazakov, 2018; Sabitova et al., 2020; Sharipov et al., 2020; Khasanov & Ahmedov, 2021). Topographic maps, Global Navigation Satellite System (GNSS) measurements, photogrammetry techniques, and airborne laser scanning are the main data sources for DEMs production used in Uzbekistan for small areas (Takhirov et al., 2020; Shukina et al., 2022). Despite the accuracy, the traditional DEM generation methods are tedious and time-consuming for using over large areas. Modern satellite remote sensing technologies have made it possible to map large areas with minimal labor and technological costs (Smith &Clark, 2005). The widespread use of publicly available satellite-based DEMs such as SRTM, ASTER, and ALOS is an example of these benefits. However, available open-source these and many others have coarser resolutions. Their accuracy is affected by measurement error, data acquisition method, the instrument’s capability, resolution, terrain type and relief, slope, roughness, land cover, and others (Mukherjee et al., 2011; Hu et al. 2017; Shetty et al., 2022). The vertical accuracy assessment of space-borne elevation data sets has been one of the major concerns for worldwide researchers (Mukherjee et al., 2013; Elkhrachy, 2018; González-Moradas & Viveen, 2020; Preety et al., 2022). For the practical application of the DEM, it is necessary to identify the influence of error sources on the vertical accuracy of the DEM and refine the models using terrestrial measurements for each area separately (Dragut & Eisank, 2011). Global DEMs have got an upsurge in their usage in Uzbekistan. But so far there are few works on assessing their accuracy and developing methods for improving it. In earlier studies, the accuracy of DEMs, such as SRTM, ASTER GDEM, and ALOS PALSAR, was assessed using various data (DEMs of higher resolution, the utilization of Differential Global Positioning System (DGPS), terrestrial measurements (Bakiev & Khasanov, 2021). It was revealed that for the territories located near the reservoirs, ASTER DEM gives a more realistic representation of steep slopes and mountain ridges compared to SRTM or ALOS (Fazilova et al., 2021). The goal of this study is to quantify and compare the vertical accuracy of open access DEMs obtained using the How to cite this item: ISSN 1794-6190 e-ISSN 2339-3459 https://doi.org/10.15446/esrj.v27n2.103801 Dilbarkhon Fazilova, Obidjon Arabov 86 Shuttle Radar Topography Mission (SRTM30), Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER GDEM2), and Advanced Land Observing Satellite (ALOS AW3D30) for the territory of the Fergana Valley in the Republic of Uzbekistan. (1) ALOS World 3D (AW3D30). The global digital surface model dataset with a horizontal resolution of approximately 30-meter mesh (1 arcsec. latitude and longitude) generated from 5 m resolution. The worldwide digital elevation model DEM and ortho-rectified image (ORI) were created using the archived information about the Panchromatic Remote Sensing Instrument for Stereo Mapping (PRISM). PRISM comprised three panchromatic radiometers that were procured along with track stereo images. It had a spatial resolution of 2.5 m in the nadir-looking radiometer and accomplished worldwide coverage, making it an appropriate potential candidate for exact worldwide DEM and ORI generation (Tadono et al., 2014). This is a non-standard version of the ALOS AW3D30 dataset provided in a WGS84 ellipsoidal vertical datum. For this study, 9 tiles of data were downloaded from https://www.eorc.jaxa.jp/ ALOS/en/aw3d30/index.htm website. The geoid undulations according to the EGM96 global geopotential model can be determined using the service of the International Center for Global Earth Models (ICGEM) (Barthelmes & Köhler, 2016). The practical application of equation (1) to determine the required normal heights is complicated by a number of factors, including random errors (noise) in determining the heights, the difference in relevance for different types of heights, and insufficient knowledge of the relationship between them, geodynamic phenomena (soil subsidence, deformation of tectonic plates near subduction zones, etc.). p ) By the extensive use of GPS technique with geodetic aims, great interest has been collimated to the precise determination of local/regional geoid with an aim to replace levelling measurements with GPS surveys. Geoid models derived from the global geopotential models (such as EGM2008, EIGEN- 6C4, EGM96) are the one alternative solution, but their accuracy needs to be estimated first for tectonic territories like Fergana Valley. There are two approaches for the transformation: gravimetrically determined geoid model, and interpolation between geometrically derived geoid heights where GPS measurements have been collocated with benchmarks. The gravimetric method offers benefits for areas with a homogeneous coverage of terrestrial data, but it is involving mathematical and computational procedures (Featherstone et al., 1998). The geometric approach has been widely used for a relatively small area, which interpolates geoid heights based on the GPS-derived heights and leveled heights at some points (Zhong, 1997; Erol & Çelik, 2004; Rabah & Kaloop, 2013; Ligas & Kulczycki, 2018; Dawod & Abdel-Aziz, 2020). The global geoid models like EGM96 can achieve the accuracy of regional or local geoid models by this method. 2. Data and method Topography of the study area generated by SRTM30 and distribution of GPS points over Fergana Valley Vertical accuracy evaluation free access digital elevation models (DEMs): case Fergana Valley in Uzbekistan Vertical accuracy evaluation free access digital elevation models (DEMs): case Fergana Valley in Uzbekistan 87 87 Vertical accuracy evaluation free access digital elevation models (DEMs): case Fergana Valley in Uzbekistan Table 1. Description of digital elevation models (DEM) Data SRTM30 v. 2.0 ASTER GDEM v.2 ALOS World 3D Source Space Shuttle Radar ASTER DAICHI/ALOS Generator/Distributor NASA/USGS METI/NASA JAXA Period of observation from February 11th, 2000 to February 22nd, 2000 March 1, 2000 - November 30, 2013 from 2006 to 2011 Spatial resolution (m) 30 30 30 Vertical datum EGM96 EGM96 EGM96 Horizontal datum WGS84 WGS84 WGS84 Data type Interferometry synthetic aperture radar Optical stereoscopy Optical stereoscopy Vertical accuracy (LE90) < 10 m <15-20 m <7m reference data (internal validation) (Polidorit & Hage, 2020). The vertical accuracy can be directly estimated by comparing the heights extracted from a DEM and their values interpolated to the location of the GPS points. In order to make a consistent comparison of GPS surveys with SRTM, ASTER, and ALOS DEMs, all measurements must refer to the same horizontal coordinate system and vertical datum. The datum of the GPS’s height is the ellipsoidal surface based on the World Geodetic System 1984 (WGS84) reference ellipsoid. For SRTM, ASTER, and ALOS DEMs, each grid cell’s horizontal and vertical coordinates referenced to WGS84 and EGM96 (Lemoine et al., 1997) were output as a text file. Then elevations were transformed to ellipsoid heights (h) relative to WGS84 using EGM96 derived geoid undulations (N) by algebraic summation: termed as voids where water or heavy shadow prevented the quantification of elevation. At a 90% confidence level, the vertical accuracy is expected to be 10 m (Rodriguez et al., 2006). Eight tiles of the SRTM30 elevation data for the study area were downloaded from https://www.usgs.gov/centers/eros website. ASTER GDEM v.2 (ASTER GDEM2). The 1-arc-second (30 m) ( ) ( ) stereoscopic product ASTER GDEM2 was created by stacking all individual cloud-masked scene DEMs and non-cloud-masked scene DEMs, then applying various algorithms to remove abnormal data. The reason for considering this model is that ASTER GDEM2 has some advantage over SRTM30 because its stereo pair has more nadir view when collecting data in very steep and rugged terrain (Abrams et al., 2010). It is used to determine the shortwave and ultra-shortwave components of the geoid, to improve of accurate geopotential models (Erol & Celik, 2004; Soycan, 2014). Early studies used the method due to its efficiency for local areas with poor knowledge of the geoid. The high accuracy of the estimation, especially for engineering surveys, is shown (Mishra & Ghosh, 2017; Jassim & Yousef, 2021). To improve its accuracy parametric models (or a corrective surface) introduced when refining heights (Fotopoulos et al., 2003). Such models include high-order polynomials with interpolation with different basis functions (Zhao et al., 2022), least squares collocation (Lyszkowicz et al., 2014), finite element method (Zaletnyik et al., 2007), Fourier series (Grigoriadis et.al., 2021), and artificial neural networks (Konakoglu & Akar, 2021). Interpolation methods (such as inverse distance The raster values (heights) of these DEMs were extracted from the coordinates of the GPS points. These heights are referred to as heights obtained from each of the DEMs.i The vertical accuracy of DEM at the scale of the study area is verified using 27 reference stations of the State Satellite Geodetic Network (SSGN) in the region, which is developing quite rapidly, considering the developed infrastructure of the country (Fazilova, 2022). To date, only these points of SSGN have been installed in the region (Fig. 1). These points are the so-called “common points,” with the values of normal heights in the Baltic system of 1977 available in the database of the International Gravimetric Bureau in Toulouse (Drewes et al., 2016). Measurements of other global navigation satellite systems (GNSS) such as GLONASS, Galileo, and Beidou are currently not available at the stations. The GAMIT/GLOBK software package version 10.71 was used to calculate the coordinates of points (Herring et al., 2018). According to Dong et al. (1998), processing was carried out according to the recommendations and standards of the International Earth Rotation Service IERS2010 (IERS, 2010). The ITRF2014 coordinate system (Altamimi et al., 2016) was chosen as the reference coordinate system for the regional solution. The coordinate component residuals’ weighted RMS (WRMS) value was 1.0– 3.2 mm and 3.2–6.5 mm for horizontal and vertical components, respectively. The quality of the DEM is a primary requirement from the various processing steps, the interpolation used for resampling, and the local properties (land cover and slope). Typically, the quality assessment includes two approaches, namely with (external) or without (internal) reference data set. 2. Data and method ASTER GDEM2 data has a vertical accuracy of ±15–20 m, depending on the region’s environmental situation (Thomas et al.2014). For this study, 7 ASTER GDEM2 elevation data tiles were downloaded from https://asterweb.jpl.nasa.gov/gdem.asp website. 2. Data and method The area taken for the study is the territory of the Fergana Valley in Uzbekistan, which is an east-west-trending intra-orogenic intermountain depression, the most seismically active and densely populated region of the country. It is located in the eastern part of the Republic of Uzbekistan and borders the Republic of Tajikistan and Kyrgyzstan. The width of the research area is 370 km by 19 km. It is bounded by the Alai and Turkestan systems mountain uplifts in the south, by the Fergana Mountain in the northeast, and by the Chatkal- Kurama Mountain in the north and northwest (Umurzakov & Rabbimkulov, 2022). The maximum topographic relief between the Fergana Valley floor, with an altitude of roughly 330 m, and the surrounding mountain peaks is about 1000 m. The main problem for the territory of the republic, which has a complex relief, is the need for better knowledge of the geoid by gravimetric methods. The geoid of the Fergana Valley is the only area that has been sufficiently well studied by classic leveling. As a result, the influence of the Tien Shan and Pamir Mountain ranges on gravitational anomalies was revealed (Ustyantsev, 2011) (Fig.1). In the work, the entire territory of the Fergana Valley is considered from the point of view of the fact that for many geodetic engineering applications, the assessment of the accuracy of DEMs has not yet been made. The study utilized three sources of DEMs generated from various data acquisition techniques, namely Shuttle Radar Topography Mission (SRTM) DEM of 30 m, Advanced Space Borne Thermal Emission and Reflection Radiometer (ASTER)-Global DEM of 30 m, and ALOS World-3D of 30 m. The important technical details of DEMS are explained in Table 1. SRTM30 v. 2.0. It can be considered to be either an SRTM30 data set enhanced with GTOPO30 or an upgrade to GTOPO30. 1-arcsecond (about 30 meters) resolution DEM, delivered in 1°×1° tiles. Systematic interferometric data were collected for each terrain segment at least twice from different angles (on ascending, north-going, and descending orbit pass) to fill in areas shadowed from the radar beam by terrain. The finished product contains ‘no-data’ Figure 1. Topography of the study area generated by SRTM30 and distribution of GPS points over Fergana Valley Figure 1. 3. Results and discussion The ellipsoidal heights had been calculated using the corrected EGM96 geoid undulations for all three DEMs at 27 points. The descriptive statistics of ellipsoidal heights from each data source range (min, max), the measures of central tendency (mean), and the dispersion (standard deviation, SD) are shown in Table 2. The improvement of EGM96 model is based on the corrector surface fitting (CSF) algorithm and consists of several steps. The first step calculates the difference between the GPS/leveling geoid heights / GPS leveling i N and the geoid height according to EGM96 96 EGM i N at each control “common point” (i=1...27) (Soycan, 2014): For all models, the average elevation was found to be varying between 471 m and 480 m and is also comparable to the reference GPS value of 486 m. At the same time, the linear correlation coefficient between the reference surface and all three DEMs is at the level of 99% and confirms at first glance that the models do not have significant discrepancies from the reference data. Next, we analyzed the differences between the DEM surfaces and the reference surface from GPS measurements. For this, the differences of the grids (GPS-SRTM30), (GPS-ASTER GDEM2) and (GPS-ALOS AW3D30) were determined (Fig. 3 (a-c)). We can observe that compared with the reference model, all the global DEMs in mountainous areas generally overestimated elevation. But the range of elevations the ALOS AW3D30 versus GPS elevations are from 1.5 m to 10.64 m, that compared to the SRTM30 and ASTER GDEM2 elevations is two times less. i (2) (2) The second step is to define the trend Ti based on the calculated differences, which is approximated by a polynomial or harmonic series. In our case, we chose a 3-parametric trend solution for a first-order polynomial: (3) (3) The vertical errors of the DEMs were quantified by comparing individual test DEM elevations and reference GPS elevations at sample points by using the following metrics. The first, root mean square error (RMSE), characterizes the difference between the DEM values ZMODEL and the reference ones (ZGPS in our case). The Mean Error (ME) will estimate of the offset from the reference model. The DEM is compared with a reference data set (external validation). In contrast, in the second case, inconsistencies are sought within the DEM itself with no Dilbarkhon Fazilova, Obidjon Arabov 88 weighting, bilinear interpolation, polynomial regression, triangulation, radial basis functions and nearest-neighbor interpolation) were considering according the application area, surface features, distribution of data and their accuracy. Early studies are devoted to assessing the accuracy of the employed calculation method of the geoid as well as the accuracy, density, and distribution of the used data. Polynomial, least squares collocation, multiquadric (MQ) methods had the best results for geoid undulation calculation. The results confirmed that the radial basis functions (RBF) method is the popular approach to interpolate irregularly spaced data. Especially RBF with multiquadric (MQ) function gives the lowest standard deviation and noise level for the prediction of gravity anomalies, distortion modeling (Soycan, 2014; Doganalp & Selvi, 2015). We use RBF with MQ function interpolator for construction of the surfaces in the work. Vertical accuracy of DEMs was assessed statistically using GPS data based on point-wisely analysis. The surface continuity is a crucial issue in local geoid modeling. In this regard, the results of this study make a significant contribution to the practical use of local geoids. Therefore, we performed the construction of the surface grid using RBF with MQ function interpolation method. Using the 1′×1′ EGM96 global geoid height (Fig. 2a) and 1′×1′ the geoid corrector grid surfaces (Fig. 2b) were constructed for the study area. The geoid height corrections range ranged from –0.66 m to 0.88 m. weighting, bilinear interpolation, polynomial regression, triangulation, radial basis functions and nearest-neighbor interpolation) were considering according the application area, surface features, distribution of data and their accuracy. Early studies are devoted to assessing the accuracy of the employed calculation method of the geoid as well as the accuracy, density, and distribution of the used data. Polynomial, least squares collocation, multiquadric (MQ) methods had the best results for geoid undulation calculation. The results confirmed that the radial basis functions (RBF) method is the popular approach to interpolate irregularly spaced data. Especially RBF with multiquadric (MQ) function gives the lowest standard deviation and noise level for the prediction of gravity anomalies, distortion modeling (Soycan, 2014; Doganalp & Selvi, 2015). We use RBF with MQ function interpolator for construction of the surfaces in the work. 3. Results and discussion In fact, the absolute vertical accuracy of LE90 at a 90% confidence level (LE90) can be estimated based on the obtained RMSE (Mukherjee et al., 2013): where 0a represent a bias, 1 2 , a a represent tilt of the geoid plane with respect to WGS84 ellipsoid, while ji, and li are the are the northings and eastings in some plane coordinate system. CSF dNi values was defined by removing the polynomial trend from the value of DNi: (4) (4) (6) The refined values of EGM96 geoid height anomalies are determined by the expression: (6) (5) (5) (7) (5) (7) b) Figure 2. (a) EGM96 geoid undulations and (b) the corrector surface for geoid fitting a) b) a) b) Figure 2. (a) EGM96 geoid undulations and (b) the corrector surface for geoid fitting Vertical accuracy evaluation free access digital elevation models (DEMs): case Fergana Valley in Uzbekistan 89 Table 2. Summary statistics of DEMs for the study area Min, m Max, m Mean, m SD, m Equation of correlation GPS and DEM Coefficient of correlation R2 GPS 330.080 836.060 485.922 150.833 - - SRTM30 324.284 823.250 476.552 147.652 y = 0.989x - 4.182 0.9996 ASTER 326.362 808.831 470.957 147.131 y = 0.985x -7.736 0.9981 ALOS AW3D30 324.771 830.344 479.847 149.187 y = 0.999x -5.914 0.9997 Table 2. Summary statistics of DEMs for the study area were about 16.4 m, 26.9 m, and 10.9 m for SRTM30, ASTER GDEM2, and ALOS AW3D30, respectively. From Fig. 3d, it can be visualized that, for the Fergana Valley the absolute vertical accuracy error (LE90) of all three DEMs is close to that declared by the manufacturers (LE90distrib), but slightly worse. These variation in the accuracy defines the role of tectonic and terrain features in measurement of elevation irrespective of their spatial resolution. The values implied that ALOS AW3D30 elevations approximated Earth’s surface elevations better than SRTM30 and ASTER GDEM2 at the region. References Abrams, M., Bailey, B., Tsu, H. & Hato, M. (2010). The ASTER Global DEM. Pho- togrammetric Engineering and Remote Sensing, 76(4), 344-348. Herring, T. A., King, R. W., Floyd, M. & McClusky, S. C. (2018). Introduction to GAMIT/GLOBK. Release 10.7. Technical report. Massachusetts In- stitute of Technology, http://geoweb.mit.edu/gg/Intro_GG.pdf (last ac- cessed September 2020) Altamimi, Z., Rebischung, P., Métivier L., & Collilieux, X. (2016). ITRF2014: A New Release of the International Terrestrial Reference Frame Modeling Nonlinear Station Motions. Journal of Geophysical Research: Solid Earth, 121(8), 6109–6131. https://doi.org/10.1002/2016JB013098 Hu, Z., Peng, J., Hou, Y., & Shan, J. 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Surfaces of residuals of ellipsoidal heights according to DEM and GPS: (a) SRTM30, (b) ASTER GDEM2, (c) ALOS AW3D30, and (d) comparative statistics of DEMs c) c) ) ) Figure 3. Surfaces of residuals of ellipsoidal heights according to DEM and GPS: (a) SRTM30, (b) ASTER GDEM2, (c) ALOS AW3D30, and (d) comparative statistics of DEMs Dilbarkhon Fazilova, Obidjon Arabov 90 Erol, B., & Çelik, R. N. (2004). Modelling local GPS/levelling geoid with the assessment of inverse distance weighting and geostatistical kriging methods, 20th ISPRS Congress, Technical Commission IV, Istanbul, Turkey, 76. https://www.isprs.org/proceedings/xxxv/congress/comm4/ papers/319.pdf (last accessed September 2022) 4. Conclusions In this work, for the first time for the territory of the Fergana Valley in Uzbekistan, the vertical accuracy of three publicly available DEMs, SRTM30, ASTER GDEM2, and ALOS AW3D30 was statistically assessed using high accuracy GPS survey data. The geometric approach using GPS/leveling data and EGM96 global geopotential model-based geoid undulations was applied for local geoid modeling. The geoid height corrections range ranged from –0.66 m to 0.87 m. Root-Mean-Square errors of ~10.0 m, ~16.4 m, and ~6.6 m was obtained for SRTM30, ASTER GDEM2, and ALOS AW3D30, respectively. It was found that compared with the reference model, all the global DEMs in mountainous areas generally overestimated elevation and the value of vertical accuracy at a 90% confidence level by 3-6 meters exceeded the declared by distributors. But the range of elevations the ALOS AW3D30 versus GPS elevations are from 1.5 m to 10.64 m, that compared to the SRTM30 and ASTER GDEM2 elevations is two times less. ALOS AW3D30 proved to be the most accurate DEM that best represents the topography of the earth’s surface and could be used for some engineering applications in Fergana Valley. 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https://openalex.org/W2804927841	https://europepmc.org/articles/pmc6059517?pdf=render	English	null	Risk of Venous Thromboembolism in Glioblastoma Patients	Curēus	2,018	cc-by	4,562	DOI: 10.7759/cureus.2678 Risk of Venous Thromboembolism in Glioblastoma Patients Gerald Lim , Clement Ho , Gloria Roldan Urgoti , Derek Leugner , Jay Easaw 1 2 3 4 5 1. Department of Oncology, Tom Baker Cancer Centre, Calgary 2. Radiation Oncology, BC Cancer Agency, Fraser Valley Centre, Surrey, USA 3. Medical Oncology, Tom Baker Cancer Center, calgary, CAN 4. EMS Data Science, AHS 5. Medical Oncology, Cross Cancer, Edmonton, CAN  Corresponding author: Gerald Lim, gerald.lim@albertahealthservices.ca Disclosures can be found in Additional Information at the end of the article Results Twenty-three out of 115 patients (20%) experienced a symptomatic VTE. This complication was not associated with overall survival at two years (p=0.06, heart rate (HR)=1.61). Hypertension and smoking were associated with VTE (p-values 0.034 and 0.048, respectively). A scoring system with the following variables was developed to predict the likelihood of developing VTE: (1) Karnofsky performance status (KPS) - 70, 1 point; KPS < 70, 2 points; (2) Age – 45 to 60, 1 point; 61 to 70, 2 points; (3) Current smoking, 1 point; (4) Hypertension, 1 point. Patients with >3 points were 5 times more likely to develop a VTE. Background Patients with cancer are at increased risk of venous thromboembolic events (VTE) with a particularly high prevalence in patients with glioblastoma (GB). We designed this current study to determine the incidence of symptomatic VTE in patients with GB undergoing first-line chemoradiotherapy and to develop a clinical score to help physicians identify those who are at the highest risk of VTE. Methods A retrospective study cohort included patients diagnosed with GBM treated with radical concurrent chemoradiotherapy between 2005 and 2010 in Southern Alberta. Descriptive statistics were used to characterize the patient population. A predictive value for VTE was assessed by comparing logistic models and using the area under the receiver operating characteristic curve. Open Access Original Article Open Access Original Article Open Access Original Article Received 02/09/2018 Review began 05/08/2018 Review ended 05/18/2018 Published 05/23/2018 © Copyright 2018 Lim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Conclusions Received 02/09/2018 Review began 05/08/2018 Review ended 05/18/2018 Published 05/23/2018 In our population, our simple scoring system allows the identification of patients with GB receiving first-line therapy, who are at the highest risk of VTE. These results require validation in an independent series. © Copyright 2018 Lim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Categories: Radiation Oncology, Neurosurgery, Oncology Keywords: radiotherapy, dvt, gbm Categories: Radiation Oncology, Neurosurgery, Oncology Keywords: radiotherapy, dvt, gbm Introduction How to cite this article Lim G, Ho C, Roldan urgoti G, et al. (May 23, 2018) Risk of Venous Thromboembolism in Glioblastoma Patients. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 VTE detection and treatments Symptomatic venous thromboembolic events, including either deep vein thrombosis and pulmonary embolism (PE) were identified using Doppler ultrasound or thin-cut computed tomography (CT) scan in accordance with institutional standards. Anticoagulation use for the treatment of symptomatic thromboembolism was confirmed using linked hospital discharge data and the provincial pharmacy network. Patient characteristics and treatments This study was approved by the Calgary Health Region Ethics Board. We conducted a population-based evaluation of patients diagnosed with GB in Southern Alberta, Canada. Patients were identified by the provincial cancer registry and their clinical charts were reviewed. We identified 115 patients diagnosed with GBM (World Health Organization (WHO) grade IV glioma) between January 2005 and December 2010 who were offered radical concurrent chemoradiotherapy using temozolomide (75 mg/m2/day x six weeks) and radiotherapy prescribed to 60 Gy in 30 divided daily fractions during the same time period. Clinical characteristics analyzed included a past history of hypertension, dyslipidemia, diabetes mellitus, performance status (KPS score) at first consultation, and the presence of paresis at diagnosis. The presence of paresis was stratified as no paresis, paresis present but allowing independent mobilization, and severe paresis impairing independent mobilization. However, since a large majority of patients had no paresis, this variable was evaluated as paresis present or absent. The association between cancer and venous thromboembolism (VTE) was described in the mid- nineteenth century by Trousseau [1-2]. Although the etiology is debated, suggested causes include the release of high levels of cytokines, acute phase proteins, and over-expression of tissue factors. Moreover, the activation of oncogenic pathways (RAS, EGFR, HER2, MET, SHH) and the loss of tumor suppressors (TP53, PTEN) alter the expression, activity, and release of coagulation factors [3]. Patients with malignancy have up to a 6.5-fold increased risk of developing VTE. The risk is particularly elevated in patients with brain cancers (HR 21.4) [4-5]. Among non-cancer causes of death, Khorana et al. identified VTE as the second-leading cause of death in cancer patients after infection [6]. VTE is of particular concern in patients with glioblastoma multiforme (GBM), where its incidence has been reported to be as high as 22% [5,7-8]. At present, there remains clinical equipoise regarding the relationship between VTE and overall survival. While one paper suggested no significant association between VTE and survival in patients with high-grade glioma [9], an epidemiological study of 9489 patients reported a 30% increased risk of death within two years of a VTE event [10]. Studies have been heterogeneous in their reporting of VTE incidences, with some identifying only symptomatic VTEs and others screening for asymptomatic cases. Most clinicians would consider symptomatic VTE to be clinically relevant, as it is not common practice to look for clots in asymptomatic individuals. This is a population-based study assessing the incidence of symptomatic VTE in patients with GBM undergoing chemoradiotherapy and the clinical factors that could predict the likelihood of a patient developing VTE. Using these data, we developed a clinical score to help physicians identify GBM patients at the highest risk of developing clots. Data analysis Clinical characteristics between groups of patients were compared using a two-sample t-test for continuous variables, and Pearson’s chi-square or Fisher's exact test for categorical variables, 2018 Lim et al. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 2 of 11 depending on sample size. Predictive value for VTE was assessed by comparing logistic models (with bootstrap) and further evaluated, using the area under the receiver operating characteristic (ROC) curve (AUC). Relative risks and odds ratios of developing VTE were estimated with 95% confidence intervals. All tests of significance were two-sided and p-values less than or equal to 0.05 were considered to be statistically significant. Overall survival (OS) was calculated from the original date of consultation to date of death or last follow-up. Time to progression (TTP) was calculated from the date of first cancer center consultation to the date of disease progression or the last date of follow-up. Survival curves and corresponding standard errors were estimated using the Kaplan-Meier method. The association of clinical characteristics with OS was evaluated using the log-rank test. Prognostic value was analyzed using the Cox proportional hazard model (with bootstrap) for OS and TTP. Results One hundred and fifteen patients with GBM received concurrent, postoperative, chemoradiotherapy between 2005 and 2010 in Southern Alberta, Canada. All had a consultation at the Tom Baker Cancer Center in Calgary. The median age was 57 years (range 23 to 83 years); 65% were male; 71% of patients underwent gross total (14%) or subtotal resection (57%); and all patients were treated with radical concurrent chemoradiotherapy (Table 1). Twenty percent of all patients experienced a symptomatic thromboembolic event (23 patients). There was no association between age and the development of VTE when age was dichotomized at the median (< 57 vs >57 years) (p=0.09). KPS score did not correlate with survival (p=0.74), likely due to the fact that patients who undertook radical concurrent chemoradiation were pre- selected for a minimum KPS of 70. A history of hypertension or smoking at diagnoses were associated with a higher likelihood of VTE with p-values of 0.034 and 0.048, respectively. The extent of surgery, dyslipidemia, and diabetes mellitus were not associated with the development of VTE or survival. Nine patients (39% of patients with VTE) had paresis; it impaired mobility in four cases. Due to the low number, the presence of paresis was analyzed as present (in any degree) vs no paresis and it was not associated with VTE or survival (Table 1). Characteristics All Patients Patients with VTE Patients without VTE Association to VTE Relative Risk for VTE Median Survival in Months Difference in Survival Experience (5yr) Hazard Ratio n(%) n(%) n(%) p-value* (95% CI) median (95% CI) p-value* (95% CI)** Total 115 (100%) 23 (100%) 92 (100%) Age at Presentation Median (range) 57 (23- 83) 61 (47- 70) 56.5 (23- 83) <57 53 (46%) 7 (30%) 46 (50%) 0.092 0.51 (0.23, 1.15) 15.6 (12.2, 20) 0.1222 0.741 (0.51, 1.09) >=57 62 (54%) 16 (70%) 46 (50%) 1.95 (0.87, 4.39) 14.2 (10.4, 17.4) 1.35 (0.92, 1.98) 018 Lim et al. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 3 of 1 2018 Lim et al. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 2018 Lim et al. Cureus 10(5): e2678. Results DOI 10.7759/cureus.2678 3 of 11 3 of 11 Gender Female 40 (35%) 8 (35%) 32 (35%) 0.591 1 (0.46, 2.15) 14.6 (11.6, 19.7) 0.4972 1.147 (0.77, 1.71) Male 75 (65%) 15 (65%) 60 (65%) 1 (0.46, 2.15) 14.3 (11.5, 18.6) 0.872 (0.59, 1.3) Surgical Resection Biopsy/other 34 (30%) 10 (43%) 24 (26%) 0.144 Baseline 14.3 (11.2, 18.8) 0.8984 Baseline Subtotal 65 (57%) 12 (52%) 53 (58%) 0.63 (0.3, 1.3) 14.2 (11.2, 19.4) 1.096 (0.71, 1.69) Gross-total 16 (14%) 1 (4%) 15 (16%) 0.21 (0.03, 1.52) 16.7 (9.2, 20.6) 1.005 (0.54, 1.86) MGMT Promoter Unmethylated 56 (49%) 12 (52%) 44 (48%) 0.709 1.15 (0.55, 2.39) 12.5 (11.2, 14.8) 0.0050 1.728 (1.17, 2.54) Methylated 59 (51%) 11 (48%) 48 (52%) 0.87 (0.42, 1.81) 19.2 (14.1, 22.6) 0.579 (0.39, 0.85) VTE No 92 (80%) 0 (0%) 92 (100%) NA NA 15.2 (12.6, 19.8) 0.3646 0.8 (0.49, 1.3) Yes 23 (20%) 23 (100%) 0 (0%) 11.6 (7.7, 16) 1.249 (0.77, 2.02) Anticoagulation Regime None 92 (80%) 0 (0%) 92 (100%) NA NA 15.2 (12.6, 19.8) 0.3067 Baseline LMWH 16 (14%) 16 (70%) 0 (0%) 9.7 (5.3, 14.1) 1.505 (0.87, 2.61) Wafarin 7 (6%) 7 (30%) 0 (0%) 16 5 (7 7 19 2) 0.874 (0 38 2018 Lim et al. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 4 of 11 2018 Lim et al. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 2.01) KPS <=70 33 (29%) 7 (30%) 26 (28%) 0.837 1.09 (0.49, 2.4) 13.4 (9.4, 19.4) 0.7449 1.072 (0.71, 1.63) >70 82 (71%) 16 (70%) 66 (72%) 0.92 (0.42, 2.03) 14.6 (12.2, 18.8) 0.933 (0.61, 1.42) Smoking No 92 (80%) 15 (65%) 77 (84%) 0.048 0.47 (0.23, 0.97) 14.8 (12.2, 18.8) 0.4810 0.844 (0.53, 1.35) Yes 23 (20%) 8 (35%) 15 (16%) 2.13 (1.03, 4.41) 14.1 (9.6, 19.4) 1.184 (0.74, 1.9) Hypertension No 85 (74%) 13 (57%) 72 (78%) 0.034 0.46 (0.23, 0.93) 14.3 (12, 17.6) 0.1813 1.351 (0.87, 2.1) Yes 30 (26%) 10 (43%) 20 (22%) 2.18 (1.07, 4.44) 15.5 (9.7, 26) 0.74 (0.48, 1.15) Dyslipidemia No 99 (86%) 20 (87%) 79 (86%) 0.893 1.08 (0.36, 3.21) 14.2 (12, 17.4) 0.5335 1.191 (0.69, 2.07) Yes 16 (14%) 3 (13%) 13 (14%) 0.93 (0.31, 2.77) 16 (3.2, 25.1) 0.84 (0.48, 1.46) Diabetes Mellitus No 109 (95%) 21 (91%) 88 (96%) 0.345 0.58 (0.17, 1.91) 14.3 (12, 17.6) 0.0798 2.266 (0.89, 5.78) Yes 6 (5%) 2 (9%) 4 (4%) 1.73 (0.52, 5.72) 14.8 (2.8, 0) 0.441 (0.17, 1.13) Paresis No 81 (70%) 14 (61%) 67 (73%) 0.261 0.65 (0.31, 1.36) 14.8 (12.6, 18.8) 0.4485 0.852 (0.56, 2018 Lim et al. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 5 of 11 1.29) Yes 34 (30%) 9 (39%) 25 (27%) 1.53 (0.73, 3.2) 11.9 (7.7, 20.2) 1.173 (0.78, 1.78) 1.29) Yes 34 (30%) 9 (39%) 25 (27%) 1.53 (0.73, 3.2) 11.9 (7.7, 20.2) 1.173 (0.78, 1.78) TABLE 1: Patient characteristics, survival, and hazard ratios VTE: venous thromboembolism 2018 Lim et al. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 TABLE 1: Patient characteristics, survival, and hazard ratios VTE: venous thromboembolism TABLE 1: Patient characteristics, survival, and hazard ratios Eight of the 115 patients included in this series were enrolled in a Phase III, double-blind, clinical trial adding placebo/bevacizumab to the first-line treatment. Four patients developed VTE. However, it was not known whether patients were receiving the active drug or the placebo. Among the 23 patients that developed VTE, two presented after the initial consultation but before starting concurrent treatment, five were receiving chemoradiotherapy and 16 patients developed VTE after the conclusion of chemoradiotherapy. It was not possible to discern from the chart review if VTE complications contributed to death in these patients. In 11 patients (48%), the time of VTE did not correlate with the documented time of progression or death (e.g. the two events were > four weeks apart). In six cases, the VTE diagnosis coincided with the time of recurrence/progression but occurred more than four weeks before death. In the remaining six cases, death (with or without the documented progression of disease) occurred within four weeks from the thromboembolic complication. Although VTE was not documented as the cause of death, it is possible that VTE contributed to death. Anecdotally, from the treating physicians, it was felt that all patients succumbed to their disease rather than an acute thromboembolic event. The median OS for patients with and without VTE was 11.6 and 15.2 months, respectively. OS at two was not associated with a diagnosis of VTE (p= 0.06) (Figure 1). 6 of 11 FIGURE 1: Kaplan Meier plot of two-year overall survival between VTE and non-VTE patients VTE: venous thromboembolism Among the 23 patients who experienced symptomatic thromboembolic events, 16 were treated with low molecular weight heparin (LMWH) and seven were treated with warfarin. The median OS for patients treated with warfarin as compared with LMWH was 16.5 vs 9.7 months with overlapping ranges and no statistically significant difference. FIGURE 1: Kaplan Meier plot of two-year overall survival between VTE and non-VTE patients VTE: venous thromboembolism FIGURE 1: Kaplan Meier plot of two-year overall survival between VTE and non-VTE patients VTE: venous thromboembolism Among the 23 patients who experienced symptomatic thromboembolic events, 16 were treated with low molecular weight heparin (LMWH) and seven were treated with warfarin. The median OS for patients treated with warfarin as compared with LMWH was 16.5 vs 9.7 months with overlapping ranges and no statistically significant difference. Among the 23 patients who experienced symptomatic thromboembolic events, 16 were treated with low molecular weight heparin (LMWH) and seven were treated with warfarin. The median OS for patients treated with warfarin as compared with LMWH was 16.5 vs 9.7 months with overlapping ranges and no statistically significant difference. A molecular tumor analysis showed almost equal distribution between methylated- and unmethylated-O-6-methylguanine-DNA methyltransferase (MGMT) promoter status (51% and 49%, respectively). This parameter was not associated with VTE occurrence but OS at five years was significantly better in patients with a methylated-MGMT promoter (HR 0.579, p=0.005), compared with patients whose tumors had unmethylated MGMT promoter (Figure 2). 7 of 11 2018 Lim et al. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 FIGURE 2: Overall survival of glioblastoma patients by methylation status Predicting the likelihood of developing a symptomatic clot FIGURE 2: Overall survival of glioblastoma patients by methylation status FIGURE 2: Overall survival of glioblastoma patients by methylation status Predicting the likelihood of developing a symptomatic clot Variable inclusion and nested model comparisons were evaluated using the Wald and log- likelihood statistics, model prediction performance was evaluated using the area under the ROC curve (AUC). For choosing the best model, statistical penalization methods and variable selection models (Enet, NEnet, Lasso; utilizing cross-validation) were compared to the stepwise and best subsets of a logistic regression model; AUC is reported for interpretability. A practical score that is simple to perform in routine clinical situations was developed. The score was based on four variables: (1) Karnofsky performance status: 70 = 1 point; < 70 = 2 points; otherwise, 0 points (2) Age: 45 to 60 years = 1 point; 61 to 70 years = 2 points; otherwise, 0 points (2) Age: 45 to 60 years = 1 point; 61 to 70 years = 2 points; otherwise, 0 points (3) Smoking at diagnosis: Yes = 1 point, No = 0 points (3) Smoking at diagnosis: Yes = 1 point, No = 0 points (4) Hypertension history: Yes = 1 point, No = 0 points (4) Hypertension history: Yes = 1 point, No = 0 points 8 of 11 2018 Lim et al. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 These scores are summed for a total score per patient (range, 0 to 6) with AUC=0.758 (95% CI: 0.668, 0.832) and p-value=0.001. For every point increase, the odds of a patient being diagnosed with a symptomatic VTE increased by 2.2 (95% CI: 1.3, 3.7; p=0.002). A validated cut point allowing the categorization of patients into low- and high-risk groups was ≤2 vs. >2 points. These two risk categories have AUC = 0.739 (95% CI: 0.649, 0.817) and p <0.001. Patients with scores >2 points (high risk of a clot) had a five-times (95% CI: 2.3, 11.1) higher risk of a symptomatic VTE compared to patients with scores ≤2. Discussion Glioblastoma multiforme is the most common primary brain tumor in adults. Despite surgery, followed by radiation therapy and chemotherapy, most patients with GBM go through a rapid and disappointing clinical course with a median survival of just over a year [11-12]. Unfortunately, the poor clinical course for these patients is worsened by a high rate of symptomatic VTEs and other complications. In this retrospective, population-based review, 20% of patients developed a symptomatic VTE. It could be useful to identify beforehand the subset of patients at highest risk of VTE. It is unclear if the development of a VTE signifies a worse prognosis for OS, but the simple predictive algorithm proposed identified patients with a five-fold higher risk of VTE. Although the median survival of patients in this study with and without VTE was 11.6 months and 15.3 months, respectively, this was not statistically significant (P=0.06). The small sample size may have prevented this difference from reaching statistical significance. Whether prophylactic anticoagulation therapy would improve the quality of life or OS needs further validation. Despite the very high incidence of VTE/PE, it would be both impractical and potentially unsafe to prophylactically anticoagulate every GBM patient [5,13]. The early terminated PRODIGE study showed that the primary prophylaxis of GB patients caused a trend towards diminishing VTE events at the cost of an increased incidence of intracranial hemorrhage and without a survival benefit [13]. However, our data suggest that it may be possible to identify GBM patients at the highest risk for thromboembolic complications, thereby allowing the targeted use of anticoagulants in a carefully selected group of GBM patients. In this study, our goal was to develop a simple practical scoring system specific to GBM patients that could identify those patients at the highest risk of developing VTE. Among all the variables examined, only smoking and hypertension independently predicted a higher risk of clot formation. However, when combined with age and KPS, these four variables produced a highly predictive score for symptomatic clot formation in our patient population. Our data suggest that a clot score greater than 2 points suggests that the patient has a five-fold increased risk of developing a symptomatic VTE. Higher risk patients, including patients with GBM, are routinely counseled about the signs and symptoms of VTEs and to avoid long periods of stasis and other activities that predispose clots. 2018 Lim et al. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 Disclosures Human subjects: Consent was obtained by all participants in this study. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. Conclusions GBM is the most common malignant primary brain tumor in adults. Patients with GBM have a poor prognosis and are at a high risk of developing symptomatic VTE. In this study, 20% of patients were found to have a symptomatic VTE. It is unclear if the diagnosis of the disease predicts poorer OS. However, a simple scoring system based on age, KPS, smoking, and hypertension histories, allowed the identification of patients with GBM receiving first-line therapy, who were at the highest risk of VTE. These results require validation in an independent series. Discussion Prophylactic anticoagulation in all GBM patients is controversial but perhaps targeting a selected subset of GBM patients with a high clotting score on our scale might be beneficial. The merits of this intervention in this subset of patients warrants further research. In this study, our goal was to develop a simple practical scoring system specific to GBM patients that could identify those patients at the highest risk of developing VTE. Among all the variables examined, only smoking and hypertension independently predicted a higher risk of clot formation. However, when combined with age and KPS, these four variables produced a highly predictive score for symptomatic clot formation in our patient population. Our data suggest that a clot score greater than 2 points suggests that the patient has a five-fold increased risk of developing a symptomatic VTE. Higher risk patients, including patients with GBM, are routinely counseled about the signs and symptoms of VTEs and to avoid long periods of stasis and other activities that predispose clots. Prophylactic anticoagulation in all GBM patients is controversial but perhaps targeting a selected subset of GBM patients with a high clotting score on our scale might be beneficial. The merits of this intervention in this subset of patients warrants further research. Our scoring system identified in this report includes just four variables: KPS, age, smoking, and hypertension. The association between known cardiovascular risk factors (i.e. metabolic syndrome, obesity, hypertension, diabetes) and VTE has been previously recognized [14]. Mechanistically, smoking and hypertension could result in endothelial injury and loss of elasticity, thus resulting in an increased risk of clot formation. Patients with poorer performance status would likely be less mobile and less independent in their functioning. The lack of mobility would result in stasis, a well-known risk factor for developing clots. Older age could contribute in such a manner in addition to having a vascular system that was less healthy and more prone to injury and subsequent clots. 9 of 11 2018 Lim et al. Cureus 10(5): e2678. DOI 10.7759/cureus.2678 This scoring system is a first attempt to create a simple practical scoring system to identify GBM patients at the highest risk of developing a blood clot. However, we appreciate that this study has a number of limitations, including its retrospective design with a low number of patients that precludes further subgroup analysis and may account for the lack of a statistical significance of some results. Also, the described population is a selected group of patients, already with good KPS, enough to be candidates for concurrent chemoradiotherapy treatment and these results cannot be extrapolated to the total population of patients with GBM. We did not collect information about the concurrent use of drugs that could alter DVT risk (i.e. ASA, clopidogrel, hormonal replacement therapy). Although there was no statistical difference in outcomes based on DVT treatment (low molecular weight heparin (LMWH) vs warfarin), the small number of patients precluded a meaningful analysis in this area. This certainly would be of particular interest and has not been well studied. Despite the high incidence of VTE in this population, patients with GBM are usually absent or underrepresented in studies evaluating anticoagulation. The recently published randomized control study of tinzaparin versus warfarin in patients with cancer does not address the issue in this population [15]. Although a prospective trial to evaluate this score would be optimal, our next goal is to validate this score using a larger dataset, which will include GBM patient cohorts from other Canadian centers. 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https://openalex.org/W3090195501	https://www.frontiersin.org/articles/10.3389/fnhum.2020.509091/pdf	English	null	Methodological Problems With Online Concussion Testing	Frontiers in human neuroscience	2,020	cc-by	10,905	Methodological Problems With Online Concussion Testing Reaction time testing is widely used in online computerized concussion assessments, and most concussion studies utilizing the metric have demonstrated varying degrees of difference between concussed and non-concussed individuals. The problem with most of these online concussion assessments is that they predominantly rely on consumer grade technology. Typical administration of these reaction time tests involves presenting a visual stimulus on a computer monitor and prompting the test subject to respond as quickly as possible via keypad or computer mouse. However, inherent delays and variabilities are introduced to the reaction time measure by both computer and associated operating systems that the concussion assessment tool is installed on. The authors hypothesized systems that are typically used to collect concussion reaction time data would demonstrate signiﬁcant errors in reaction time measurements. To remove human bias, a series of experiments was conducted robotically to assess timing errors introduced by reaction time tests under four different conditions. In the ﬁrst condition, a visual reaction time test was conducted by ﬂashing a visual stimulus on a computer monitor. Detection was via photodiode and mechanical response was delivered via computer mouse. The second condition employed a mobile device for the visual stimulus, and the mechanical response was delivered to the mobile device’s touchscreen. The third condition simulated a tactile reaction time test, and mechanical response was delivered via computer mouse. The fourth condition also simulated a tactile reaction time test, but response was delivered to a dedicated device designed to store the interval between stimulus delivery and response, thus bypassing any problems hypothesized to be introduced by computer and/or computer software. There were signiﬁcant differences in the range of responses recorded from the four different conditions with the reaction time collected from visual stimulus on a mobile device being the worst and the device with dedicated hardware designed for the task being the best. The results suggest that some of the commonly used visual tasks on consumer grade computers could be (and have been) introducing signiﬁcant errors for reaction time testing and that dedicated hardware designed for the reaction time task is needed to minimize testing errors. Keywords: reaction time, reaction time variability, online cognitive testing, online concussion testing, intraindividual reaction time variability, concussion, concussion testing ORIGINAL RESEARCH published: 01 October 2020 doi: 10.3389/fnhum.2020.509091 Methodological Problems With Online Concussion Testing Jameson Holden 1, Eric Francisco 1, Anna Tommerdahl 1, Rachel Lensch 1, Bryan Kirsch 1, Laila Zai 2, Alan J. Pearce 3, Oleg V. Favorov 4, Robert G. Dennis 1,4 and Mark Tommerdahl 1,4* 1 Cortical Metrics LLC, Carrboro, NC, United States, 2 Lucent Research, Denver, CO, United States, 3 College of Health Science and Engineering, LaTrobe University, Melbourne, VIC, Australia, 4 Department of Biomedical Engineering, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States Edited by: Edited by: Srikantan S. Nagarajan, University of California, San Francisco, United States Edited by: Srikantan S. Nagarajan, University of California, San Francisco, United States Reviewed by: Paul Edward Kosnik, Tissue Genesis, United States Magdalena Ietswaart, University of Stirling, United Kingdom Correspondence: Mark Tommerdahl mark_tommerdahl@med.unc.edu Specialty section: This article was submitted to Health, a section of the journal Frontiers in Human Neuroscience Received: 31 October 2019 Accepted: 26 August 2020 Published: 01 October 2020 Citation: Holden J, Francisco E, Tommerdahl A, Lensch R, Kirsch B, Zai L, Pearce AJ, Favorov OV, Dennis RG and Tommerdahl M (2020) Methodological Problems With Online Concussion Testing. Front. Hum. Neurosci. 14:509091. doi: 10.3389/fnhum.2020.509091 Reviewed by: Paul Edward Kosnik, Tissue Genesis, United States Magdalena Ietswaart, University of Stirling, United Kingdom Correspondence: Mark Tommerdahl mark_tommerdahl@med.unc.edu BACKGROUND The methods used by Merkel, visual stimulation and tactile response (Merkel, 1885), are still commonly used due to the simplicity of implementation, though it is noteworthy that the individual data obtained by Merkel ranged between 152 and 201 msec, which is not the case for most contemporary studies. A study by Woodley et al. (2013) postulated that as a human race, we are getting “dumber.” The basic premise of the study was that reaction times are getting slower, and that this contradicts a number of other studies that had demonstrated that, based on performance on IQ-tests, we are actually getting a bit smarter. The purpose of this report is not to weigh in on whether or not humans are getting dumber as a species, but rather to focus on the accuracy of reaction time testing, how it has changed historically and if these changes are the result of altered scientiﬁc methodology that could be inadvertently leading to inaccurate scientiﬁc ﬁndings in the literature. p y Many of the studies spanning the 150-year time frame investigated changes in reaction time that resulted from a number of neurological disorders or insults. For example, reaction times have been demonstrated to be altered by changes introduced by neurological insults such as TBI/mTBI (Ruesch, 1944; van Zomeren and Deelman, 1976; MacFlynn et al., 1984; Stuss et al., 1989; Ponsford and Kinsella, 1992; Collins and Long, 1996; Zahn and Mirsky, 1999; Warden et al., 2001; Collins et al., 2003; Sarno et al., 2003; Willison and Tombaugh, 2006; Niogi et al., 2008; Gould et al., 2013; Eckner et al., 2015), PTSD (Ruesch, 1944), pharmaceuticals (Edwards and Cohen, 1961; Ancelin et al., 2006), aging (Benton, 1977; Sherwood and Selder, 1979; Fozard et al., 1994; Lajoie and Gallagher, 2004; Der and Deary, 2006; Zhang et al., 2011; Woods et al., 2015), dementia (Ancelin et al., 2006), Parkinson’s (Evarts et al., 1981; Goodrich et al., 1989), schizophrenia (Schwartz et al., 1989), ADHD (Meere et al., 1992; reviewed in Tamm et al., 2012; Puts et al., 2017), sleep deprivation (Lorenzo et al., 1995), caﬀeine (Cheney, 1934), alcohol (Hernández et al., 2006), autism spectrum disorders (Puts et al., 2014; Ferraro, 2016), and diabetes (Patil and Phatale, 2015). BACKGROUND Casual observation of the data plotted in Figure 1 suggests that the methods underlying the represented scientiﬁc literature are becoming much more variable and consequently, less accurate. There are numerous online tests that are routinely used for assessments of individuals with concussion and/or used in concussion research, and the majority of these assessment tools have components that speciﬁcally address the reaction time and/or reaction time variability of the individual that is being tested. However, these contemporary concussion computerized online assessment tools predominantly rely on whatever computer systems that they are downloaded and run on. Administration of the reaction time test by these online assessments typically use the computer’s monitor and mouse to deliver a stimulus (such as a character on the monitor) and receive a response (button press of the computer mouse), respectively. As the prevalence of online concussion testing that relies on consumer grade computers has increased while the utilization of laboratory research tools has decreased, the authors sought to determine if that could have an impact on performance and accuracy of the reaction time test. g q y Reports in the literature describing and/or utilizing reaction time tests date back to the nineteenth century. In 1849, Hermann von Helmholtz used electric stimulation to investigate nerve conduction velocity, ﬁrst examining the conduction velocity in the legs of frogs before modifying his methods to accommodate human subjects. Helmholtz stimulated the skin at two separate locations and measured the time required for the human subject to respond via hand signal to the stimulation at each location. By measuring the distance between the two points of stimulation and the diﬀerence in their associated reaction time, he deduced a fairly accurate estimate of nerve conduction velocity. Donders expanded on the concept to include central as well as peripheral nervous system processing in which he stimulated either skin, eye or ear and had the subjects respond with their hands (Donders, 1868, 1969). Time recorded was based on a chronograph, and although the early methods may seem crude or cumbersome by contemporary methods, the results from these early experiments inspired numerous investigations utilizing reaction time over the subsequent 150 plus years. Abbreviations: CPU, Central Processing Unit; VCA, Voice Coil Actuator; LCD, Liquid Crystal Display; IQ, Intelligence Quotient; FET, Field Eﬀect Transistor; msec, milliseconds; mTBI, mild traumatic brain injury; USB, Universal serial bus. Citation: October 2020 \| Volume 14 \| Article 509091 Frontiers in Human Neuroscience \| www.frontiersin.org 1 Methodological Problems With Concussion Testing Holden et al. BACKGROUND See Table 1 (see Appendix A: Supplementary Tables) for complete references used to obtain each data point. FIGURE 1 \| Results of reaction time testing reported in the literature. Reaction time values are categorized by modality of measurement (visual reaction time, auditory reaction time, and tactile reaction time) and plotted in the corresponding year collected. See Table 1 (see Appendix A: Supplementary Tables) for complete references used to obtain each data point. to suﬀer. Laboratory equipment of the mid-nineteenth century was speciﬁcally designed for laboratory use and was probably as accurate at performing reaction time testing as many of today’s computer-based reaction time tests, if not more so. Reaction time tests suﬀer from latencies that are introduced at points aside from core processor timing in their protocols as well. For example, these can be introduced by the commodity human interface peripherals. USB and wireless mice and keyboards introduce latency in their communication protocols at several points, including pre-transmission buﬀering, transmission, and post-transmission buﬀering before transfer to the CPU for processing. Most computer screens have a refresh rate of 60 Hz, and a screen ﬂash can occur up to 17 msec before or after the “stimulus delivery” time that is recorded by the CPU. Touch screens on both mobile and desktop devices have a built-in latency related to the sensing mechanism, usually by capacitance. For smooth operation, touch sensing requires a certain amount of signal processing both in hardware and software or ﬁrmware, because touch signals typically involve a certain amount of “integration” at or near the sensor. This is necessary both for noise mitigation, to eliminate spurious signals, as well as to detect the proximity of a ﬁnger or stylus by capacitive coupling. For example, capacitive sensing can be done in several diﬀerent ways, but these all invariably involve the rate of charge or discharge of a capacitor which is modiﬁed by the proximity of a ﬁnger or stylus that changes the value of the capacitor being charged, and thus the RC time constant, by a few percent. Styli may be standardized for a speciﬁc device, but ﬁngers are not, and thus it is necessary to set thresholds and make decisions in ﬁrmware whether or not a touch event has occurred, or not, for a wide range of contact conditions. All of this integration and processing takes time, even when done by distributed processing. BACKGROUND The same arguments can be modiﬁed and applied to older force sensing screen interfaces which had the added computational burden of calculating a force centroid to determine where force was being exerted on the plane of the display, and similarly for any other touch sensing strategy such as resistive or others. Layered on top of this is the ﬁrmware task of interpreting what type of touch is being detected, whether there are one or multiple touch points. Once the signal has been cleaned, ﬁltered, and interpreted by the peripheral touch sensing device, it can then be placed in the communication buﬀer, where it waits its turn for CPU priority. Because of all the variables involved, and diﬀerent strategies employed by touch sensing peripheral hardware, it is not possible to calculate the hardware The question we sought to address was whether or not modern computing methods introduce problems to the reaction time measure. There are inherent delays predicted to be introduced by both software and hardware. Many contemporary reaction time tests are administered through a computer program that calculates the time elapsed between stimulus delivery and the subject response (typically the click of a mouse). The majority of these tests use either a visual (e.g., screen ﬂash) or auditory (loud beep) stimulus, as these stimuli can be delivered using commodity-grade human-computer interfaces such as computer monitors, mice, keyboards, and touch screens. Reaction time tests that employ a diﬀerent mode of stimulation (such as a tactile stimulus) require additional hardware, which may be connected to the test computer by a physical or wireless connection. The reaction time test is contingent on the CPU timing accuracy of the testing computer, which can vary based on which programs are running in the background and the inherent processing speed of the chip. Also of great concern is the operating system (OS) timing cycle and task priority structure, which typically manages many tasks, including system overhead unrelated to the reaction time test in progress. BACKGROUND The widespread utilization of the reaction time test across many decades of research and its utility in many diﬀerent clinical and clinical research venues led us to ponder how the accuracy of this measure might have changed historically. Contemporary users of the reaction time metric might assume that using modern and faster computer technology automatically leads to more accurate reaction time measures. The fallacy of this assumption is that the modern computer technology commonly deployed for reaction time testing is not designed to be laboratory equipment, which causes the accurate timing of external events There have been many reaction time studies over the past 150 years. The graph in Figure 1 is a summary of the data points obtained from healthy subjects across a collection of those publications. Each plotted data point is the overall average obtained for healthy controls in each of the selected studies (which is inclusive of the 16 studies used by Woodley et al., 2013 plus additional reports that focused on reaction time). The data demonstrate not only an upward drift of reaction time, but a larger range of reaction times, with the progressive degradation of reaction time appearing to begin in the 1970s and 1980s. Thus, the question that the authors think should be asked is not whether we are getting worse at reaction time testing, but could there be inconsistencies introduced by the reaction time testing itself? inconsistencies introduced by the reaction time testing itself? Note that while early reaction time studies (between 1850 and 1950) demonstrated human performance in the 150–200 msec range, the range reported post-2000 extends from 150 to 400 msec. This enormous shift suggests either a very diﬀerent population that is being tested or a very diﬀerent strategy for measuring reaction time. Since the dumbing down of the human species is really not something that the authors believe can be accurately measured (other than observations of how much time some generations spend utilizing social media), we targeted addressing the actual methods that were being used. October 2020 \| Volume 14 \| Article 509091 Frontiers in Human Neuroscience \| www.frontiersin.org 2 Methodological Problems With Concussion Testing Holden et al. FIGURE 1 \| Results of reaction time testing reported in the literature. Reaction time values are categorized by modality of measurement (visual reaction time, auditory reaction time, and tactile reaction time) and plotted in the corresponding year collected. BACKGROUND While this division of attention is seldom apparent to the user, it typically introduces delays of ∼15 msec, or more when the computational demand is high, such as in the presence of malware or background tasks, or as a result of widely employed networking prioritization such as “audio prioritization.” In this case, the delays in other functions can even become clearly apparent to the user, being on the order of hundreds of milliseconds or more. Even at its minimum, this CPU latency is typically between 2 and 20 msec, which will signiﬁcantly and ambiguously alter reaction time test results. In addition to OS latency, diﬀerent device driver ﬁrmware can introduce latencies diﬀering by tens of milliseconds or more between drivers, even with identical hardware (Plant and Turner, 2009). These computer hardware and software variations can introduce variable timing delays of up to 100 msec (Neath et al., 2011). October 2020 \| Volume 14 \| Article 509091 Frontiers in Human Neuroscience \| www.frontiersin.org 3 Methodological Problems With Concussion Testing Holden et al. latency. As a result, current “lag” from touch screen-to-display varies from 50 to 200 msec (Ng et al., 2012). latency. As a result, current “lag” from touch screen-to-display varies from 50 to 200 msec (Ng et al., 2012). latency. As a result, current “lag” from touch screen-to-display varies from 50 to 200 msec (Ng et al., 2012). FIGURE 2 \| Dedicated mechanical apparatus. g The objective of this study was to determine if there are signiﬁcant and measurable diﬀerences introduced to reaction time measures that are collected with diﬀerent types or categories of hardware currently used in commercially available reaction time tests. More speciﬁcally, how would a visual reaction time test performed on a computer laptop or mobile device with consumer grade hardware (most common method) compare with a tactile reaction time test delivered with laboratory grade hardware (less commonly performed). If equipment and/or operating systems do in fact introduce errors into the reaction time test, then it would be hypothesized that diﬀerent methods requiring signiﬁcantly diﬀerent hardware or software would generate very diﬀerent reaction times and reaction time variability for individuals taking the reaction time test. In order to directly investigate the diﬀerences introduced by various testing strategies, the human element was removed from this study and automated/dedicated hardware was used to perform the reaction time tasks. BACKGROUND Four modes of reaction time testing were evaluated robotically to compare the potential contributions of diﬀerent user interfaces to the reaction time test. FIGURE 2 \| Dedicated mechanical apparatus. Simulation #1: Visual Stimulus and Response via Mobile Device p An analog light sensor (Adafruit ALS PT19) was mounted 2 mm above a mobile touchscreen (Nextbit Robin, Android 7.1.1 “Nougat”), which was programmed to ﬂash from black to white at random intervals (4–6 s). The light sensor was conﬁgured with a triggering threshold of 800 mV. In order to simulate the response interval of a reaction time test, a dedicated mechanical apparatus was designed that was triggered to respond automatically at a ﬁxed interval (100 msec) after the ﬂash was detected by the light sensor. The mechanical apparatus (Figure 2) was assembled on a standard breadboard and was comprised of a linear voice coil actuator (VCA), a 555 Timer conﬁgured for monostable output (110 ± 1 msec), an N-channel ﬁeld-eﬀect transistor (FET), and a 5 V power supply. A capacitive sensor was mounted to the VCA probe tip of the apparatus and placed 2.6 mm above the touchscreen in order to simulate a button press by a human (Protocol 1 of Figure 3). After the programmed delay the VCA would receive a 10 ms pulse from the 555 timer which caused the VCA to actuate. The capacitive sensor reached its expected triggering point (initial screen contact) at the middle point of the pulse, theoretically adding 5 msec to the 100 msec programmed delay. Thus, the expected true reaction time for the system was 105 msec. MATERIALS AND METHODS mechanical response to either a computer mouse, a touchscreen or a dedicated device (Brain Gauge; Cortical Metrics, LLC). Experiments were conducted with four diﬀerent conditions in order to observe results obtained with variable stimulus and response protocols for a reaction time test with a non-human interface. The stimuli delivered were visual/optical (simulation of a visual stimulus) with a mobile device, visual/optical with a computer monitor, and tactile/mechanical (simulation of a mechanical stimulus) with a dedicated hardware device (mechanical stimulus delivered with the Brain Gauge; Cortical Metrics, LLC). The Brain Gauge is a commercially available tactile stimulator that both delivers vibrotactile stimuli and can be used as a response device and was well-suited for this study. Originally developed for research laboratory use with good resolution, the Brain Gauge maintained technical speciﬁcations as it evolved to a commercial product. The response methods used were tactile/mechanical via touchscreen (simulation of ﬁnger press on touch screen), tactile/mechanical via computer mouse (simulation of ﬁnger response via computer mouse), and tactile/mechanical via dedicated hardware device (simulation of ﬁnger response via Brain Gauge). Apparatus and Device Setup Four simulations were performed, and diﬀerent conﬁgurations were used to deliver those simulations. In each case, simulation of an individual taking a reaction time test was performed by detection of a visual or mechanical stimulus via electronic switch to simulate stimulus detection and delivery of a mechanical stimulus to simulate a ﬁnger depressing a response device. The conﬁgurations were assembled with a standard breadboard. Each of the following tasks ran for N = 100 trials and each simulation was conducted with the same CPU (MacBook Pro 2017). Trials were performed for either a visual or mechanical stimulus simulation. Detection simulation was performed by Simulation #2: Visual Stimulus and Response via Computer Mouse The analog light sensor was placed 2 mm in front of an LCD monitor (60 Hz, 1080p, Dell) and was programmed to ﬂash from black to white at random intervals (4–6 s). The light sensor was conﬁgured with a triggering threshold of 800 mV. Frontiers in Human Neuroscience \| www.frontiersin.org October 2020 \| Volume 14 \| Article 509091 4 Methodological Problems With Concussion Testing Holden et al. RE 3 \| Reaction time simulations. Protocols 1 (Nextbit Robin) and 2 (MacBook Pro 2017) delivered mechanical stimuli in response to a visual stimulus. Protocols 4 (MacBook Pro 2017) delivered mechanical stimuli in response to a tactile stimulus. FIGURE 3 \| Reaction time simulations. Protocols 1 (Nextbit Robin) and 2 (MacBook Pro 2017) delivered mechanical stimuli in response to a visual stimulus. Protocols 3 and 4 (MacBook Pro 2017) delivered mechanical stimuli in response to a tactile stimulus. FIGURE 3 \| Reaction time simulations. Protocols 1 (Nextbit Robin) and 2 (MacBook Pro 2017) delivered mechanical stimuli in response to a visual stimulus. Protocols 3 and 4 (MacBook Pro 2017) delivered mechanical stimuli in response to a tactile stimulus. Metrics, LLC) in order to detect a mechanical stimulus of a simulated reaction time test. A 1.5 mm stimulus was used to depress the switch above the actuation point and trigger the mechanical response simulator circuitry. The VCA probe tip of the mechanical apparatus was positioned above a computer mouse to simulate a subject’s responding digit in a resting state. The stimulus pattern, programmed delay and switch response were identical to the conditions in Simulation #1. The mechanical apparatus from Simulation #1 was modiﬁed to simulate a controlled human reaction time of 100 msec after the mechanical switch was triggered. Expected true reaction time for the system was 105 msec. The VCA of the mechanical apparatus was positioned directly above the left button of the USB computer mouse to simulate a subject’s response. The mechanical response was simulated as in Simulation #1 and was used to simulate a ﬁnger press 100 msec after the ﬂash triggered the light sensor. Expected true reaction time for the system was 105 msec. RESULTS Four simulated reaction time tests were performed. In each case a stimulus (visual or tactile) was delivered and detected electronically, and a response was made mechanically either via touchscreen, USB mouse or with a dedicated testing device (Brain Gauge; Cortical Metrics, LLC). The expected true reaction time for all four experimental groups was 105 msec. Results reported below have subtracted out the commanded 555 timer delay of 100 msec which was held constant using identical hardware for all testing protocols. A reported latency value of 5.0 msec would indicate the system achieved the expected results after adding back the 100 msec 555 timer delay. demonstrated signiﬁcant inaccuracies when compared to the tactile based testing. As demonstrated in Figure 5, relative contributions of latencies can be inferred from the various combinations of individual components. Protocols 1 and 2 have the highest overall latencies and variabilities and are the only protocols using a visual display. Since both the Nextbit Robin and Macbook Pro displays are clocked at 60 Hz, both have multiple processing cores, and given that all direct user input on both macOS and Linux is handled by kernel interrupts at the lowest level, diﬀerences in numerical computational power between the devices should not be a factor for such simple user input tasks. Therefore, the authors conclude that the primary contributor to the observed diﬀerence in latency between Protocols 1 and 2 is the touchscreen digitizer. Other touchscreen digitizers may exhibit less latency than observed in Protocol 1, but it is highly unlikely that any capacitive digitizer could outperform a simple USB mouse in a consumer-grade device. At best, the observed latencies for other 60 Hz touchscreen devices would approach those observed in Protocol 2. Reaction time to a simulated visual stimulus in which a touchscreen was used as the response device generated the highest latency of 399 ± 16.3 msec. When the same visual stimulus simulation was coupled with a response from a USB Mouse, reaction time latency was signiﬁcantly improved to 80.1 ± 8.0 msec. Reaction time to a tactile stimulus simulation utilizing the same USB mouse for a response device demonstrated a latency of 30.7 ± 2.6 msec. Reaction time to the tactile stimulus simulation with response on the dedicated tactile device had the smallest latency error of 5.6 ± 0.25 msec. Average latency errors are plotted in Figure 4. Simulation #3: Tactile Stimulus and Response via Computer Mouse p p A mechanical switch (Cherry MX Red) was mounted above the probe tip of a tactile stimulator (Brain Gauge; Cortical October 2020 \| Volume 14 \| Article 509091 Frontiers in Human Neuroscience \| www.frontiersin.org 5 Methodological Problems With Concussion Testing Holden et al. RESULTS Perhaps more signiﬁcantly than the latency on each task was the variability. In Table 2 (see Appendix A: Supplementary Tables), note the variability and range of latencies. This variability is prominently noticeable in Figure 5 which displays the data point-by-point and Figure 6 which directly compares system variability. The fundamental deﬁciency of commodity-grade computer interfaces for use in high-ﬁdelity human performance research has been a concern for decades among researchers who value accuracy over simplicity. While there are a number of reports that demonstrate that many researchers have developed specialized laboratory equipment for the accurate measurement of human performance, a large and growing cohort of researchers are more frequently using less specialized equipment. From a high-altitude perspective, it appears that there was a time in the scientiﬁc study of human performance when researchers actually built and understood their instruments, tested and calibrated them carefully, knew their strengths and limitations, and factored these into the analysis of their results. But with the advent and ubiquity of low-cost commodity-grade human interface devices and displays designed to give the perception of smooth operation while being increasingly simple to use and low in cost, the use of these devices as if they were scientiﬁc-grade instruments Simulation #4: Tactile Stimulus and Response With Dedicated Hardware Device FIGURE 4 \| Comparison of the latency errors of four different methods of reaction time testing. The mechanical detection system was arranged as in Simulation #3. The VCA probe tip was mounted above the response tip of the dedicated hardware reaction time device, depressing the device’s tip by 1.5 mm. The VCA response was identical to the task of Simulation #3. The mechanical apparatus from Simulation #3 was used to simulate a controlled human reaction time of 100 ms after the mechanical switch was triggered. Expected true reaction time for the system was 105 ms. Frontiers in Human Neuroscience \| www.frontiersin.org DISCUSSION In this study, we demonstrated that reaction time testing, simulated robotically, shows profound diﬀerences in performance when stimuli are delivered either by visual or tactile modalities, and that there are signiﬁcant diﬀerences in performance when the responses to those stimuli are delivered mechanically via either touchscreen, USB mouse, or a dedicated device that was designed for the task. In other words, a comparison was made in reaction time performance between consumer-grade computer interfaces and laboratory equivalent research tools with no human factors involved. The consumer-grade based testing that used visual stimuli In this study, we demonstrated that reaction time testing, simulated robotically, shows profound diﬀerences in performance when stimuli are delivered either by visual or tactile modalities, and that there are signiﬁcant diﬀerences in performance when the responses to those stimuli are delivered mechanically via either touchscreen, USB mouse, or a dedicated device that was designed for the task. In other words, a comparison was made in reaction time performance between consumer-grade computer interfaces and laboratory equivalent research tools with no human factors involved. The consumer-grade based testing that used visual stimuli October 2020 \| Volume 14 \| Article 509091 Frontiers in Human Neuroscience \| www.frontiersin.org 6 Holden et al. Methodological Problems With Concussion Testing FIGURE 5 \| Direct comparison of data from the four reaction time testing methods with averaged offset subtracted. Raw data is plotted with offset of median latency subtracted, a technique used by many reaction time assessments to adjust for systematic latencies. All data is plotted on the same scale. The visual task simulations had signiﬁcantly greater variabilities than the tactile simulations. FIGURE 5 \| Direct comparison of data from the four reaction time testing methods with averaged offset subtracted. Raw data is plotted with offset of median latency subtracted, a technique used by many reaction time assessments to adjust for systematic latencies. All data is plotted on the same scale. The visual task simulations had signiﬁcantly greater variabilities than the tactile simulations. literature and that this may contribute to what is now recognized as the “replication crisis.” The replication crisis appears to span most of biomedical research, even in cancer and drug development research (Prinz et al., 2011; Begley and Ellis, 2012) in which careful replication exercises demonstrate that as much as 75 to 89% of academic research, published by the best laboratories in top journals, may simply not be reproducible. Frontiers in Human Neuroscience \| www.frontiersin.org DISCUSSION Multiple reports have noted the importance of reaction time assessment in monitoring mTBI and concussion (Ruesch, 1944; van Zomeren and Deelman, 1976; MacFlynn et al., 1984; Stuss et al., 1989; Ponsford and Kinsella, 1992; Collins and Long, 1996; Zahn and Mirsky, 1999; Warden et al., 2001; Collins et al., 2003; Sarno et al., 2003; Willison and Tombaugh, 2006; Niogi et al., 2008; Gould et al., 2013; Eckner et al., 2015). More recently, investigators have recognized that reaction time variability is a better indicator for cognitive function than reaction time alone, suggesting that it is much more sensitive to neurological disorders such as concussion (Cole et al., 2018). of scripts. Plant states, “In sum, accuracy has continued to decrease but our conﬁdence in the equipment and the perception of accuracy has risen as computers have become faster and ubiquitous.” The signiﬁcance of this for concussion research is that the inaccuracies introduced to reaction time measures could make it diﬃcult to replicate studies. A study conducted on one computer system will introduce both latency and variability errors—some of which will be unique to the computer system that the online testing is being run on. Even if the computer systems are replicated from one study site to another, the variability of the system will contribute to inaccuracies in the study and make it diﬃcult to replicate. Additionally, these same errors make the reaction time measure untrustworthy as a clinical outcome measure (which could also be signiﬁcantly diﬀerent for an individual testing at multiple locations), and it makes reaction time variability not viable as an individual outcome measure. Although group averages will continue to show general trends (i.e., concussed individuals having reaction times slower than non-concussed individuals) with suﬃciently high numbers of subjects that yield statistical signiﬁcance, an individual measure of reaction time will simply not have the necessary reliability to be counted on as a clinical metric. If the goal of clinical research is to develop and/or improve outcome measures to monitor an individual’s status, then inaccurate measures that are not replicable are a disservice to all. DISCUSSION awareness on the part of authors of such systematic errors, which leads to erroneous or under-reporting of details of the experimental apparatus used to inaccurately collect the data. Thus, by adding inaccurate data and inadequate methodological detail, the replicability crisis is exacerbated, since the data could not be reproduced independently. Systematic errors of this type lead both to the publication of bad data, and to the wider problem of scientiﬁc reproducibility. For a scientiﬁc measure to be valid, it must be both accurate and precise. The problem with commodity-grade computer interfaces is that they may be precise while not being accurate, or they may lack both precision and accuracy because they typically introduce constant or variable non-zero timing oﬀset biases that cannot simply be overcome by “taking more samples” and relying upon the central limit theorem as suggested by Ulrich and Giray (Ulrich and Giray, 1989). Using this approach with any form of systematic bias, additional samples will only render a result that is more precisely inaccurate. The paradox faced by researchers is that while modern commodity-grade human computer interface devices and networking increasingly gain the veneer of smooth glitch-less operation, precise timing tasks in the background are compromised in increasingly subtle ways that are more diﬃcult to detect, quantify, predict, and eliminate. FIGURE 6 \| Comparison of system errors potentially introduced from the four different testing modalities. Note the indicated normative range of reaction time variability for human subjects (5–20 ms; indicated by “+”) is well below the errors introduced by the visual reaction time testing modalities (e.g., “” for systems that use a visual stimulus and touchscreen device such as an iPad and “” for systems that provide a visual stimulus and mouse or keyboard response with a desktop computer or laptop). Could improvements to the accuracy and precision of reaction time testing increase the reliability of computerized assessments? In the authors’ opinion, this is a resounding yes. To address this question, consider the example of mild traumatic brain injury (mTBI), which is just one of many neurological disorders that demonstrate an altered reaction time. DISCUSSION Many factors contribute to the replication crisis across medical research. Plant suggests that within the ﬁeld of psychology, this is likely due in part to hardware and software problems that contribute to timing errors and reproducibility problems between diﬀerent laboratories. Plant further points out that faster hardware has not improved timing accuracy, rather over the years it has apparently gotten worse, and that most researchers simply do not know what their timing accuracy actually is. Further complicating the issue is the fact that web-based studies have become increasingly common and have introduced several new sources of inaccuracy, including server load and caching has become alarmingly widespread, while the understanding of how well they work and how accurate and reliable they are has dwindled signiﬁcantly. This trend was ﬁrst pointed out with respect to the use of computer mice by Beringer (1992), more recently described by Plant et al. (2002, 2003), then by Plant et al. (2004), and then again in 2009 (Plant and Turner), the latter after the use of mobile “smart” devices had begun its exponential rise. In their 2009 paper, Plant and Turner updated their earlier ﬁndings and observed that the trend had not improved. They noted that millisecond precision is a very diﬀerent thing from millisecond accuracy. Even with newer human interface technologies, timing accuracy has not enjoyed the same priority and improvements over time as cost reduction. They conclude that, “It is important to note that the fact that hardware and software produce answers that “look accurate” does not mean that those answers are valid.” In 2016 Plant (Plant, 2016) again emphasizes that millisecond timing accuracy errors are prevalent throughout the psychology October 2020 \| Volume 14 \| Article 509091 Frontiers in Human Neuroscience \| www.frontiersin.org 7 Methodological Problems With Concussion Testing Holden et al. FIGURE 6 \| Comparison of system errors potentially introduced from the four different testing modalities. Note the indicated normative range of reaction time variability for human subjects (5–20 ms; indicated by “+”) is well below the errors introduced by the visual reaction time testing modalities (e.g., “” for systems that use a visual stimulus and touchscreen device such as an iPad and “**” for systems that provide a visual stimulus and mouse or keyboard response with a desktop computer or laptop). Frontiers in Human Neuroscience \| www.frontiersin.org How Good an Outcome Measure Is Reaction Time Testing for Concussion? Although there have been numerous reports that used reaction time as an outcome measure for concussion studies, the results & conclusions from those ﬁndings vary widely. Some reports described little or no diﬀerence between the reaction time of concussed and non-concussed individuals (Iverson et al., 2004; Straume-Naesheim et al., 2005), and some have even reported that concussed individuals have faster reaction times than non- concussed individuals (Lynall et al., 2018; Iverson et al., 2019; Norman et al., 2019). In one study, comparison of reaction time measures showed no correlation between the metric obtained with several diﬀerent computerized methods (Schatz and Putz, 2006) even though the methods were all targeting simple reaction time from the same cohort. The authors’ interpretation of these ﬁndings is that they reﬂect what could happen with poorly administered and inaccurate testing methods. On the other hand, a number of reports have demonstrated utility for reaction time as a concussion assessment (Warden et al., 2001; Willison and Tombaugh, 2006; Cole et al., 2018; Danna-Dos-Santos et al., 2018; Lange et al., 2018). Norris et al. (2013) described the reaction time metric as having prognostic utility and others have proposed that reaction time could be used in the absence of baseline measures by just comparing to normative values (Schmidt et al., 2012), and the authors share that opinion based on our recent work. For example, note the ROC curve in Figure 7 (reproduced from Favorov et al., 2019) that shows the area under the receiver operating characteristic (ROC) curve to be 0.69 for predicting concussed status based on tactile reaction time with the same conﬁguration described in the methods section in this report. While this is not a perfect outcome measure by itself, it indicates good sensitivity and speciﬁcity and makes a good argument to use the reaction time metric as an aid in the assessment of concussed individuals. More signiﬁcantly, the reaction time variability metric (also referenced as intraindividual variability in some reports and an obvious by-product of collecting reaction time), appears to be a very good predictor of concussion in the cohort studied (note AUC of 0.91). Other investigators have also pointed out that reaction time variability could be an extremely useful tool in assessments of neurological disorders (Collins et al., 1999; MacDonald et al., 2003, 2006) including concussion (Cole et al., 2018). DISCUSSION From a broader perspective, systematic instrumentation errors that result from the misuse and misunderstanding of basic research test equipment leads directly to one and/or two outcomes: (1) data with signiﬁcant absolute errors, which may or may not be internally replicable enough to allow the use of data variability as a useful metric, and subsequently adds more erroneous data to the body of peer-reviewed literature, and (2) lack of The evaluation of individuals who have sustained mild traumatic brain injury has been growing in prominence in the public forum, with much of this debate arising from the widespread inadequacy of the methods commonly used to assess cognitive function and the neurological insults that are caused by mTBI. One of the measures that is commonly obtained by most online cognitive assessment tools is simple reaction time, yet very few of these online assessment tools have the capacity to evaluate the metric accurately, much less the capacity to evaluate reaction time variability. Reaction time variability has a normative range of 10–20 msec, which simply cannot be measured by systems that have variable latency ranges of 84 msec. Normative reaction time is in the 200–220 msec range (Zhang et al., 2011; Favorov et al., 2019; Pearce et al., 2019), so introduction of this amount of error is also signiﬁcant. Additionally, many of these assessments are performed on multiple computers and operating systems for the same subject, which can lead to errors caused by inconsistencies October 2020 \| Volume 14 \| Article 509091 8 Methodological Problems With Concussion Testing Holden et al. between diﬀerent systems. As mentioned above, reaction time variability appears to be a more important measure for mTBI assessment than reaction time, and given the extreme sensitivity of this measure to the timing errors introduced by commodity grade computing, the need for improved accuracy is signiﬁcant. FIGURE 7 \| ROC curves for reaction time and reaction time variability from a recent study comparing concussed to non-concussed individuals. Reproduced with modiﬁcation and with permission from (Favorov et al., 2019). Frontiers in Human Neuroscience \| www.frontiersin.org How Good an Outcome Measure Is Reaction Time Testing for Concussion? tactile reaction time predictive of ﬁndings in other studies? In a separate study that compared observations from individuals with persistent post-concussion symptoms (PPCS) and healthy controls with both tactile reaction time (collected with the Brain Gauge) and visual reaction time (collected with CogState, a commercial program), there was a diﬀerence in the mean visual reaction time and the mean tactile reaction time of the healthy controls of slightly over 80 msec (Pearce et al., 2020). Does this mean that subtracting the delay introduced with the visual reaction time task will then provide an accurate measure? The answer is a resounding no. The increased variability of the visual reaction time measure—which is eﬀectively random— leads to inaccuracies that make it ineﬀective. In that same study, there was a statistically signiﬁcant diﬀerence of the tactile reaction time collected from healthy controls and individuals It is possible that the diﬀerences in tactile vs. visual reaction times could be partially accounted for by biological diﬀerences, but it appears to be, from the authors point of view, primarily technology based. Two of the authors (Oleg Favorov and Mark Tommerdahl) had several discussions with the late Steve Hsiao (Johns Hopkins University) in the 1990s about diﬀerences between the somatosensory and visual systems. Steve was of the opinion that object recognition takes place in diﬀerent sensory modalities (such as touch and vision), but projects to the same decision center (Hsiao, 1998), and any biological diﬀerences between visual and tactile reaction time should be limited. This may seem surprising to many, simply because most reported visual reaction times are slower than reported tactile reaction times (e.g., the afore mentioned Pearce et al., 2020 study reported that visually based reaction times were 95 msec slower than tactile times for healthy controls), and based on the data presented in this report, the diﬀerences in reaction time that are often reported between visual and tactile are most likely methodological. Technological diﬀerences aside, the tactile component of the somatosensory system may also be a better vehicle for delivering reaction time testing and probably aﬀords a vehicle for delivering better assessments and research results with lower inter-subject variability. From an engineering perspective, there is a much better signal-to-noise ratio for inputs through the somatosensory system to be much higher ﬁdelity simply because of the lack of environmental noise. How Good an Outcome Measure Is Reaction Time Testing for Concussion? Interestingly, one report stated that although this variability had been demonstrated in a number of reports to be a good indicator of neurological dysfunction, it stated that concussion was not in the same category as other brain insults because reaction time variability was not altered in the concussed individuals that they studied (Sosnoﬀet al., 2007). The methods of that report stated that an online cognitive test was administered on a computer—no details as to resolution were given—and given the results of this FIGURE 7 \| ROC curves for reaction time and reaction time variability from a recent study comparing concussed to non-concussed individuals. Reproduced with modiﬁcation and with permission from (Favorov et al., 2019). report, the authors suspect that the resolution of that experiment was inadequate to obtain results accurate enough to evaluate reaction time variability. There has been a growing interest in the long-term impact of concussions and the symptoms that often linger post-concussion. Research into persistent post-concussion symptoms (PPCS) has mostly investigated the ongoing eﬀects on cognitive functioning, such as working memory, attention and concentration, and executive functioning. However, interest in the sensory-motor system in quantifying ongoing concussion symptoms has emerged in the last decade (De Beaumont et al., 2007, 2011; Johansson et al., 2009; Pearce et al., 2014, 2015), and accurate measures of reaction time and reaction time variability make it possible to detect an altered state in this population. Recently we demonstrated in people with PPCS that those with ongoing self-reported symptoms had signiﬁcantly slower reaction times and had signiﬁcantly higher reaction time variabilities compared to both those who had fully recovered from a concussion and age-matched healthy controls (Pearce et al., 2019). Additionally, the study reported that reaction time variability co-varied with fatigue of the individuals with PPCS and thus highlights the impact that a sensitive measure could have in post- concussion assessments. Although this report focuses on the technological diﬀerences in commercially available tactile vs. visual methodology in reaction time testing, it does prompt speculation on the diﬀerences that would be based on biological diﬀerences. In this paper, we did not use any data from human subjects, so that those diﬀerences would not have impact on the results. October 2020 \| Volume 14 \| Article 509091 9 Methodological Problems With Concussion Testing Holden et al. with PPCS, but there was no diﬀerence detected with the visual reaction time. CONCLUSIONS The results suggest that some of the commonly used visual tasks on consumer grade computers could be (and have been) introducing signiﬁcant errors for reaction time testing and that dedicated hardware designed for the reaction time task is needed to minimize testing errors. How Good an Outcome Measure Is Reaction Time Testing for Concussion? There was also a signiﬁcant diﬀerence with tactile reaction time variability, but the visual reaction time variability could not be evaluated. However, there have been multiple publications that examined the results from reaction time testing with the tactile based approach described here, and these results demonstrated that reaction time and reaction time variability can be very useful for testing concussed individuals. Favorov et al. (2019) demonstrated that reaction time testing with the Brain Gauge can diﬀerentiate concussed from non-concussed individuals: using the ROC (Receiver Operating Characteristic) curve analysis, they found the area under the curve AUC = 91% for reaction time variability and AUC = 69% for reaction time, which suggests that reaction time variability is much more sensitive to that condition. Similarly, another study showed reaction time and reaction time variability (with the Brain Gauge) to diﬀerentiate 3 groups of individuals—healthy controls, individuals that had recovered from concussion and individuals with chronic symptoms of concussion (Pearce et al., 2019). Data from a large cohort of individuals with sports concussion were examined and also demonstrated that diﬀerences in mean values of reaction time and reaction time variability (with the Brain Gauge) are statistically highly signiﬁcant between healthy control and post- concussion populations (p < 10−15; Tommerdahl et al., 2020). The diﬀerence in reaction times of the tested groups was ∼30% while the diﬀerence in reaction time variability was over 80%, again strongly suggesting that reaction time variability is a much more sensitive measure for detecting alterations of information processing speed due to concussion than reaction time. Reports of visual reaction time testing have simply not demonstrated such levels of discriminability, which leads to the question: how do tactile based results with the Brain Gauge directly compare with those obtained from visual reaction time testing? Using the same methods described in this report (that were done robotically), comparison of tactile reaction time with the Brain Gauge was compared with visual reaction time with healthy controls. From the data presented in this paper, the expected diﬀerence in the tactile vs. visual reaction times should be ∼80 msec, and the results in the study with healthy controls was slightly over that value (∼85 msec; Kim et al., 2020), and the measured reaction time variability for that cohort was 16 msec vs. 81 msec (tactile vs. visual). This ﬁnding leads to the next question: is that diﬀerence in visual vs. How Good an Outcome Measure Is Reaction Time Testing for Concussion? Inputs via somatosensation do not have to compete with other inputs, which is in sharp contrast to the testing environment that most visual tests contend with. As pointed out by Leonard (1959), “..stimulation of the ﬁngers seems to be the answer” to obtaining an accurate measure of pure reaction time with minimal distractions. 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EF and RL analyzed the data and generated the ﬁgures. BK built supporting hardware for the project. JH wrote the software. BK and JH wrote the methods and results section. BK, EF, and JH conducted the experiments. AT provided background research and provided signiﬁcant input to the introduction and discussion. RGD and MT wrote the introduction and discussion. AJP and OVF provided important contributions to the discussion. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnhum. 2020.509091/full#supplementary-material REFERENCES A., Sarkar, R., et al. (2008). Extent of microstructural white matter injury in postconcussive syndrome correlates with impaired cognitive reaction time: a 3T diﬀusion tensor imaging study of mild traumatic brain injury. Am. J. Neuroradiol. 29, 967–973. doi: 10.3174/ajnr.A0970 Schmidt, J. D., Register-Mihalik, J. K., Mihalik, J. P., and Kerr, Z. Y., Guskiewicz, K. M. (2012). 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Psychol. 42, 1–12. doi: 10.1111/j.2044-8317.1989.tb01111.x October 2020 \| Volume 14 \| Article 509091 Frontiers in Human Neuroscience \| www.frontiersin.org 12 Holden et al. Methodological Problems With Concussion Testing Zhang, Z., Francisco, E., Holden, J., Dennis, R., and Tommerdahl, M. (2011). Somatosensory information processing in the aging population. Front. Aging Neurosci. 3:18. doi: 10.3389/fnagi.2011. 00018 van Zomeren, A. H., and Deelman, B. G. (1976). Diﬀerential eﬀects of simple and choice reaction after closed head injury. Clin. Neurol. Neurosurg. 79, 81–90. doi: 10.1016/0303-8467(76)90001-9 Warden, D. L., Bleiberg, J., Cameron, K. L., Ecklund, J., Walter, J., et al. (2001). Persistent prolongation of simple reaction time in sports concussion. Neurology 57, 524–526. doi: 10.1212/WNL.57.3.524 Conﬂict of Interest: JH, EF, AT, RL, BK, RD, and MT were employed by company Cortical Metrics LLC. LZ was employed by company Lucent Research. Willison, J., and Tombaugh, T. N. (2006). Detecting simulation of attention deﬁcits using reaction time tests. Arch. Clin. Neuropsychol. 21, 41–52. doi: 10.1016/j.acn.2005.07.005 The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Woodley, M. A., Te Nijenhuis, J., and Murphy, R. (2013). Were the Victorians cleverer than us? The decline in general intelligence estimated from a meta- analysis of the slowing of simple reaction time. Intelligence 41, 843–850. Frontiers in Human Neuroscience \| www.frontiersin.org October 2020 \| Volume 14 \| Article 509091 REFERENCES doi: 10.1016/j.intell.2013.04.006 Copyright © 2020 Holden, Francisco, Tommerdahl, Lensch, Kirsch, Zai, Pearce, Favorov, Dennis and Tommerdahl. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Woods, D. L., Wyma J. M., Yund, E. W., William, Herron, T. J., and Reed, B. (2015). Factors inﬂuencing the latency of simple reaction time. Front. Hum. Neurosci. 9:131. doi: 10.3389/fnhum.2015.00131 Zahn, T. P., and Mirsky, A. F. (1999). Reaction time indicators of attention deﬁcits in closed head injury. J. Clin. Exp. Neuropsychol. 21, 352–367. doi: 10.1076/jcen.21.3.352.924 October 2020 \| Volume 14 \| Article 509091 Frontiers in Human Neuroscience \| www.frontiersin.org 13
https://openalex.org/W4315471816	https://www.frontiersin.org/articles/10.3389/fimmu.2022.1088886/pdf	English	null	The application basis of immuno-checkpoint inhibitors combined with chemotherapy in cancer treatment	Frontiers in immunology	2,023	cc-by	9,334	TYPE Mini Review PUBLISHED 10 January 2023 DOI 10.3389/fimmu.2022.1088886 TYPE Mini Review PUBLISHED 10 January 2023 DOI 10.3389/fimmu.2022.1088886 TYPE Mini Review PUBLISHED 10 January 2023 DOI 10.3389/fimmu.2022.1088886 TYPE Mini Review PUBLISHED 10 January 2023 DOI 10.3389/fimmu.2022.1088886 OPEN ACCESS Ming-Yan Shi †, Han-Ge Liu †, Xiao-Hong Chen, Ye Tian, Zhi-Nan Chen* and Ke Wang* Ming-Yan Shi †, Han-Ge Liu †, Xiao-Hong Chen, Ye Tian, Zhi-Nan Chen* and Ke Wang* National Translational Science Center for Molecular Medicine & Department of Cell Biology, Fourth Military Medical University, Xi’an, China Immuno-checkpoint inhibitors (ICIs) bring a promising prospect for patients with cancers, which restrains the growth of tumor cells by enhancing anti- tumor activity. Nevertheless, not all patients beneﬁt from the administration of ICIs monotherapy. The partial response or resistance to ICIs is mainly due to the complex and heterogenous tumor microenvironment (TME). The combined therapy is necessary for improving the efﬁcacy of tumor treatment. Chemotherapy is reported not only to kill tumor cells directly, but also to stimulate effective anti-tumor immune responses. Several combined therapies of ICIs and chemotherapeutic agents have been approved for the ﬁrst-line treatment of cancers, including PD-1/PD-L1 inhibitors. This review summarizes the potential mechanisms of the combined therapy of ICIs and chemotherapeutic agents in inducing immunogenic cell death (ICD) and reprogramming TME, and elucidates the possible anti-tumor effects of combined therapy from the perspective of metabolic reprogramming and microbiome reprogramming. COPYRIGHT © 2023 Shi, Liu, Chen, Tian, Chen and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. OPEN ACCESS OPEN ACCESS EDITED BY Jian Zhang, Southern Medical University, China REVIEWED BY Jake ODonnell, The University of Queensland, Australia Dalil Hannani, Grenoble (TIMC-IMAG), France Jian Qiao, University of Texas Southwestern Medical Center, United States CORRESPONDENCE Ke Wang wangke@fmmu.edu.cn Zhi-Nan Chen znchen@fmmu.edu.cn †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology RECEIVED 03 November 2022 ACCEPTED 20 December 2022 PUBLISHED 10 January 2023 CITATION Shi M-Y, Liu H-G, Chen X-H, Tian Y, Chen Z-N and Wang K (2023) The application basis of immuno- checkpoint inhibitors combined with chemotherapy in cancer treatment. Front. Immunol. 13:1088886. doi: 10.3389/fimmu.2022.1088886 COPYRIGHT © 2023 Shi, Liu, Chen, Tian, Chen and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice No use distribution or OPEN ACCESS EDITED BY Jian Zhang, Southern Medical University, China REVIEWED BY Jake ODonnell, The University of Queensland, Australia Dalil Hannani, Grenoble (TIMC-IMAG), France Jian Qiao, University of Texas Southwestern Medical Center, United States CORRESPONDENCE Ke Wang wangke@fmmu.edu.cn Zhi-Nan Chen znchen@fmmu.edu.cn †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology RECEIVED 03 November 2022 ACCEPTED 20 December 2022 PUBLISHED 10 January 2023 CITATION Shi M-Y, Liu H-G, Chen X-H, Tian Y, Chen Z-N and Wang K (2023) The application basis of immuno- checkpoint inhibitors combined with chemotherapy in cancer treatment. Front. Immunol. 13:1088886. doi: 10.3389/fimmu.2022.1088886 COPYRIGHT © 2023 Shi, Liu, Chen, Tian, Chen and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. immuno-checkpoint inhibitors, chemotherapy, tumor microenvironment, immunogenic cell death, anti-tumor activity The application basis of immuno-checkpoint inhibitors combined with chemotherapy in cancer treatment OPEN ACCESS EDITED BY Jian Zhang, Southern Medical University, China REVIEWED BY Jake ODonnell, The University of Queensland, Australia Dalil Hannani, Grenoble (TIMC-IMAG), France Jian Qiao, University of Texas Southwestern Medical Center, United States *CORRESPONDENCE Ke Wang wangke@fmmu.edu.cn Zhi-Nan Chen znchen@fmmu.edu.cn †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology RECEIVED 03 November 2022 ACCEPTED 20 December 2022 PUBLISHED 10 January 2023 CITATION Shi M-Y, Liu H-G, Chen X-H, Tian Y, Chen Z-N and Wang K (2023) The application basis of immuno- checkpoint inhibitors combined with chemotherapy in cancer treatment. Front. Immunol. 13:1088886. doi: 10.3389/fimmu.2022.1088886 COPYRIGHT © 2023 Shi, Liu, Chen, Tian, Chen and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Introduction Malignant tumors, as a type of incurable diseases, have threatened human health seriously owing to the immunosuppressive tumor microenvironment. The tumor immunotherapies dramatically make a monumental breakthrough for cancer treatment and bring signiﬁcant improvement for patient survival by boosting effective immune response to eliminate malignant cells (1, 2). Oncolytic virus therapies, cancer vaccines, cytokine therapies, adoptive cell transfer therapies, and ICIs are included, and ICIs, which include inhibitors of the programmed cell death protein 1 (PD-1), Frontiers in Immunology frontiersin.org 01 Shi et al. Shi et al. 10.3389/ﬁmmu.2022.1088886 10.3389/ﬁmmu.2022.1088886 programmed cell death-ligand 1 (PD-L1), and the cytotoxic T lymphocyte-associated protein 4 (CTLA-4), have been broadly used in clinical applications and contribute to prolonged survival for lung cancer patients (2–5). In this review, we summarize the synergetic effects of chemotherapy with ICI therapy in tumor treatment from the perspective of inducing ICD, remodeling TME, metabolic reprogramming, and microbiome reprogramming (Figure 1). In addition, further researches need to be conducted to explore the novel mechanisms of above-mentioned therapy in cancers, which may provide a solid foundation for future clinical applications. However, not all patients beneﬁt from immune checkpoint blockade therapy (6, 7). Based on response to ICIs, three broad population of patients are identiﬁed, which include responders, those that acquire resistance, and those that never respond (8– 10). Unfortunately, only 20% of NSCLC patients response to ICIs, which shows a relatively lower clinical effect compared to other cancers (7). The complex and heterogenous TME is reported to be involved in the response to ICIs (11, 12). Based on the status of T cell inﬁltration, TME is classiﬁed as the immune-inﬂamed phenotype, the immune-excluded phenotype, and the immune-desert phenotype (13, 14). The reactivation and clonal-proliferation of antigen-experienced T cells in the TME are necessary for an effective anti-tumor response after ICI administration. Nevertheless, the tumor-associated macrophages (TAMs), cancer-associated ﬁbroblasts (CAFs), myeloid-derived suppressor cells (MDSCs), and regulated T cells (Tregs) in TME have an inhibitory impact on the inﬁltration and activation of effector T cells (15). As a result, ICIs combined with other therapies that activate the immune effects of TME seems to be a better choice for tumor treatment. As of December 2021, 4,897 clinical trials are conducted to test the efﬁcacy of PD-1/PD-L1 inhibitors. Among them, 83% are ICIs combined with other therapies, such as chemotherapy, radiotherapy, and other immuno-oncology therapies (16). Introduction Several clinical trials show that ICIs combined with chemotherapy have a better clinical effect compared to ICIs monotherapy (17–19). For example, pembrolizumab, a humanized monoclonal antibody against PD-1, plus platinum- based chemotherapy signiﬁcantly improved overall survival rates of NSCLC patients with a PD-L1 ≥50% and negative for genomic alterations in the EGFR and ALK genes, compared with pembrolizumab monotherapy (19). Meanwhile, ICIs combined with chemotherapy also prolong the NSCLC patient survival, compared with chemotherapy (20–23), which have been approved for the ﬁrst-line treatment in advanced NSCLC patients (24). Inducing immunogenic cell death Several studies have shown that chemotherapeutic agents have the ability of inducing ICD in animal experiments, including anthracyclines, cyclophosphamide, oxaliplatin, pemetrexed, and paclitaxel (25–30). For example, anthracyclines, including doxorubicin, idarubicin and mitoxantrone, are identiﬁed as ICD inducers in the mouse models of colorectal cancer (27), and cyclophosphamide can also induce ICD as shown in glioma mouse models (30). In addition, single-agent pemetrexed or docetaxel can induce ICD in 16 NSCLC patients, with increasing plasma concentration of soluble calreticulin (31). ICD is a form of regulated cell death, which activates an adaptive immune response in immunocompetent hosts (32, 33). The hallmarks of ICD include the exposure and release of numerous damage- associated molecular patterns (DAMPs), the phosphorylation of eukaryotic translation initiation factor 2 subunit-a (eIF2a), and the activation of type I IFN signaling and autophagy. Among them, DAMPs play a vital role in stimulating adaptive immune response, which contain increasing extracellular ATP, surface-exposed calreticulin, and released high mobility group box protein 1 (HMGB1) (34, 35). Increasing extracellular ATP, which provides a ‘ﬁnd me’ signal, attracts antigen-presenting cells through binding with the purinergic receptors (36, 37) and stimulates dendritic cell (DC) maturation (38–41). Moreover, ATP triggers the formation of the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex (42), which promotes the secretion of IL-1b and IL-18 (43), to stimulate adaptive FIGURE 1 Chemotherapeutic agents enhance the anti-tumor activity of ICIs through several mechanisms, including inducing immunogenic cell death, changing the proportion and activity of immune cells in TME, immune cell metabolic reprogramming, and microbiome reprogramming. herapeutic agents enhance the anti-tumor activity of ICIs through several mechanisms, including inducing immunogenic cell death, g the proportion and activity of immune cells in TME, immune cell metabolic reprogramming, and microbiome reprogramming. frontiersin.org 02 Frontiers in Immunology Shi et al. 10.3389/ﬁmmu.2022.1088886 immune response (44). In addition, the P2X7 receptor (P2X7R), is expressed on various immune cells, and its expression sensitizes cells to enhanced ambient ATP concentrations, which modulates energy metabolism and T cell growth and differentiation (45). The exposure of calreticulin from endoplasmic reticulum to cell surface depends on the rapid phosphorylation of eIF2a and ERp57 (46), and provides an ‘eat me’ signal to promote phagocytosis by DCs, provoking the adaptive immune response (27, 47). Inducing immunogenic cell death In summary, the use of chemotherapeutic agents impairs the immunosuppressive role of TME by reducing the number of immunosuppressive cells, which enhances the anti- tumor activity. to cell apoptosis (65). Moreover, cyclophosphamide also decreases the amount of intra-tumoral Tregs in NSCLC mouse model (66), which is also identiﬁed in patients with recurrent prostate cancer (67). In addition, the amount of MDSCs, another immunosuppressive cell that helps tumor cells evade immune destruction (68, 69), is decreased after the administration of docetaxel, gemcitabine and 5-ﬂuorouracil in mouse experiments (70–73). As shown in mouse models of melanoma (B16F10), gemcitabine signiﬁcantly reduces the immunosuppressive state by decreasing the size of MDSCs and Tregs (70). Moreover, the number of circulating MDSCs in patients with pancreatic cancer decreases after the administration of gemcitabine, which provides precise clinical evidence (74), which is also validated in glioblastoma patients (75). The combination of gemcitabine and cisplatin reduce the amount of Tregs and regulatory B cells in in nasopharyngeal carcinoma patients (76). In addition, cyclophosphamide plus gemcitabine combination chemotherapy reduces the immunosuppressive state through decreasing the number of Tregs and MDSCs, enhancing anti- tumor immune response in colon carcinoma-bearing mice (63). In summary, the use of chemotherapeutic agents impairs the immunosuppressive role of TME by reducing the number of immunosuppressive cells, which enhances the anti- tumor activity. In addition, several animal studies have validated that some chemotherapeutic agents, including platinum drugs and docetaxel, have the ability to promote the inﬁltration of CD8+ T cells, enhancing their anti-tumor effects (53, 60, 77–79). It has been reported that the administration of cisplatin and docetaxel increases intra-tumoral CD8+ T cell inﬁltration in a phase I/II study of neoadjuvant chemotherapy for resectable NSCLC (80). The increasing CD8+ T cell inﬁltration contributes to enhancing the anti-tumor activity and limits the tumor progression (81). Meanwhile, further studies explaining the mechanisms that platinum drugs increase the inﬁltration of CD8+ T cells have been conducted. For example, oxaliplatin enhances the secretion of CXCL9, CXCL10, and CXCL11 from tumor cells, which attracts CD8+ effector T cells through interacting with CXCR3 (82, 83), and subsequently promotes T cell inﬁltration in tumor tissues. In addition, neoadjuvant chemotherapy increases the inﬁltration of tissue resident memory T cells (TRMs) in resectable NSCLC patients, which will provide long-term anti- tumor immune response (84). Inducing immunogenic cell death TRMs express high levels of inhibitory receptors, such as PD-1 and Tim-3, and it has been validated that TRMs show a signiﬁcant expansion and enhancing cytotoxic capacity after the administration of PD-1 inhibitors (85–88). Moreover, cisplatin can increase the inﬁltration of CD8+ T cells via activating cGAS-STING signaling in K-ras-driven tumor cells (77), and enhance the killing effects of CD8+ T cells through Fas/Fas ligand interactions in NSCLC mouse model (89), which may provide an inﬂammatory environment to enhance the anti-tumor activity of ICIs. Inducing immunogenic cell death However, mutant calreticulin with loss of the KDEL sequence is secreted to extracellular space, and may inhibit the phagocytosis of dying cancer cells by DCs through saturating binding sites on DCs (48). Moreover, the interaction between calreticulin and toll-like receptor 4 (TLR4) expressed on tumor cell surface promotes the secretion of TNFa and CCL19, which facilitates the migration and maturation of DCs, to limit the tumor progression in vivo (49). HMGB1, released from dying cancer cells, binds with TLR4 expressed on DCs to strengthen the antigen-presenting activity of DCs through activating the PI3K/AKT/mTOR signaling pathway, and promotes anti-tumor immune response of T cells (50, 51). Different HMGB1 isoforms exert different effects on immune response, and the reduced form is responsible for the activation of DCs (52). In addition, the administration of carboplatin, cisplatin, and gemcitabine increases the PD-L1 expression on tumor cells, and shows a better efﬁcacy when combining with ICIs in NSCLC mouse models (53–55), in which the role of cisplatin has been validated in human NSCLC tumor samples (56). Moreover, cisplatin increases the expression level of MHC class I antigen on tumor cells, and subsequently augment CTL-mediated attack to tumor cells (57, 58). In summary, chemotherapy-induced ICD promotes the cross- presentation of tumor antigen to CD8+ cytotoxic T lymphocytes (CTLs), which limits tumor progression effectively. to cell apoptosis (65). Moreover, cyclophosphamide also decreases the amount of intra-tumoral Tregs in NSCLC mouse model (66), which is also identiﬁed in patients with recurrent prostate cancer (67). In addition, the amount of MDSCs, another immunosuppressive cell that helps tumor cells evade immune destruction (68, 69), is decreased after the administration of docetaxel, gemcitabine and 5-ﬂuorouracil in mouse experiments (70–73). As shown in mouse models of melanoma (B16F10), gemcitabine signiﬁcantly reduces the immunosuppressive state by decreasing the size of MDSCs and Tregs (70). Moreover, the number of circulating MDSCs in patients with pancreatic cancer decreases after the administration of gemcitabine, which provides precise clinical evidence (74), which is also validated in glioblastoma patients (75). The combination of gemcitabine and cisplatin reduce the amount of Tregs and regulatory B cells in in nasopharyngeal carcinoma patients (76). In addition, cyclophosphamide plus gemcitabine combination chemotherapy reduces the immunosuppressive state through decreasing the number of Tregs and MDSCs, enhancing anti- tumor immune response in colon carcinoma-bearing mice (63). ICIs combined with chemotherapy in various cancers Considering the limitations of ICIs monotherapy in controlling tumor progression, a large amount of studies are devoted to the safety and effectiveness of ICIs combined with standard-of-care chemotherapies. ICIs combined with chemotherapy has been approved for the treatment of certain cancer types by FDA, and more than 600 ongoing clinical trials are devoted to exploring or optimizing for various oncological indications. In patients with NSCLC, pembrolizumab plus a platinum and pemetrexed provides a better prognosis compared to pembrolizumab monotherapy (113), owing to the immune activation of these chemotherapy drugs. Similar effects are also observed in patients with untreated locally incurable recurrent or metastatic head and neck squamous cell carcinoma treated with pembrolizumab plus a platinum and 5-ﬂuorouracil (114, 115). In addition, atezolizumab plus carboplatin and nab- paclitaxel prolongs the overall survival and progression-free survival in patients with advanced non-squamous NSCLC (116). Neoadjuvant carboplatin and paclitaxel chemotherapy increases the amount of central memory CD8+ T cell in peripheral blood of patients with advanced serous ovarian cancer, enhancing antigen processing and presentation (117). However, the further studies are needed to investigate the corresponding mechanisms of neoadjuvant carboplatin and paclitaxel chemotherapy in inhibiting NSCLC progression. Although ICIs combined with chemotherapy signiﬁcantly prolongs the survival of patients with tumors, it is necessary for us to evaluate the toxicity of the combination therapies. ICIs combined with chemotherapy may cause hematological, gastrointestinal, and renal toxicity, and contribute to hypothyroidism, hyperthyroidism, pneumonitis, hepatitis, Changing the proportion and activity of immune cells in TME Therefore, the microbiome reprogramming induced by chemotherapy may provoke the anti-tumor effects of ICIs. However, the inﬂuence of chemotherapy on local microbiome needs to be investigated further, and the impacts of chemotherapy on ICI administration are required to be validated in clinical researches from the perspective of microbiome reprogramming. Immune cell metabolic reprogramming As a crucial hallmark of cancer, metabolism reprogramming provides a favorable immunosuppressive microenvironment for the tumor progression (101–103). Chemotherapy usually inﬂuences patients’ nutritional status, and the serum of patients with lung cancer is accompanied by metabolic alterations, including glycolysis and lipid metabolism, phosphatidylcholine biosynthesis as well as amino acid metabolism (104). What’s more, metabolic reprogramming is closely involved in the activation and of T cells. For example, the activation of T cells needs higher levels of glycolysis and mitochondrial respiration (105). Thus, chemotherapy may inﬂuence the immune effects of T cells through metabolic processes. Pemetrexed has been validated to increase mitochondrial function of T cells in colon cancer mouse model, which is necessary for the activation of T cells (26, 106). Nevertheless, there is little researches about the inﬂuence of other chemotherapeutic agents on metabolic reprogramming of immune cells, and further metabolomics study is necessary to explore this inﬂuence in preclinical and clinical studies. Changing the proportion and activity of immune cells in TME Chemotherapeutic agents have been validated that they can interact directly with immune cells to stimulate anticancer immunity through several mechanisms, which changes the inﬁltration and activity of immune cells in TME, including the depletion of immunosuppressive cells, the activation of immune effector cells, and promoting the proliferation of immune cells. Tumor-inﬁltrating Tregs promote tumor progression by inhibiting endogenous cytotoxic T cell responses (59), and it has been reported that some chemotherapy drugs can decrease the amount of Tregs (60–63). For example, the frequencies of intra-tumoral Tregs decreases signiﬁcantly after the pretreatment of paclitaxel and cisplatin in a murine lung carcinoma model (64). Paclitaxel selectively decreases the size of Treg population in peripheral blood of patients with NSCLC, which may promote the upregulation of CD95 on Tregs, leading Frontiers in Immunology frontiersin.org 03 Shi et al. Shi et al. 10.3389/ﬁmmu.2022.1088886 Chemotherapeutic agents also increase the inﬁltration of APCs in tumor tissues, such as DCs and macrophages, and enhances the anti-tumor immune response. Anthracycline- based chemotherapy increases the intra-tumoral inﬁltration of DCs in ﬁbrosarcoma and breast cancer mouse models (90, 91). A prospective study suggests that the responsiveness of DCs recovers after anthracycline-based neoadjuvant chemotherapy in breast cancer patients (92). Except for above mentioned ATP signaling, CCL2/CCR2 axis may also be required for the intratumoral recruitment of DCs (90). In addition, platinum (IV) complexes increase the inﬁltration of M1 macrophages by decreasing the expression of CD47 in lung cancer mouse model, which is overexpressed on tumor cells and limits the antigen- presenting activity of APCs (93, 94). DCs play a vital role in maintaining CD8+ T cell function within tumors, and promote ICIs mediated anti-tumor immunity (95, 96). Furthermore, DCs may license PD-1 blockade via CD28 costimulation (97), and conventional DCs express genes correlated with CXCL9, which is related to PD-1 inhibitor response (98–100). In summary, chemotherapy-induced APCs increase in tumor tissues may contribute to ICI-enhanced anti-tumor activity. various mechanisms (107), and is closely correlated with the efﬁcacy of ICIs in cancer treatment (108–110). Chemotherapy has been validated to change the proportion of gut microbiome. The abundance of the Firmicutes phylum and Enterobacteriaceae increase after the administration of pemetrexed in the patient-lung-derived tumor xenograft mouse models (111). The selected species of Gram-positive bacteria are induced into the secondary lymphoid organs by cyclophosphamide, and stimulates the memory Th1 immune responses, which promotes the anti-tumor immune response (112). Discussion ICIs have made great contributions to the survival of patients with cancers. However, the low response rate of patients to ICIs prompts us to explore the possibility of ICIs combination with other therapies. As a routine therapy, chemotherapy attracts much attention because of its immune stimulation activity, and ICIs combination with chemotherapy achieves great effects in clinical trials. FDA has approved several combination therapies for the treatment of advanced NSCLC in the ﬁrst-line setting (122). To clarify the mechanism how chemotherapy promotes curative effect of ICIs is beneﬁcial for the application of the combination therapies. As mentioned above, chemotherapeutic agents-induced ICD and their immune stimulation activity are considered as the main mechanism of combination therapy. However, the impacts of chemotherapy are so complex that chemotherapy may inﬂuence ICI-mediated anti-tumor responses through various routes. In addition, the administration approaches of ICIs combination with chemotherapy are necessary to be improved. Firstly, a proper combination therapy can maximize the clinical beneﬁt and minimize the adverse drug reactions. As mentioned above, different chemotherapy drugs stimulate effective anti- tumor immune responses through different mechanisms, and cancer patients may reap more beneﬁts with appropriate combination therapies after evaluating the tumor conditions, including PD-L1 expression level, immune cell inﬁltration, and tumor mutation burden. However, there is little clinical trials about comparing the effectiveness of combination therapies of different chemotherapeutic drugs and ICIs. Secondly, chemotherapeutic regimens and ICIs are administrated simultaneously in most clinical trials. Nevertheless, it has reported that the sequence of administrating chemotherapeutic agents and ICIs has an impact on the efﬁciency of the combined therapies in several animal experiments. The administration of anti‐CTLA‐4 antibody after injecting cyclophosphamide signiﬁcantly inhibits the tumor progression in the CT26 colon carcinoma model, while the reverse administration sequence leads to the apoptosis of anti-tumor CD8+ T cells (130). Apart from this, the time interval from chemotherapy to immunotherapy may also inﬂuence the response to ICIs. In the long rest period group, the frequency of the Th1 subset and PD-1 + CD8+ T cells are signiﬁcantly higher than that in the short rest period group, which provides a novel perspective for the application of combination therapies (131). However, there is no more research about appropriate sequence and proper time interval, and it needs more researches to study the inﬂuence of time interval on anti-tumor effects. Microbiome reprogramming Gut microbiome is a complex ecosystem that regulates the interaction of the human and their environment, which has a potential impact on anti-tumor immune responses through frontiersin.org Frontiers in Immunology 04 10.3389/ﬁmmu.2022.1088886 Shi et al. severe skin reactions, colitis, and infusion reactions (118–121). However, the current understanding of adverse reactions of combined therapies is incomplete, and it is necessary to describe the preferable adverse reactions of different combinations, which is beneﬁcial for balancing the safety and efﬁciency of the corresponding treatment. DAMPs in breast cancer, which leads to a pro-inﬂammatory signaling (129). The inﬂuence of chemotherapy on diverse CAFs and their association with the immunotherapy efﬁcacy has not been explored comprehensively, and further researches are needed. Except for the inﬂuence of chemotherapeutic agents on novel cellular components in TME, chemotherapy-induced microbiome reprogramming also contributes to the combination of ICIs and chemotherapy. Although the impacts of chemotherapy on gut microbiome have been explained partly in animal experiments, the overall landscape of gut microbiome reprogramming after treating with different chemotherapeutic agents needs to be described in more preclinical and clinical researches. Beyond that, the inﬂuence of chemotherapy on local microbiome also needs to be investigated further, and the impacts of chemotherapy on ICI administration are required to be validated in clinical researches from the perspective of microbiome reprogramming. Author contributions All authors have made a direct contribution to the work, and approved it for publication. Funding colon cancer (132). Metronomic low dose cyclophosphamide enhance anti-tumor immune response by selectively reducing the amount of Treg cells in tumor patients (133), while high dose cyclophosphamide completely eradicates the hemopoietic cell (134). In addition, a novel administration mode called medium- dose intermittent chemotherapy provokes a striking response depending on the activation of a sustaining anti-tumor immune response (66, 135). Therefore, appropriate dosage for combined therapies is necessary to be investigated in further studies. This work was supported by Science and Technology Project of Shaanxi Province (2021PT-047), National Natural Science Foundation of China (82002425, 82273226), and the Young Elite Scientist Sponsorship Program by Cast of China Association for Science and Technology (2020QNRC001). References 12. Wu T, Dai Y. Tumor microenvironment and therapeutic response. Cancer Lett (2017) 387:61–8. doi: 10.1016/j.canlet.2016.01.043 1. Esfahani K, Roudaia L, Buhlaiga N, Del Rincon SV, Papneja N, Miller W HJr. A review of cancer immunotherapy: From the past, to the present, to the future Curr Oncol (2020) 27:S87–97 doi: 10.3747/co.27.5223 12. Wu T, Dai Y. Tumor microenvironment and therapeutic response. Cancer Lett (2017) 387:61–8. doi: 10.1016/j.canlet.2016.01.043 1. Esfahani K, Roudaia L, Buhlaiga N, Del Rincon SV, Papneja N, Miller W HJr. A review of cancer immunotherapy: From the past, to the present, to the future Curr Oncol (2020) 27:S87–97 doi: 10.3747/co.27.5223 13. Chen DS, Mellman I. 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Pembrolizumab plus chemotherapy or anlotinib vs. pembrolizumab alone in patients with previously treated EGFR-mutant NSCLC. Front Oncol (2021) 11:671228. doi: 10.1016/S0140-6736(20)32531-9 5. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Discussion Lastly, proper dose may be the most important respect to effectively stimulate the maximum anti-tumor immune response and minimize adverse reactions. For example, low dose cisplatin and oxaliplatin increase the number of circulating CD4+ and CD8+ T cells, while high dose regimens decrease the size of lymphocyte in a mouse model of With the development of sequencing technology, the landscape of immune cells within tumor tissues has been gradually revealed, and more and more cellular components have been recognized. For example, B cells represent a vital component of inﬁltrating immune cells in a variety of solid tumors, and play a dual role in modulating anti-tumor immune response (123). Chemotherapy reduces the amount of adenosine-producing B cells, which may reduce potential immunosuppression in TME (124). However, there is little research about the inﬂuence of chemotherapy on B cell subpopulations and their activity. Moreover, cancer-associated ﬁbroblasts (CAFs) account for more than 50% of stroma cells in TME, and various CAF populations have been identiﬁed based on the results of single- cell sequencing analyses, including the cancer-associated myoﬁbroblasts (myCAFs), inﬂammatory-like CAFs (iCAFs), and the antigen-presenting CAFs (apCAFs) (125). CAFs are involved in regulating tumor immune response and the efﬁcacy of immunotherapy through several routes (126). For example, CAFs increase the ratio of FoxP3+ (Tregs) and CD8+ tumor- inﬁltrating lymphocytes via IL-6 in TME, and IL-6 blockade enhances the immunotherapy efﬁcacy in esophageal cancer models (127). Several studies have reported that CAFs protect tumor cells from apoptosis induced by chemotherapy, while insulin-like growth factors secreted by CAFs enhanced the anti- tumor effects of osimertinib in mice model (125, 128). Moreover, CAFs can activate the NLRP3 inﬂammasome through sensing Frontiers in Immunology frontiersin.org 05 Shi et al. 10.3389/ﬁmmu.2022.1088886 Conﬂict of interest In summary, ICIs combination with chemotherapy has shown a better anti-tumor response and provides a more beneﬁcial survival, compared to ICIs monotherapy. This beneﬁt is supported by a strong cancer biology rationale, which induce a better immune response. Hence, the evaluation of the panoramic dynamic immune landscape of TME will be helpful to understand tumor pathogenesis and provide novel approaches for cancer treatment. The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. References Immunosuppression by mutated calreticulin released from malignant cells. Mol Cell (2020) 77:748– 760.e9. doi: 10.1016/j.molcel.2019.11.004 27. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, et al. 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https://openalex.org/W2200872911	https://www.mdpi.com/2071-1050/8/1/99/pdf?version=1453360244	English	null	How to Monitor Sustainable Mobility in Cities? Literature Review in the Frame of Creating a Set of Sustainable Mobility Indicators	Sustainability	2,015	cc-by	11,515	Beatriz Junquera 1,* and Jesús Ángel Del Brío 2 Received: 7 October 2015; Accepted: 15 January 2016; Published: 21 January 2016 Academic Editor: Giuseppe Ioppolo Received: 7 October 2015; Accepted: 15 January 2016; Published: 21 January 2016 Academic Editor: Giuseppe Ioppolo 1 Facultad de Economía y Empresa, Universidad de Oviedo, Avda. del Cristo s/n, Oviedo 33071, Asturias, Spain 1 Facultad de Economía y Empresa, Universidad de Oviedo, Avda. del Cristo s/n, Oviedo 33071, Asturias, Spain 2 Escuela Politéncia, Universidad de Oviedo, Campus de Viesques, 33071 Gijón, Asturias, Spain; delbrio@uniovi.es 1 Facultad de Economía y Empresa, Universidad de Oviedo, Avda. del Cristo s/n, Oviedo 33071, Asturias, Spain 1 Facultad de Economía y Empresa, Universidad de Oviedo, Avda. del Cristo s/n, Oviedo 33071, Asturias, Spain 2 Escuela Politéncia, Universidad de Oviedo, Campus de Viesques, 33071 Gijón, Asturias, Spain; delbrio@uniovi.es p 2 Escuela Politéncia, Universidad de Oviedo, Campus de Viesques, 33071 Gijón, Astu delbrio@uniovi.es 2 Escuela Politéncia, Universidad de Oviedo, Campus de Viesques, 33071 Gijón, Asturias, Spain; delbrio@uniovi.es * Correspondence: beatriz@uniovi.es; Tel.: +34-985-104-972; Fax: +34-985-103-708 * Correspondence: beatriz@uniovi.es; Tel.: +34-985-104-972; Fax: +34-985-103-708 Abstract: The aim of the current study is to evaluate new preventive command-and-control environmental regulation’s competitive effects on automobile manufacturers and their suppliers. The methodology that we have used is a case analysis, and its main aim is to study an unfamiliar situation. Therefore, we have chosen cases from each of the groups: two suppliers and one manufacturer. The new regulation obliges automobile companies to deeply modify their process technologies and their relationships with their suppliers (toughening requirements and strengthening long-term relationships) and to require their workers to train in environmental matters. Complying with regulation by suppliers will be possible if product and process designs are modiﬁed. However, only organisational actions, which include workers’ training in environmental and quality matters and activities to recover value in factories, are capable to achieve it. In any case, these factories have already been affected by trade-offs between environmental and other more classic manufacturing objectives, especially quality. Keywords: environmental management; manufacturing strategy; manufacturer-supplier relationships; quality management sustainability sustainability sustainability Sustainability 2016, 8, 99; doi:10.3390/su8010099 1. Introduction With the ratiﬁcation of the Kyoto Protocol by Russia, industrialised countries that signed the Protocol accounted for 55% of all CO2 emissions, which came into effect in 2004. In the European Union more than anywhere else, even prior to the Protocol, a process began to develop new regulation to ensure compliance with what would eventually become the targets set by the Protocol: for example, Directive 2000/53/CE on Management of Vehicles at the End of their Useful Life. As a consequence, said regulation shows effective guidelines for the correct environmental management of vehicles at the end of their useful life, but also preventive measurements which should be taken into consideration at any automobile’s design and manufacturing and when suppliers produce new components. Environmental regulation always induces changes at the business level. Nevertheless, the most traditional approaches for environmental regulation modiﬁes a company’s business strategy only slightly. However, regulation of the type of Directive 2000/53/CE relating to vehicles at the end of their useful life, forces not only a group of companies, but also all participants in the value chain of every industrial sector, to alter their “way of doing things”. This especially affects the manufacturing area, where environmental issues exercise greater inﬂuence. Literature has independently studied the most relevant effects of preventive regulation and command-and-control regulation. Nevertheless, preventive command-and-control environmental regulation had not been studied yet. In fact, Directive 2000/53/CE is the ﬁrst preventive command-and-control environmental regulation, so its effects have not been studied yet. As a result, this has become a remarkable topic. Consequently, we will focus on this gap. That is to say, we will study how the new preventive command-and-control regulation is able to inﬂuence and to modify a business’ strategy, and, especially, its manufacturing strategy. It is a very new environmental regulation, whose inﬂuence on business strategy, as well as on manufacturing strategy, and the relationship between regulation and strategy, may lead to important effects on organisational performance. In addition, this paper analyses the role of the supply chain within this new regulatory framework. The literature has shown the role of any supply chain in managing environmental issues. Akyuz and Erkan (2010) [12] reveal that the supply chain is still a fruitful research area. Moreover, further research is necessary. 1. Introduction Especially in the ﬁrst periods of environmental development in a region or a country, the role played by Public Administrations is key (Brío et al., 2002) [1]. Later, governments develop other kinds of tools, which demand companies to internalise those costs derived from environmental damage which they had caused earlier. Environmental instruments with this aim are called economic instruments. Some examples are environmental taxes, tradable emission rights or ﬁnancial aids (OECD, 1991; Ledyard and Szakaly-Moore, 1994) [2,3]. There are other kinds of tools which help companies to improve their image by supporting their reputation on environmental management (Cascio et al., 1996) [4]: certiﬁcations and eco-labels. This kind of regulation is known as preventive environmental regulation, because it leads companies to avoid pollution. However, previous laws, the so-called command-and-control regulation or traditional command-and-control regulation, can be deﬁned as the direct regulation in an industry or activity which states what is permitted and what is illegal (McManus, 2009) [5]. It imposes limits only to the permitted level of pollution. In fact, the ﬁrst tool used by Pubic Administrations in any country is this command-and-control regulation (Jennings and Zandbergen, 1995; O’Connor, 1998; Hillary and Thorsen, 1999) [6–8]. In order to comply with this kind of regulation, companies must develop new technologies to avoid polluting. As a result, papers on the environmental activity in companies have usually been focused www.mdpi.com/journal/sustainability Sustainability 2016, 8, 99; doi:10.3390/su8010099 www.mdpi.com/journal/sustainability 2 of 17 Sustainability 2016, 8, 99 exclusively on technological issues, whereas organisational aspects are often ignored: actions to reduce CO2 emissions (Medina and Tarlock, 2010) [9], an industry’s dynamics (BenDor, 2012) [10] or even issues linked to social problems, such as dependence by older adults (Nakanishi and Black, 2015) [11], among others. Recently, the European Union has elaborated a new kind of command-and-control regulation, the so-called preventive command-and-control environmental regulation described in this paper. It establishes preventive objectives when a new design is established to reduce and to eliminate the use of dangerous substances in manufacturing, to facilitate reutilization, recycling and revaluing and to reduce environmental pollution. This new initiative, which requires technological and organisational solutions, is used to comply with preventive regulation. They are also linked in order to guarantee the success of companies’ environmental strategies. Nevertheless, this preventive command-and-control environmental regulation shows what is permitted and what is illegal too, so, in this sense, it is similar to command-and-control regulation. 2. Theoretical Approach The environmental Directive 2000/53/CE studied in this paper establishes measures to prevent waste generated by vehicles, to regulate its collection and decontamination at the end of their useful life, in addition to other treatments, with the aim of improving the environmental management’s efﬁcacy during the vehicle life cycle (DOCE, 2000) [17]. According to our deﬁnition, it is a command-and-control environmental law in its preventive version. The aim of the directive studied in this paper is to establish measures to prevent waste generated by vehicles, to regulate its collection and decontamination at the end of their useful life, in addition to other treatment, with the goal of improving the effectiveness of environmental management during the vehicle life cycle. In this way, this directive establishes preventive measures, from the start of the vehicle’s design stage, to reduce and to eliminate the use of dangerous substances in its manufacturing and to facilitate reutilisation, recycling and valorisation of its components and to reduce the environmental pollution produced by vehicles. That is to say, this directive is preventive command-and-control environmental regulation, although in a preventive manner. The central ﬁgure in this regulation is the user, who becomes obligated to hand over the vehicle at the end of its useful life—either directly or via a system for its delivery—to an authorised treatment centre charged with the removal of contaminants. In order to facilitate the user’s compliance with this duty and to apply the principle of the manufacturer’s responsibility, the latter must be responsible for the vehicles which it has produced or sold, guaranteeing the suitability of the reception facilities. The abovementioned directive establishes preventive measures which various elements in a vehicle have to comply with, so that the use of dangerous substances is reduced to a minimum. To this effect, the use of lead, mercury, cadmium and hexavalent chrome is prohibited in vehicle materials and components, with some exceptions and conditions. (a) Designing and manufacturing vehicles and the elements composing them in such a way as to facilitate their dismantling, decontamination, reutilisation of parts and the valorisation of the vehicles at the end of their useful life and to encourage the use of new models for recycling materials and components. 1. Introduction More recently, Vanichchinchai and Igel (2011) [13] suggest that, in future, research ino the automotive assemblers and downstream business partners could be studied together with the upstream suppliers in order to investigate the relationship along the whole supply chain. Huscroft et al. (2013) [14] have addressed that environmental issues are key to study reverse logistics successfully. Some alternative papers had focused their study on the automotive industry (Hsu, Tan and Laossirihongthong, 2014) [15]. However, research has not analysed the green supply chain speciﬁcally within the automotive industry so far. The aim of the present paper is to undertake a case analysis on the reaction of an industrial sector, the automotive sector, to comply with this new preventive command-and-control environmental regulation. We analysed the role of companies scrapping vehicle companies in a previous paper (Brío et al. 2006) [16]. In this research, we study the consequences of preventive command-and-control 3 of 17 Sustainability 2016, 8, 99 environmental regulation for component suppliers and automotive manufacturers, as well as the relationships between them. The structure of this paper will be broken down as follows. The theoretical approach will ﬁrst be shown, followed by the methodology according to which this study has been carried out. Finally, the conclusions and implications of this paper will be set out. 2. Theoretical Approach (a) Designing and manufacturing vehicles and the elements composing them in such a way as to facilitate their dismantling, decontamination, reutilisation of parts and the valorisation of the vehicles at the end of their useful life and to encourage the use of new models for recycling materials and components. (b) Using codiﬁcation standards which allow workers and managers to identify those components suitable for reutilisation or valorisation. (c) Providing people who work with vehicles at the end of their useful life with the correct information for their dismantling, which allows the identiﬁcation of the different components and the location of dangerous substances. (d) Informing consumers about environmental management criteria used both at the stages of vehicle design and manufacturing, as well those used to ensure their correct environmental treatment at the end of their useful life. (d) Informing consumers about environmental management criteria used both at the stages of vehicle design and manufacturing, as well those used to ensure their correct environmental treatment at the end of their useful life. The newness of the compulsory introduction of preventive approaches in environmental management brings with it implications of great signiﬁcance to the companies. In fact, Porter and Van der Linde (1995) [18] have observed that the more complex the environmental approaches are, the less autonomous the environmental actions become. Consequently, environmental issues are linked more closely to other functional areas, especially to manufacturing department (Oglethorpe and Heron, 2010) [19]. Sustainability 2016, 8, 99 4 of 17 As preventive regulation, Directive 2000/53/CE induces a change in the way of dealing with business strategy as a whole, as we have already observed. This is particularly relevant in the manufacturing area, the department most affected by environmental regulation. The lack of preventive environmental laws has led to only a few studies about its effects on business competitiveness and, as a result, about the aspects that could foster its compliance. In any case, the new European directive which we have analysed in this paper has triggered the beginning of a new era when research about preventive command-and-control environmental regulation has become a priority. Firstly, Aggeri (1999) [20] has studied cooperative movements in the automotive industry, although these papers have been used to study only manufacturers. 2. Theoretical Approach Other studies have analysed environmental issues in automotive industry (Das et al., 1995; Bellmann and Khare, 1999, 2000; Mildenberger and Khare, 2000) [21–24], but they have not studied the implications derived from companies’ environmental actions, especially on decisions isolated from manufacturing strategy. On the other hand, these papers are descriptive and are not focused on the analysis of the implications derived from new command-and-control environmental laws, but with a preventive bias. Nevertheless, they are an exceptional theoretical support to approach the current paper. In fact, Mildenberger and Khare (2000) [24] have analysed environmental issues in the automotive industry, focusing on the German case. Likewise, Bellmann and Khare (1999) [22] have discussed the potential implications derived from environmental regulation on the automotive industry and, lately, to analyse its economic implications (Bellmann and Khare, 2000) [23]. Therefore, one of the main obstacles in carrying out this paper is the lack of speciﬁc literature. However, we have found another kind of literature which could be the support our study of the issue which we are working on. Once we have achieved this point, we have to consider, on the one hand, institutional approaches and strategic literature, which, together with contributions stemming from economic theory, have produced several win-win results for the analysis of environmental issues. On the other hand, and related to the inﬂuence of manufacturing strategy development to ease the compliance with the new regulation, we have used the literature focused on the study of manufacturing strategy, and especially those papers which have made an effort to try to integrate environmental issues. In fact, Barbera and McConnell (1990) [25] had defended the differences between several kinds of effects derived from environmental regulation on business productivity. The ﬁrst one is the so-called direct effect (the cost which companies have to deal with to comply with environmental regulation). The second effect, addressed by Barbera and McConnell (1990) [25], is the so-called indirect effect, which is linked to the inﬂuence the change could have on the productive factors to manufacture goods. That is, environmental regulation can improve productivity using manufacturing processes, which use less inputs and take advantage of productive processes which consume a smaller amount of inputs or using waste, either by selling it as derived products or reducing operative costs as a consequence of a more efﬁcient use of materials and energy (Porter and van der Linde, 1995; Shen, 1995) [18,26]. 2. Theoretical Approach In short, the literature recognizes that environmental regulation can improve or damage business competitiveness from the beginning. From an institutional point of view, Hoffman and Ventresca (1999) [27] support their analysis on three issues linked to institutions (regulative, normative and cognitive) and take into account the implications derived from this argument. Hoffman et al.’s (1999) conclusions match with the Porter hypothesis in the sense that environmental protection allows win-win alternatives for companies [28]. Hoffman (1999) [29] tries to check his theories empirically by applying this analysis to the chemical industry in the United States, a very similar paper to Hoffman’s (2005) [30]. Within the same debate, the strategic literature has analysed environmental regulation’s inﬂuence on business competitiveness (Shrivastava, 1995) [31]. Although there are no conclusive results, some authors have explained the differences perceived between companies with a different kind of relationship between environmental performance and business performance supporting their arguments on the resource-based view (Russo and Fouts, 1997) [32]. According to Porter Hypothesis, 5 of 17 Sustainability 2016, 8, 99 Marcus and Geffen (1988) [33] have analysed, focusing their reasoning on pollution prevention for electricity generation, how the system characteristics together manage the paths which imply the acquisation of new competencies and a change in the interaction with other groups and stakeholders in the company. A comprehensive view of these papers allows us to guess whether the same regulation is able to foster such different effects on several companies or not. In general, environmental approaches allow us to divide companies into two groups: those with command-and-control approaches and those with preventive approaches. Those companies, where the main method is to use specialized equipment to ﬁght against pollution, may suffer from too many costs. Simultaneously, it is unproductive, because it is not able to generate value (Andersson and Wolff, 1996) [34]. The philosophy on which this approach is supported is that the only force to manage an environmental policy is to minimize tangible costs derived from pollution. That is to say, any company beyond compliance would not accept beneﬁts derived from internalising those costs (Russo and Fouts, 1997) [32]. On the other hand, the so-called Porter hypothesis maintains that strict and preventive environmental regulation triggers the discovery and introduction of cleaner technologies and environmental improvements, an innovative effect which leads to more efﬁcient manufacturing, processes and products (Porter and van der Linde, 1995) [18]. 2. Theoretical Approach From this point of view, the more additional improvements a kind of environmental regulation fosters, the more said regulation can contribute to balance earlier costs derived from regulation, avoiding ﬁnes, among other sanctions (Greeno and Robinson, 1992; Shrivastava, 1995a; Hart, 1997) [35–37]. Additional contributions have found out several aspects which could inﬂuence the relationship between compliance with environmental regulation and business competitiveness. Dean and Brown (1995) [38] address the fact that environmental regulation can create entry obstacles because of the increase of new investment or the change of the minimum efﬁcient scale as a consequence of entering new environmental technologies. On the other hand, they suggest that environmental regulation could decrease some entry barriers, because incumbents companies simultaneously choose environmental technologies and manufacturing technologies, avoiding, in this way, potential conﬂicts. Supporting the conclusions derived from our review of the literature, we have not been able to obtain conclusive results, perhaps because not every kind of environmental regulation affects business competitiveness in the same way. We should consider the speciﬁcities linked to this new kind of environmental regulation, the so-called preventive command-and-control environmental regulation, which has not been studied yet, because it fosters companies to adopt preventive actions, that is, those that interact with product and process designs. In fact, they may ease (or make more difﬁcult) the implementation of preventive environmental practices. That is, the speciﬁc inﬂuence of a rule on business competitiveness could depend on its intrinsic characteristics. The newness derived from the directive which we have analysed in this paper fosters the most important implications from a qualitative point of view. Moreover, Porter and Van der Linde (1995) [18] found that the more complex environmental approaches become, the less autonomous environmental actions are and the more linked to other functional areas in the company they are, especially to manufacturing department. The new preventive approaches lead to a change in the way of dealing with manufacturing management, so that eco-manufacturing management was born in Angell and Klassen’s (1999) terms and in Newman and Hanna’s (1996) words [39,40]. It is deﬁned as the integration between environmental principles in decision making, which affects the transformation of resources into useful goods (Gupta and Sharma, 1996) [41]. 2. Theoretical Approach Nevertheless, the method of adopting integrated decisions into manufacturing strategy in a company is a key issue, because Newman and Hanna (1996) [42] have shown how developing a manufacturing strategy is an uncrossable constraint to improving a company environmentally (Ioppolo et al., 2012; Ioppolo et al., 2014) [43,44]. However, including environmental issues into production function is not an easy task. Moreover, preventive environmental protection may change the hierarchy of manufacturing objectives in a company and the way that companies make several manufacturing decisions. This kind of literature has studied the main implications derived from implementing preventive environmental actions, although 6 of 17 Sustainability 2016, 8, 99 voluntarily, never with a mandatory character. However, it is very helpful to know those implications and constraints derived from its implantation. Florida (1996) [45] shows the results of a survey carried out in the United States in industrial companies, whose aim was to explain the relationship between advanced manufacturing systems and innovative approaches for environmentally-conscious manufacturing. Klassen and Whybark (1999) [46] found that improving environmental performance is possible only after implementing preventive technologies in a company’s environmental technology portfolio. Sarkis (1995) [47] shows a strategic framework to manage environmentally-responsible programs and projects in industrial companies. Later, Sarkis (2001) [48] analyses the role of manufacturing into corporate sustainability, dealing with problems derived from manufacturing function speciﬁcally. In general, this kind of literature should be classiﬁed as prescriptive and supported on anecdotic evidence, although some aspects deﬁning environmentally responsible manufacturing can be perceived. For example, Richards (1994) [49], Sarkis (1995) [47], Zhang et al. (1997) [50], Gungor and Gupta (1999) [51], Nagel and Meyer (1999) [42] and Melnyk et al. (2003) [52] emphasise the importance of value recovery activities, whereas Sarkis (2001) [48] includes aspects related to decisions to implement a manufacturing strategy. Go et al. (2011) [53] have studied said effects on end-of-life vehicles in the automotive industry. Other authors have studied environmentally responsible approaches for responsible manufacturing from an ecological view (Florida, 1996; Zhang et al., 1997) [45,50], including job safety (Zhang et al., 1997) [50]. However, two papers must be addressed (Newman and Hanna, 1996; Angell and Klassen, 1999) [39,40], although, in general, only a few papers can be said to have focussed their main aim on checking empirically how the level of development in manufacturing decisions improves environmental development. 3. Methodology The methodology which we have used is a case analysis because our aim is to study an unfamiliar situation (Voss et al. 2002) [60]: the implications of the new preventive command-and-control environmental regulation on the manufacturing activity in companies. Using this methodology is the most suitable option when the key questions are what (description), how and why (explanation) in the generation of a theory (Snow and Thomas 1994; Meredith 1998) [61,62]. As a result, this methodology is the best choice according to the main objective of this research. A ﬁrst key decision relating to case analysis is referring to how many and which cases to choose for the analysis. To solve this issue we have considered the previous literature. Eisenhardt (1989) [54] suggests that from 4 to 10 cases is an adequate number, although Dyer and Wilkins (1991) [63] maintain that two at maximum is sufﬁcient. As part of this debate, Voss et al. (2002) [60] maintain that the optimum number of cases to analyse depends on the desired results. The objective of this paper is to study the consequences of preventive command and control environmental regulation between component suppliers and automotive manufacturers and the relationships between them. Taking into account the suggestions from the previous literature and the speciﬁc characteristics of our study, which include two kinds of groups (component suppliers and automotive manufacturers), we have decided to analyse three companies: two suppliers (X and Y) and one automobile manufacturer (P). At the same time, to avoid problems with external validity (a speciﬁc problem of any case analysis), we have carried out a detailed review of the literature, from which we have been able to extract the most relevant information. With a similar aim, we used the guidelines of the Spanish Association for the Environmental Treatment of End-of-Life Vehicles (SIGRAUTO), whose members are companies and institutions involved in the process of a right environmental management of vehicles at the end of their useful life. They gave us sufﬁcient criteria to choose the most suitable cases and to deﬁne the measures related to the phenomenon to be studied (Yin 1984) [64]. The ﬁeld work was carried out during the ﬁrst half of 2011. We designed a questionnaire according to our conclusions from the review of the literature and SIGRAUTO members’ suggestions. 2. Theoretical Approach 7 of 17 Sustainability 2016, 8, 99 (c) Are there any relationships between complying with preventive command-and-control regulation in automotive regulation and integrating environmental issues into manufacturing strategy? (c) Are there any relationships between complying with preventive command-and-control regulation in automotive regulation and integrating environmental issues into manufacturing strategy? (d) Is there any relationship between the way companies in the automotive industry deﬁne their environmental objectives and their level of environmental development? (d) Is there any relationship between the way companies in the automotive industry deﬁne their environmental objectives and their level of environmental development? (e) What are the main changes that complying with new preventive command-of-control regulation requires? (e) What are the main changes that complying with new preventive command-of-control regulation requires? (f) What kind of manufacturing decisions inﬂuence the compliance with the new preventive command-of-control regulation? (f) What kind of manufacturing decisions inﬂuence the compliance with the new preventive command-of-control regulation? 2. Theoretical Approach In fact, some of them have partially been analysed (Florida, 1996) [54], although without taking into account their links to a manufacturing strategy. On the other hand, we should consider those empirical studies published which have analysed relationships between the supply chain and environmental actions. Handﬁeld et al. (1997) [55], in a case analysis for the furniture industry, found the main inﬂuence areas on environmental performance in a company, after showing the weaknesses of the literature published until that moment. Young (2000) [56], in another case analysis, also shows the importance of supply function if the aim is to achieve an environmentally solid, proﬁtable and efﬁcient waste management, although the paper does not go beyond this. Some of these studies even referred, completely or partially, to the automotive industry. For instance, the case analysis by Handﬁeld et al. (2001) [57], whose main aim is to study the integration of environmental issues into product design, includes an automotive company. Hammond et al. (1998) [58] have carried out a study in the automotive industry which, among other conclusions, has shown speciﬁc issues. Nevertheless, instead of their ﬁndings, no paper has achieved relevant conclusions about how preventive environmental activities and manufacturing strategy are integrated together (Brío et al., 2002) [59]. The reason for these results is that they are linked to actions assumed voluntarily by companies, not to the new regulation impact. In this paper, we are going to work withing this gap. To sum up, preventive command-and-control environmental regulation may require a different way of management and, especially, a stronger integration of environmental issues into manufacturing strategy. However, it is a very new kind of regulation, and research about it is too scarce, so that many questions remain unanswered. Consequently, we have approached the following research questions to be answered after this case analysis: (a) Does automotive suppliers and manufacturers’ attitude towards a strict compliance with environmental regulation depend on their own environmental development? (a) Does automotive suppliers and manufacturers’ attitude towards a strict compliance with environmental regulation depend on their own environmental development? (b) Do the stakeholders play a key role to foster companies towards more developed environmental approaches? (b) Do the stakeholders play a key role to foster companies towards more developed environmental approaches? Description of the Companies Analysed X is a company founded in 1980 in Madrid. It manufactures electrical equipment for automobiles (horns and relays). The company is a division of a German multinational and leader in the manufacturing of lighting, electronic and electro-mechanical systems for the automobile industry. We visited its factory located in Madrid, with a workforce of 268 people. The company manufactures nine million horns and twenty-two million relays per year, with sales of approximately 36 million euros. Y is a company which operates in the transmission product line as part of a multinational group which began operations in Paris in 1923 manufacturing brake linings and transmission systems. On 28 May 1980 the group’s companies adopted the same name to operate as one brand. The group currently operates in 27 countries and has a workforce of 51,200 employees in 121 factories, 61 R&D facilities and 10 distribution centres. The group is structured according to product lines: transmissions, heating systems, air conditioning, lighting systems, electrical systems, windscreen wipers, security systems, ﬁre detection systems, connection and electronic systems, drive shafts and engines and, ﬁnally, services. The case study was carried out in the transmission product line of the group’s Spanish division, founded in 1968, where clutches and power steering systems are manufactured. Employees currently number approximately 300, including management, administrative and shop ﬂoor staff. Between vehicle manufacturers only plant P was analysed. The reasons for this were various: (a) this type of plant is much less common in Spain than those belonging to other types; (b) not all vehicles must be considered, but rather only those affected by the directive, which fulﬁl certain characteristics; and (c) the company studied in this section was among that group of companies which have been the least challenged by the approval of the directive, as, in reality, their principal task is assembly, so that the environmental harm which they could potentially cause is less than in the other groups of companies included in our analysis, such as suppliers. This plant, with a workforce of over 3000, is part of a multinational group. P is composed of three companies. Two of them are commercial companies marketing two brands and with headquarters in Spain. The third is a manufacturing company with two factories in Spain, one founded in 1958, in which several models are manufactured. This plant works on drawing of metal parts, soldering, paint and assembly. Description of the Companies Analysed In 2002, it produced 483,000 vehicles, of which 87% were exported. The workforce currently numbers 10,000 workers. The second manufacturing plant, where the analysis was undertaken, started operations in 1952. In 1970 was acquired by one multinational and in 1980 by another. Finally, due to a merger, in 2001 it became known as the company it is today. At this latter manufacturing plant, activities of assembly, bodywork and painting are carried out and several models are produced. Manufacturing amounts to 123,000 vehicles, of which 60% is exported. The plant has a production capacity of 38.17 vehicles per hour and works in three shifts. The company employs 3,143 people in its plant in Madrid, divided among, 2275 shop ﬂoor workers, 237 teams and 631 technical and administrative staff. 3. Methodology After that, two researchers visited the companies analysed and interviewed a manager with responsibility for environmental issues in every case. Nevertheless, to avoid the problem of internal validity which customarily affects any case analysis, we have used methodological triangulation, so that the information obtained from interviews could be complemented by observation and review of several documents. That is, triangulation or obtaining information from these companies leads us to use various sources, the following ones: their websites (when these were available), direct observation by the interviewer and the ﬁlling out of a questionnaire using literally the words by the person considered by the company to be most qualiﬁed to talk about environmental issues. We obtained qualitative and quantitative information. The process is similar to other analysis carried out in the automotive industry (Lu et al., 2014) [65]. Since conclusions drawn from the literature and a preliminary inspection of the sector lead us to see envisage substantial differences regarding to the consequences of this new kind of regulation on the groups analysed (component suppliers and automobile manufacturers), we have opted for carrying out two different questionnaires. Both of them have, however, identical objectives and even 8 of 17 Sustainability 2016, 8, 99 have identical questions and some of them are similar, although with a differient focus depending on the position of each company in the supply chain. Our review of the literature was also used to choose the questions in the study, as well as the observations undertaken in the sector by the authors of the paper and SIGRAUTO’s recommendations. We describe our three cases below. 4. Case Analysis The literature has already evaluated which kind of regulation beneﬁts/harms an industry’s environmental leaders compared to its competitors (Nehrt 1998) [66], reaching conclusions very similar to those expressed above. That is to say, companies in the best position from an environmental viewpoint demand a stricter control of regulatory compliance on the part of the authorities, extending their coverage to companies which do not form part of their direct competition. In fact, although not referring to the automotive sector, other studies have shown how the degree of environmental development in companies affects the role required of the public authorities as regards environmental protection (Brío et al. 2002) [1]. López-Gamero, Molina-Azorín and Claver-Cortés (2010) [67] have studied the potential of environmental regulation to change environmental approaches in companies, although they have independently focused on command-and control versus another kind of regulation. However, no paper has studied preventive and command-and-control regulation. In this paper, we have found that relationship for the new kind of regulation in the automotive industry. As a result, we can conclude the ﬁrst ﬁnding of our analysis: Finding 1. The more environmentally developed suppliers and manufacturers in the automotive industry are, the more they demand a stricter compliance with regulation of Public Administrations because environmental development is a competitiveness factor in that context. Not only environmental regulation is important for automobile industry, but also external pressure perceived by the companies inﬂuences, to a fair degree, the development of their environmental strategy. Some empirical studies (Kim and Lee, 2012) [68] had recognised signiﬁcant relationships between stakeholder pressures and environmental logistics practices, although they were not focused on the automotive industry. The analysed automotive manufacturer has acknowledged that it cannot allow public repercussions over any environmental irregularity, while X recognises that it does not suffer from any strong external pressure in environmental terms, while at Y the situation is not as easy. Other studies (Vastag et al. 1996; Henriques and Sadorsky 1999) [69,70] have already analysed external inﬂuence on companies’ environmental responses, but none of them has focused on the automotive industry. As a result, this study conﬁrms what had been found out in other industries. That is, as well as new environmental regulation, pressures by external stakeholders inﬂuence environmental actions by companies in the automotive industry. Finding 2. The more active stakeholders are in an industry, the more they lead companies to more developed environmental approaches. 4. Case Analysis Under this heading, the results shown above are analysed. Our results include several topics: regulation compliance and its relationships with environmental objectives and their involvement with their manufacturing strategy. After that, we differentiated between structural and infrastructural aspects linked to manufacturing strategy content. Our ﬁrst ﬁnding is related to their attitude to regulation. Given that both supplier plants are highly advanced in environmental compliance, they require the public administrations a strict control over the compliance by the others with the new regulation. However, this is not referred only to their direct competitors, but also to those companies selling substitute products. For example, one of the 9 of 17 Sustainability 2016, 8, 99 suppliers (Y) afﬁrms that “several vehicle recyclers sell material without any regulatory compliance”. From the viewpoint of the automotive manufacturer, the companies which have traditionally worked with environmental issues are able to obtain a competitive advantage from their position in this area. That is the case with the automotive manufacturer which we have analysed. Over recent years they have invested heavily in Research and Development aimed at improving the environmental performance of its products (a particle ﬁlter for its diesel and HDI engines, which are more energy efﬁcient and emit less CO2). Y is not of the opinion that the new regulation will harm it, or at least, the impact will never amount to that produced by other, less environmentally developed companies. suppliers (Y) afﬁrms that “several vehicle recyclers sell material without any regulatory compliance”. From the viewpoint of the automotive manufacturer, the companies which have traditionally worked with environmental issues are able to obtain a competitive advantage from their position in this area. That is the case with the automotive manufacturer which we have analysed. Over recent years they have invested heavily in Research and Development aimed at improving the environmental performance of its products (a particle ﬁlter for its diesel and HDI engines, which are more energy efﬁcient and emit less CO2). Y is not of the opinion that the new regulation will harm it, or at least, the impact will never amount to that produced by other, less environmentally developed companies. The companies we have analysed show a high level of environmental development and, as a result, one of their competitive advantages is supported on that environmental superiority, so that they demand a strict control by public authorities to comply with this kind of regulation. 4. Case Analysis How have these companies been able to comply with this new regulation? A key element is the degree of involvement of environmental objectives in the manufacturing strategy. This issue has already been discussed in the literature. Although Handﬁeld et al. (1997) [55], also through case analyses, deny equal priority for the classic manufacturing objectives and those for the natural environment, Angell and Klassen (1999) [39] say that they should be given equal treatment. However, no paper had studied this phenomenon in the automotive industry or its role to comply with preventive command and control regulation (we have to remember this regulation newness). In our analysis, X is a plant which we can qualify as one that “goes beyond mere compliance” (according to words by the interviewed manager). Our own observation has conﬁrmed this sentence: X not only complies with environmental 10 of 17 Sustainability 2016, 8, 99 regulation, but also it has achieved ISO 14001 certiﬁcation and evaluates all operating aspects which could cause impact. In this case, the environmental objective is important, although not to the same degree as those more classic (cost, quality, ﬂexibility and delivery times). Supplier Y has made even greater progress: by making signiﬁcant investments and progress in fulﬁlling the regulation’s requirements. Without a doubt, at Y the natural environment protection is given a similar level of priority to the most classic manufacturing objectives. The analysed automotive manufacturer has a continuous process to introduce environmental product innovations. All the interviewed managers agree that complying with preventive command and control environmental regulation requires a strong integration of environmental objectives into their manufacturing strategies. Actually, companies in automotive supply chain carried out this process of integration before complying with the new kind of regulation. This may be the ﬁrst factor that explains how preventive command and control regulation inﬂuences whether a company has a competitive advantage supported on its environmental situation or not. Finding 3. Complying with preventive command-and-control regulation in the automotive industry implies to integrate environmental issues into manufacturing strategy. y p g g gy Talking about environmental objectives is not a simple task because it is a multidimensional concept and, as a result, there are different kinds of environmental performance measurements and companies choose those that are better adapted to their characteristics. In fact, different environmental protection approaches result in diverse environmental performance measurement (Illinitch et al. 1998) [71]. 4. Case Analysis Both X and Y have systems to measure their environmental activity. Those at X try to evaluate the level of compliance with regulation. Y, on the other hand, focuses more on the preventive aspects linked to environmental management. The analysed automotive manufacturer has systematic measurements about its environmental performance and is audited annually by an external body about said indicators. Previous research considers this situation as an environmental development indicator (Brío and Junquera 2003) [72]. That is, we have interviewed managers belonging to companies with a different level of environmental performance. Our automotive manufacturer shows the highest level, while X is the least developed company if we consider its environmental performance. In any case, the measurements used show us every company’s environmental approach because of the close linking between environmental strategy and measurement system. gy y Finding 4. How companies in the automotive industry deﬁne their environmental objectives shows their level of environmental development. The introduction of the most preventive environmental policies has substantially modiﬁed their manufacturing strategy. As a result, we have shown greater formalisation of some procedures, the modiﬁcation in other cases and the generation of new procedures. Said issues have been seen by Kitazawa and Sarkis (2000) [73] in their case studies. However, it had not been analysed in the automotive industry. In our case analysis, X and Y show the new environmental requirements have led to an increased formalisation of procedures to begin the process of raising employees’ awareness. X and Y had been certiﬁed with ISO 9001 earlier, as well as with QS 9000 and UDA6-1 and, as a result, some processes were modiﬁed and other ones were created. Likewise, the analysed automotive manufacturer points out the high level of process formalisation because of the new environmental requirements when the company was certiﬁed with ISO 14001. Finding 5. Complying with new preventive command-and-control environmental regulation requires companies to increase their procedure formalisation. In the short term, preventive command-and-control environmental regulation leads to important competitive effects in the short term derived from the adaptation of a company’s activity to the new regulation. That is to say, it makes costs higher. The company must implement technical and organisational actions, whose combination is very useful for the development of its manufacturing strategy (Angell and Klassen 1999) [39]. 4. Case Analysis In our cases, X and Y have carried out technical and organisational changes, by modifying design (in the case of the latter company in a more speciﬁc way), to avoid the use of lead in the clutches, as well as hexavalent chrome. Among organisational changes, 11 of 17 Sustainability 2016, 8, 99 X uses the International Material Data System (IMDS) to inform manufacturers of the basic substances contained in all its products via the Internet and Y has implemented a process of quality control subsequent to the elimination of lead in its clutches. Likewise, both plants have taken the following measures: distribution of written documents, formalisation of processes to identify the regulatory requirements, extremely thorough internal environmental audits, planning of environmentally measurable objectives, programmes to achieve environmental objectives, periodic review of objectives to achieve continuous improvement, release of environmental reports, as well as the assignment and training of human resources in environmental matters. Preventive command-and-control environmental regulation also inﬂuences the application of environmental tools for Design for the Environment (DfE) at the analysed automotive manufacturer. Manufacturers are obliged to introduce innovations facilitating compliance with preventive regulation. The analysed automotive manufacturer expects, during the period 2010–2020, the ﬁrst vehicles with a fuel cell. Our results strengthen Berry and Rondinelli’s (1998) results, among other authors [74], who had spoken about its inﬂuence on design of preventive regulation, but had not examined its implications. However, our study shows new changes in structural manufacturing decisions, which have affected technologies to improve and minimise any product’s environmental impact. The analysed automotive manufacturer was committed, together with other European automobile manufacturers, to controlling greenhouse gases (140 grams of CO2 in 2007). This challenge was and is a corporative priority and is being carried out through innovative technology in its diesel and petrol engines, a ﬁeld in which the company is a benchmark, as its diesel HDi engines, their respective decontaminant systems and the direct petrol injection engines, designed and built by the corporation, associate environmental protection with manufacturing decisions. Environmental consequences are more evident in infrastructural manufacturing decisions. Preventive regulation affects relationships with components suppliers. X and Y have toughened the relationship with their suppliers as a result of the stricter demands from their clients: ISO 14001, environmental criteria selection and the use of plastics. These results are in accordance with that suggested by the literature. 4. Case Analysis Azzone and Noci (1998) [75,76], for example, have already demonstrated the importance of the relationship with suppliers for the environmental development of any company. Cramer and Schot (1993) [77] refer speciﬁcally to the requirements of ecological purchases. On the other hand, some papers had studied the role of logistics and supply chain process integration as a source of competitive advantage (Mellat-Parast and Spillan, 2014) [78], although the previous literature has not analysed what is speciﬁcally happening in the automotive industry. The analysed automotive manufacturer demonstrates that preventive command-and-control environmental regulation helps them strengthen their long-term relationship with them, as any legal non-compliance would lead to disastrous consequences for its public image as an innovator in environmentally-friendly products. Geffen and Rothenberg (2000) [79] show in case studies that, as a supplier learns more about the manufacturer’s productive process, they discover the kind of environmental actions which are best adapted to the clients’ needs. The role of employees is a key element in compliance with preventive command-and-control environmental regulation. Workers’ training, according to the literature (Gupta and Sharma 1996; Sarkis, 2001) [41,48], is a key element for the application of preventive command-and-control regulation. Likewise, this training is also the result of the implementation of environmental management systems and, especially, of ISO 14001 certiﬁcation. The analysed automotive manufacturer has organised informal meetings for the implementation of environmental actions, as well as suggestion boxes, encouraging their use with the aim of engendering environmental improvements. All this has been made possible thanks to the decision of upper management to support environmental preservation. Conﬁrming the results of our paper, Taylor and Welford (1993) [80] showed the importance of informal meetings about the natural environment at IBM (New York, NY, USA). Handﬁeld et al. (2001) [57] address the importance of individual contributions to the success of environmental objectives. Additionally, in the case of suppliers, this training results from the implementation of 12 of 17 Sustainability 2016, 8, 99 environmental management systems and especially of ISO 1400 certiﬁcation, as those interviewed have emphasised. In fact, both X and Y have underlined the importance of training their workforce in its compliance. It is at Y, however, where training is planned in more detail: it is planned in modules according to the hierarchical level to which it is being given (‘cascading’ environmental training). 4. Case Analysis That is, our results match with Antonioli, Mancinelli and Mazzanti’s (2013) [81] ones in the sense that environmental innovation is an element of organisational change, especially for human resource management and they are necessary to deal with the new environmental regulation. Quality control and, more speciﬁcally, certiﬁcation (ISO 9000, but also other types) are very important in the three companies, but only X, however, acknowledges its advantages in the application of preventive command-and-control environmental approaches. This contradicts the previous literature, which believes in the advantages of applying knowledge in quality control for environmental development (Klassen and McLaughlin 1993) [82], especially as regards ISO 9000 (Beechner and Koch 1997) [83]. An explanation is perhaps that this knowledge is more helpful when a company’s environmental objectives do not go beyond mere compliance with the regulation. Y is a company with more developed environmental approaches than X. Likewise, the analysed automotive manufacturer has a long tradition to invest heavily in R&D aimed at improving the environmental impact of its products. On the other hand, our case analysis lets us ﬁnd out some obstacles to comply successfully with this kind of regulation. We can see the importance of communication between suppliers and manufacturers to explain the difﬁculties to adapt to preventive command-and-control environmental regulation, as Geffen and Rothenberg (2000) [79] had addressed earlier. This idea was already suggested in the literature for manufacturing strategy in general (Fine 1998; Fawcett, Waller and Fawcett 2010) [84,85]. We have analysed the reasons which inﬂuence this lack of communication too: in automotive sector, as X and Y point out, the environmental requirements do not encourage closer ties with clients, because automobile manufacturers have great bargaining power. They only use any new requirements to call for stricter compliance from their suppliers. In any case, sometimes said inadequate communication leads to any efﬁciency problems. That is to say, from the aforementioned, we can conclude that only a combination of technological and organisational measures (product design, process technology, closer client-manufacturer relationships, a strong quality management and employees’ involvement, among others) enables compliance with preventive command-and-control environmental regulation successfully. Finding 6. Complying with new preventive command-and-control regulation requires changes manufacturing strategy and its implementation, mainly product and process design, relationships supplier-manufacturer, the role of employees and quality control systems. However, a lack of close client-manufacturer relationships could hinder the compliance with preventive command-and-control regulation. 4. Case Analysis To sum up, the literature had shown that consequences of environmental regulation on economy and businesses are multiple and complex (Gurtoo and Anthony, 2007) [86]. We have added that complying with preventive command-and-control regulation means to transform business strategy and, as a result, functional strategies, but especially manufacturing strategy. That is, the companies with the aim of complying with said regulation have to modify the implantation of their manufacturing strategies. 5. Conclusions With Russia’s ratiﬁcation of the Kyoto Protocol in November 2004, the industrialised countries which had signed the Treaty accounted for 55% of CO2 emissions, set as the point at which obligatory compliance would enter into effect. However, prior to this, signatory countries, especially in the European Union, had already begun drawing up legislation to comply with the goals set by the Protocol. Such is the case of Directive 2000/53/CE on Vehicle Management at the End of their Useful Life. The aim of this paper is to evaluate, by means of case studies, the consequences of said Directive. 13 of 17 Sustainability 2016, 8, 99 While it is true that actions required aimed at achieving compliance are the most difﬁcult ones for companies to put into practice, only measures with a preventive nature will allow the achievement of a competitive advantage from the natural environment management. This problem is especially relevant in manufacturing processes (Sanchez-Rodrígues, Potter and Naim 2010) [87], those most affected by environment problems at the companies. For this reason, we have analysed the effects which the compliance with preventive regulation may exercise on the future of a company’s manufacturing strategy, both in terms of its content and its process. As regards content, we have distinguished between the analysis of environmental protection as a production goal and the actions needed to develop its attainment. These latter may be of a structural type (installations, process technology, capacity and vertical integration) or infrastructural (new products, the role of employees, quality management, planning and control systems, relationships with clients and other members of the value chain, relationships with environmentally-aware stakeholders, systems to measure environmental performance and the environmental department’s organisation). In this paper we have shown how this process is necessary in the automotive industry to comply with preventive command-and-control environmental regulation. After an in-depth review of the literature, which studies the competitive effects stemming from environmental regulation and the effects linked to its inﬂuence on manufacturing strategy, we decided that the most suitable methodology was a case analysis. Given that our aim was to study regulation and its consequences evaluated by component suppliers and vehicle manufacturers, we chose subjects to study out of each group: two from the ﬁrst and one from the second. 5. Conclusions From the case studies and the review of the literature, we have been able to conﬁrm that, in general, both groups represent two sides of the same coin. However, the heterogeneity of the companies forming each group in terms of their ability to respond to the environmental challenge depends on factors such as the environmental strategy, the degree to which this is integrated into manufacturing strategy and communication between the various levels of the supply chain. Compliance with this new type of regulation usually affects some manufacturing decisions, although not all, and in different ways and intensity depending on whether supplier or manufacturer. Our results have important implications for research. It is the ﬁrst paper which has studied how new preventive command-and-control environmental regulation inﬂuences business strategy in the automotive industry. On the other hand, our results are useful for managers because they show what conditions let companies adapt their environmental approaches to the new environmental approaches successfully. Although this research has enabled us to make the progress shown previously, we cannot avoid its limitations: ﬁrstly, as regards external validation, which problem should be avoided with subsequent research, using surveys based on larger samples. As has been explained, this will be one of our objectives in future studies. A second limitation is based on the fact that the companies studied here are those that, out of a large number, were willing to cooperate (especially, among the parts suppliers). Therefore, the overview given here may show an optimistic bias, as it is reasonable to suppose that those not taking part felt to be at a disadvantage. On the other hand, some studies (Ramanathan et al., 2010) [88] have suggested the signiﬁcance of environmental regulation for the improvement of economic performance in several industrial sectors. As a result, future research should approach how these improvements can be achieved not only in the automotive industry, but in the other sectors. 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W2157436384.txt	https://respiratory-research.biomedcentral.com/track/pdf/10.1186/rr22	en		Mimicking microbial 'education' of the immune system: a strategy to revert the epidemic trend of atopy and allergic asthma?	Respiratory research	2,000	cc-by	2,917	http://respiratory-research.com/content/1/3/129 Commentary Mimicking microbial ‘education’ of the immune system: a strategy to revert the epidemic trend of atopy and allergic asthma? DASRS, Pomezia (Rome), and †Institute of Experimental Medicine, CNR, Rome, Italy Received: 30 June 2000 Revisions requested: 30 August 2000 Revisions received: 8 September 2000 Accepted: 8 September 2000 Published: 25 October 2000 commentary Paolo Maria Matricardi† and Sergio Bonini† Respir Res 2000, 1:129–132 The electronic version of this article can be found online at http://respiratory-research.com/content/1/3/129 © Current Science Ltd (Print ISSN 1465-9921; Online ISSN 1465-993X) Abstract Introduction microbes of their ecosystem alter the immune balance at mucosal level between type 1 (Th1, Tcl) and type 2 (Th2, Tc2) immunity, thereby predisposing to atopic diseases [5,6], including allergic asthma. Partially deprived of appropriate microbial stimulation, type 1 immune mechanisms would no longer downregulate the hypersensitivityand allergy-causing type 2 response to a sufficient degree [1,5–11]. Although the hygiene hypothesis remains a hypothesis, it raises questions. Must we go back to living in ‘dirty’ primary research Allergic asthma is on the increase in Western countries, as are efforts to identify the reasons for this increase [1]. This trend is part of a generalized increase in prevalence of atopic conditions that are characterized by mucosal eosinophilic inflammation, such as allergic rhinoconjunctivitis and atopic eczema [1,2]. The allergy ‘epidemic’ appears to parallel the overly hygienic conditions that are typical of affluent societies [3–5]. Hence, the hygiene hypothesis has been suggested. According to this hypothesis, changing interactions between humans and reports Keywords: allergy, asthma, DNA immunostimulatory sequences, epidemiology, lactobacilli, lipopolysaccharide, mycobacteria, prevention, therapy review Deficient microbial stimulation of the immune system, caused by hygiene, may underly the atopy and allergic asthma epidemic we are currently experiencing. Consistent with this ‘hygiene hypothesis’, research on immunotherapy of allergic diseases also centres on bacteria-derived molecules (eg DNA immunostimulatory sequences) as adjuvants for allergen-specific type 1 immune responses. If we understood how certain microbes physiologically ‘educate’ our immune system to interact safely with environmental nonmicrobial antigens, we might be able to learn to mimic their beneficial actions. Programmed ‘immunoeducation’ would consist of safe administration, by the correct route, dose and timing, of those microbial stimuli that are necessary to ‘train’ the developing mucosal immune system and to maintain an appropriate homeostatic equilibrium between its components. Overall, this would result in a prevention of atopy that is not limited to certain specific allergens. Although such a strategy is far beyond our present potential, it may in principle revert the epidemic trend of atopy and allergic asthma without jeopardizing the fight against infectious diseases. Respiratory Research Vol 1 No 3 Matricardi and Bonini conditions? Are allergy and asthma an unavoidable price that westernized societies must pay for the decline in morbidity and mortality from infectious diseases? Will ‘poor’ hygiene cure asthma? The answer to this provocative question is ‘no’. Many respiratory infections induce wheezing, and many cases of wheezing that are linked to recurrent respiratory infections are labelled ‘asthma’ or are perceived as such [12]. Some respiratory viruses (eg respiratory syncytial virus) induce asthma in predisposed individuals [13], whereas others (eg rhinoviruses) exacerbate pre-existing atopic bronchial inflammation [14]. The rising trend in severe asthma cases in poor, and hence supposedly less hygienic, urban areas of US cities (‘inner city asthma’) appears to refute the hygiene hypothesis [15]. Therefore, the words ‘dirty’ or ‘hygiene’ are too generic to be used to label environments that facilitate or protect against allergy. We must still learn whether and what kind of hygienic measures cause atopy, and generic antihygienic procedures would obviously facilitate the spread of infectious diseases. By contrast, the traditional lifestyle that is typical of anthroposophic [16] and farming communities [17] appears highly protective, given the very low prevalence of respiratory allergies in these groups. However, it remains to be established whether the lower prevalence of atopy is due to higher exposure to microbes or to other hallmarks of a rural lifestyle [18]. Less indirect support for the hygiene hypothesis came from epidemiological studies of Italian military cadets [19,20], which showed that exposure to food-borne and orofaecal infections, but not to air-borne viruses, was inversely associated with respiratory allergies. These serological studies support the notion that a high turnover of ingested microbes (mainly saprophytic, commensal and pathogen bacteria) at mucosal surfaces, in particular the gut mucosa, may ‘educate’ our mucosal immune system to interact safely with nonmicrobial antigens [21–24]. This would explain why the children of farmers and anthroposophic communities are protected against bronchial allergy and other atopic diseases [16,17,22,25]. Interestingly, the concentration of exogenous lipopolysaccharide in house dust was inversely related to atopy among infants at risk for asthma [26]. This suggests that not only ingested bacteria, but also bacterial immunostimulating substances from inhalable sources could also afford protection against allergy. Given these premises, we may ask if children living in US inner cities are exposed to a sufficiently diversified set of bacteria and if they eat sufficiently contaminated food. It would be interesting to determine the magnitude of exposure to orofaecal and food-borne infections [22], and to saprophytic bacteria-contaminated soil [11], in inner cities. It is tempting to speculate that allergic children living in those ‘unhygienic’ areas inhale and ingest a different kind, variety and amount of bacteria compared with children of farmers and anthroposophic families, who have access to natural soil and eat only biologically treated food (fresh vegetables and farm products). Bacteria and bacterial substances that may prevent atopy To exert an atopy-preventing effect, it is thought microbes must be present where and when allergen uptake, processing and presentation to T cells occur [9,10]. Appropriate bacteria would act as natural Th1 ‘adjuvants’ during the priming of T cells against newly encountered environmental antigens. Facultative and professional antigen-presenting cells (dendritic cells) may be the target of a microbial bystander effect, that may dictate the pattern of accessory molecules and cytokines, and modulate the outcome (Th1- or Th2-like) of the allergen-specific T-cell response [9,27]. At least five kinds of bacteria or bacterial substances are being examined for their proven or alleged atopy-preventing effect, and these are discussed below. Immunostimulatory sequences Short immunostimulatory DNA sequences with CpG motifs are 20 times as frequent in bacterial DNA as in mammalian DNA [28]. Immunostimulatory sequences are among the strongest of the known Th1-stimulating adjuvants; they suppress IgE responses and eosinophilic recruitment in vivo, and prevent allergic asthma in animal models [29]. Adjuvant DNA immunostimulatory sequences can provide part of the immunostimulatory effects of Freund’s adjuvant without the severe inflammatory and toxic side effects attributed to the paraffin oil and mycobacterial cell wall products [30]. Mycobacterium vaccae An inverse association between atopy and reactivity to tuberculin among Japanese children suggested that exposure to environmental mycobacteria inhibits atopic sensitization through stimulation of type 1 immunity [31], as suggested by animal experimental models [32]. Humans have been always exposed to mycobacteria, mainly through natural soil dust and contaminated food. Rook and Stanford [11] proposed that mycobacteria might therefore have influenced the evolution of the immune system because they have been ubiquitous throughout mammalian phylogeny. Consequently, the concrete paving of modern cities and food hygiene may have deprived westernized populations of a fundamental stimulus for the maturation of their mucosa-associated lymphoid tissue, thus contributing to the asthma epidemic [6,11,20]. http://respiratory-research.com/content/1/3/129 Lipopolysaccharide Oral bacterial extracts ‘Immunoeducation’: a novel strategy or an utopian goal? Bacteria or bacterial products are already being tested against allergic diseases. Encouraging preliminary data are coming from animal studies in which DNA immunostimulatory sequences are used as adjuvants with allergen for allergen-specific immunotherapy, and from trials with Homeostasis of the immune system is so complex and microbial exposure is so diversified that, at the present state of our knowledge, ‘immunoeducation’ is far beyond our reach. More feasible and specific immunological therapies or prophylactic measures may emerge from the ongoing studies referred to above. Ultimately, although poor hygiene will never cure asthma, the hygiene hypothesis may result in new strategies in the fight against the allergy and asthma epidemic. Acknowledgements We are indebted to Jean Ann Gilder for revising and editing the text. References 1. 2. 3. 4. 5. 6. 7. 8. 9. Woolcock AJ, Peat JK: Evidence for the increase of asthma worldwide. Ciba Found Symp 1997, 206:122–139. Von Mutius E, Martinez FD, Fritzsch C, Nicolai T, Roell G, Thiemann HH: Prevalence of asthma and atopy in two areas of West and East Germany. Am J Respir Crit Care Med 1994, 149:358–364. Strachan DP: Hay fever, hygiene and household size. Br Med J 1989, 299:1259–1260. Cookson WOCM, Moffatt MF: Asthma: an epidemic in the absence of infection. Science 1997, 275:41–42. Martinez FD, Holt PG: Role of microbial burden in aetiology of allergy and asthma. Lancet 1999, 354:12–15. Romagnani S: Regulation of the development of type 2 T-helper cells in allergy. Curr Opin Immunol 1994, 6:838–846. Martinez FD: Role of viral infections in the inception of asthma and allergies during childhood: could they be protective? Thorax 1994, 49:1189–1191. Strachan DP: Epidemiology of hay fever: towards a community diagnosis. Clin Exp Allergy 1995, 25:296–303. Holt PG: Environmental factors and primary T-cell sensitisation to inhalant allergens in infancy: reappraisal of the role of infections and air pollution. Pediatr Allergy Immunol 1995, 6:1–10. primary research Oral bacterial extracts have been claimed to stimulate immune responses against recurrent airway infections, but their therapeutic or preventive effect is far from proven [38]. Some allergists administer these preparations empirically as coadjuvants of oral allergen vaccines but, to our knowledge, scientific trials to evaluate this approach have not been performed. Conclusion reports Allergic 2-year-old Swedish and Estonian children were colonized less often by lactobacilli, and harboured higher counts of aerobic bacteria (coliforms, Staphylococcus aureus) than did nonallergic children [21]. Lactobacilli from human gastrointestinal mucosa are strong stimulators of IL-12 production by mononuclear cells in vitro [35] and of major histocompatibility class II molecules in vivo [36]. The ingestion of fermented vegetables, which are rich in lactobacilli, has been associated with a lower risk of atopy and allergies in children exposed to an anthroposophic lifestyle [16]. Evidence that gut flora may affect the induction of oral tolerance has promoted calls for clinical trials aimed at establishing whether lactobacilli-containing preparations are useful in allergic diseases [37]. Conversely, if we understood how microbes ‘educate’ our immune system we could perhaps learn to safely mimic their beneficial effect. Programmed ‘immunoeducation’ would consist of safe administration, by the correct route and at the correct dose and time schedule, of the variety of microbial stimuli that are required by the mucosal immune system during its development, and that are necessary to maintain an appropriate equilibrium between its components. This approach may be helpful in preventing atopy with a more ‘physiological’ stimulation, without the need of immunizing against all of the allergens that are potentially encountered during a whole lifetime. review Lactobacillus spp M vaccae (SRL172) administered before the pollination period in patients affected by seasonal respiratory allergies [39]. These strategies are aimed at reducing allergic reactivity in patients and at treating or preventing sensitization to single specific allergens. Hopefully, they may therefore improve our potential for immunotherapy or immunoprophylaxis of sensitization toward some selected allergens [40]; however, we would continue to observe a high prevalence of atopic diseases among populations following a hygienic lifestyle, because it would be difficult to immunize against any potential environmental or food allergenic molecules. commentary Endotoxin from the cell wall of Gram-negative bacteria promotes survival and maturation of dendritic cells [33]. It is among the most potent stimuli for production of IL-12, a key molecule in type 1 immunity, by these cells [34]. Lipopolysaccharide from gut commensal flora may provide a major stimulus for postnatal maturation of dendritic cell function at both peripheral tissue sites and central lymphoid compartments [9]. Thus, deficient stimulation by Gram-negative bacteria in the gastrointestinal tract may result in deficient maturation of antigen-presenting cells and lower propensity to develop Th1 responses toward environmental antigens [9,23]. Inhalation of ‘exogenous’ endotoxin from house dust may protect against atopy [26], but it should be noted that Gram-negative bacteria that colonize the gastrointestinal tract are by far a greater ‘endogenous’ source of endotoxin. Interestingly, the gut microflora of infants growing in developing countries is characterized by early colonization, high turnover rate and high strain diversity of Gram-negative bacteria (ie Escherichia coli), suggesting a higher exposure to endogenous lipopolysaccharide as compared with infants reared in developed countries [24]. Respiratory Research Vol 1 No 3 Matricardi and Bonini 10. Matricardi PM: Infections preventing atopy: facts and new questions. Allergy 1997, 52:879–882. 11. Rook GAW, Stanford JL: Give us this day our daily germs. Immunol Today 1998, 19:113–116. 12. Folkerts G, Busse WW, Nijkamp FP, Sorkness R, Gern JE: Virusinduced airway hyperresponsiveness and asthma. Am J Respir Crit Care Med 1998, 157:1708–1720. 13. Stein RT, Sherrill D, Morgan WJ, Holberg CJ, Halonen M, Taussig LM, Wright AL, Martinez FD: Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet 1999, 354:541–545. 14. Johnston SL, Bardin PG, Pattemore PK: Viruses as precipitants of asthma symptoms. III Rhinoviruses: molecular biology and prospects for future intervention. Clin Exp Allergy 1993, 23:237–246. 15. Platts-Mills TAE: Changes in the 20th century environment: do they explain the increase in asthma? In Proceedings of the NIAID Symposium ‘Asthma and Environment’. 1998 March 14; 54th AAAAI Annual Meeting, Washington. 1998:3–32. 16. Alm JS, Swartz J, Lilja G, Scheynius A, Pershagen G: Atopy in children with an anthroposophic lifestyle. Lancet 1999, 353:1485–1488. 17. Braun-Fahrlander C, Gassner M, Grize L, Neu U, Sennhauser FH, Varonier HS, Vuille JC, Wuthrich B: Prevalence of hay fever and allergic sensitization in farmer’s children and their peers living in the same rural community. SCARPOL team. Swiss Study on Childhood Allergy and Respiratory Symptoms with Respect to Air Pollution. Clin Exp Allergy 1999, 29:28–34. 18. Strachan DP: Lifestyle and atopy. Lancet 1999, 353:1457–1458. 19. Matricardi PM, Rosmini F, Ferrigno L, Nisini R, Rapicetta M, Chionne P, Stroffolini T, Pasquini P, D’Amelio R: Cross sectional retrospective survey of the prevalence of atopy among Italian military students with antibodies against hepatitis A virus. Br Med J 1997, 314:999–1003. 20. Matricardi PM, Rosmini F, Riondino S, Fortini M, Ferrigno L, Rapicetta M, Bonini S: Exposure to foodborne and orofecal microbes versus airborne viruses in relation to atopy and allergic asthma: epidemiological study. Br Med J 2000, 320:412–417. 21. Bjorksten B, Naaber P, Sepp E, Mikelsaar M: The intestinal microflora in allergic Estonian and Swedish 2-year-old children. Clin Exp Allergy 1999, 29:342–346. 22. Matricardi PM, Bonini S: High bacterial turnover rate preventing atopy: a solution to inconsistencies impinging on the hygiene hypothesis? Clin Exp Allergy 2000, 30:1506–1510. 23. Wold AE: The hygiene hypothesis revised: is the rising frequency of allergy due to changes in the intestinal flora? Allergy 1998, 53:20–25. 24. Adlerberth I, Jalil F, Carlsson B, Mellander L, Hanson LA, Larsson P, Khalil K, Wold AE: High turnover rate of Escherichia coli phenotypes in the intestinal flora of infants in Pakistan. Epidemiol Infect 1998, 121:587–598. 25. Matricardì PM, Rosmini F, Rapicetta M, Gasbarrini G, Stroffolini T: Atopy, hygiene and anthroposophic lifestyle [letter]. Lancet 1999, 354:430. 26. Gereda JE, Leung DY, Thatayatikom A, Streib JE, Price MR, Klinnert MD, Liu AH: Relation between house-dust endotoxin exposure, type 1 T-cell development, and allergen sensitisation in infants at high risk of asthma. Lancet 2000, 355:1680–1683. 27. Holt PF, Sly PD, Bjorksten B: Atopic versus infectious diseases in childhood: a question of balance? Pediatr Allergy Immunol 1997, 8:53–58. 28. Roman M, Martin-Orozco E, Goodman JS, Nguyen MD, Sato Y, Ronaghy A, Kornbluth RS, Richman DD, Carson DA, Raz E: Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nature Med 1997, 3:849–854. 29. Kline JN, Waldschmidt TJ, Businga TR, Lemish JE, Weinstock JV, Thorne PS, Krieg AM: Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma. J Immunol 1998, 160:2555–2559. 30. Spiegelberg HL, Orozco EM, Roman M, Raz E: DNA immunization: a novel approach to allergen-specific immunotherapy. Allergy 1997, 52:964–970. 31. Shirakawa T, Enomoto T, Shimazu SI, Hopkin JM: The inverse association between tuberculin responses and atopic disorder. Science 1997, 275:77–79. 32. Erb KJ, Holloway JW, Soibeck A, Moll H, Le Gros G: Infection of mice with Mycobacterium bovis-Bacillus Calmette-Guerin (BCG) 33. 34. 35. 36. 37. 38. 39. 40. suppresses allergen-induced airway eosinophilia. J Exp Med 1998, 187:561–569. Rescigno M, Granucci F, Citterio S, Foti M, Ricciardi-Castagnoli P: Coordinated events during bacteria induced maturation of dendritic cells. Immunol Today 1999, 20:200–203. Le J, Lin JX, Henriksen-Destefano D, Vilcek J: Bacterial lipopolysaccharide-induced interferon-gamma production: roles of interleukin 1 and interleukin 2. J Immunol 1986, 136:4525–4530. Hessle C, Hanson LA, Wold AE: Lactobacilli from human gastrointestinal mucosa are strong stimulators of IL-12 production. Clin Exp Immunol 1999, 116:276–282. Herias MV, Hessle C, Telemo E, Midtvedt T, Hanson LA, Wold AE: Immunomodulatory effects of Lactobacillus plantarum colonizing the intestine of gnotobiotic rats. Clin Exp Immunol 1999, 116:283–290. Famularo G, De Simone C: Oral bacteriotherapy. Immunol Today 1998, 19:486–487. Malling HJ: Bacterial vaccines: anything but placebo. Allergy 2000, 55:214–218. Holgate ST: Science, medicine, and the future: allergic disorders. Br Med J 2000, 320:231–234. Holt PG: Immunoprophylaxis of atopy: light at the end of the tunnel? Immunol Today 1994, 15:484–489. Authors’ affiliations: DASRS, RMAS, Laboratory of Immunology and Allergy, Pomezia (Rome), Italy (Paolo Maria Matricardi), and Institute of Experimental Medicine, CNR, Rome, Italy (Paolo Maria Matricardi and Sergio Bonini) Correspondence: Paolo M Matricardi, MD, DASRS – RMAS, Lab di Immunologia ed Allergologia, Aeroporto Pratica di Mare, 00040 Pomezia (Rome), Italy. Tel: +39 336 782 508; Fax: +39 06 7725 5269; e-mail: matricardi.pm@mclink.it
https://openalex.org/W4309215663	https://www.frontiersin.org/articles/10.3389/fpsyg.2022.993122/pdf	English	null	Fertility decision of Iranian women during the COVID-19 pandemic and home quarantine: A cross-sectional study in Iran	Frontiers in psychology	2,022	cc-by	4,421	OPEN ACCESS OPEN ACCESS EDITED BY Changiz Mohiyeddini, Oakland University William Beaumont School of Medicine, United States REVIEWED BY Federica Cavazzoni, University of Milano-Bicocca, Italy Linda G. Kahn, New York University, United States CORRESPONDENCE Parvin Abedi parvinabedi@ymail.com SPECIALTY SECTION This article was submitted to Health Psychology, a section of the journal Frontiers in Psychology RECEIVED 13 July 2022 ACCEPTED 26 October 2022 PUBLISHED 15 November 2022 CITATION Afshari P, Abedi P and Beheshtinasab M (2022) Fertility decision o Iranian women during the COVID-19 pandemic and home quarantine: A cross- sectional study in Iran. Front. Psychol. 13:993122. doi: 10.3389/fpsyg.2022.993122 COPYRIGHT © 2022 Afshari, Abedi and Beheshtinasab. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication i this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Poorandokht Afshari , Parvin Abedi and Maryam Beheshtinasab Midwifery Department, Reproductive Health Promotion Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Background: Fertility decision is defined as the mutual decision of partners toward having children, which may be equally controlled by the two partners or dominantly powered by the female partner. This study aimed to evaluate fertility decision of women during the COVID-19 pandemic in Iran. Methods: This cross-sectional study was conducted on 600 women (300 pregnant and 300 non-pregnant) during the COVID-19 pandemic. A demographic questionnaire and the Attitudes toward Fertility and Childbearing Scale were used to collect the data. Independent t-test, Chi-square test, and logistic regression were used for analyzing data. Results: The mean ± SD age of participants with negative and positive attitude toward fertility was 28.96 ± 5.88 and 28.39 ± 6.2, respectively. Almost half of the studied women postponed their pregnancy to after the pandemic. The total score of fertility decision in women with positive attitudes toward fertility was 95.43 ± 18.51 compared to 46.73 ± 17.2 in women with negative attitudes toward fertility (p < 0.0001). None-employed women had 0.595 times the odds of having a positive attitude toward fertility (95% CI: 0.423–0.836). Women who were not pregnant had 1.5 times the odds of having a positive attitude toward childbearing (95% CI: 1.067–2.112). TYPE Original Research PUBLISHED 15 November 2022 DOI 10.3389/fpsyg.2022.993122 TYPE Original Research PUBLISHED 15 November 2022 DOI 10.3389/fpsyg.2022.993122 TYPE Original Research PUBLISHED 15 November 2022 DOI 10.3389/fpsyg.2022.993122 COVID-19, pandemic, fertility decision, home quarantine, Iran COPYRIGHT © 2022 Afshari, Abedi and Beheshtinasab. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Data collection tools A demographic questionnaire and the Attitudes toward Fertility and Childbearing Scale were used to collect the data. The demographic questionnaire included questions regarding age, occupation, education, age, husband’s occupation and educational attainment, number of children, infection with COVID-19, hospitalization, and vaccination. The Attitudes toward Fertility and Childbearing Scale (AFCS) was initially developed by Soderberg et al. and has 27 questions in three subscales including the importance of fertility for the future, childbearing as a barrier at the present time, and social identity (Soderberg et al., 2013). It is scored based on a 5-item Likert scale, with 5 indicating “strongly agree” and 1 indicating “strongly disagree.” A higher score indicates more positive attitudes toward fertility and childbearing. OPEN ACCESS In addition, women who were not hospitalized during the pandemic had 0.520 times the odds of having a positive attitude toward fertility (95% CI: 0.342–0.790). Conclusion: The results of this study showed that half of the women postponed their pregnancy to after the pandemic. Also, employed women, women who were not pregnant, and women who were not hospitalized during pandemic were more likely to have positive attitudes toward fertility during the COVID-19 pandemic. Policymakers should devise some strategies to clarify the benefits and harms of pregnancy during crises such as COVID-19 pandemic. COVID-19, pandemic, fertility decision, home quarantine, Iran Frontiers in Psychology 01 frontiersin.org frontiersin.org 10.3389/fpsyg.2022.993122 Afshari et al. Sampling The following formula was used for sampling: ( ) 2 1 /2 2 Z 1 α − × − = p p n d 1−α/2 = 1.96, p = 50%, d = 0.4, N = 600. ( ) 2 1 /2 2 Z 1 α − × − = p p n d 1−α/2 = 1.96, p = 50%, d = 0.4, N = 600. 1−α/2 = 1.96, p = 50%, d = 0.4, N = 600. Introduction pandemic. The design of this study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences (Ref. ID: IR.AJUMS.REC.1400.334). All women provided written informed consent prior to data collection, and participants were assured about confidentiality of data. Fertility decision is defined as mutual decision of partners toward having children, which may be equally controlled by the two partners or dominantly powered by the female partner (Stein et al., 2014). Factors such as age, education, economic status, and desired number of children can influence fertility decision of couples (Adhikari, 2010). While factors such as higher educational attainment, availability of contraceptive methods, and Gross Domestic Products (GDP) may be negatively correlated with fertility decision, religion seems to have a positive impact on it (Götmark and Andersson, 2020). Iran is a country located in the Middle East and has a population of 83,992,949 (Worldometer, 2022a). Evidence shows that fertility rate is under replacement in Iran (1.5 children per woman during reproductive age; Hosseini et al., 2021). A recent study in Iran showed that those participants with a higher age-gap with their husbands and with having more children had more negative attitudes toward fertility, and those who had husbands with higher level of education, and higher score of social and emotional factors were more positive toward fertility (Jafarzadeh Esfehani et al., 2019). Inclusion/exclusion criteria Eligible participants were pregnant or non-pregnant monogamous women in reproductive age (18–40 years), who had basic literacy. Women with a history of infertility and medical contraindication for pregnancy were excluded from the study. Setting The public health centers are classified into the west and east banks of Karun River in Ahvaz city, and in this study, three centers from each bank were chosen. Eligible women in these centers received a phone call and then a link to access a website that was provided for them. Like all other countries across the globe, Iran faced the COVID-19 pandemic in early 2020. According to statistics, 7.2 million people have so far been affected with COVID-19 infection and 141,000 individuals have died from this infection (Worldometer, 2022b). In addition to mortality, the COVID-19 pandemic has changed many aspects of everyday life including couples’ fertility decisions. For example, according to Albeitawi et al., around 80% of Jordanian women believed that pregnancy during the pandemic is dangerous, and infertile women did not like to use assisted reproductive technologies for pregnancy (Albeitawi et al., 2022). Also, Ghaznavi et al. found that the number of births in Japan declined during the pandemic (Ghaznavi et al., 2022). No study has directly addressed fertility decision of couples during the COVID-19 pandemic in Iran, a study by Peivandi et al. reported that Iranian infertile couples were not willing to continue their infertility treatment because of fear of transmission of infection to the fetus (Peivandi et al., 2022). Confirming the results of Peivandi et al., Kamath et al. in India found that almost one-third of infertile couples paused their treatment and postponed it until after pandemic (2021). Also, another study in the United States showed that out of 16,000 couples who decided to conceive before COVID-19 pandemic, almost 70% decided to postpone their pregnancy temporarily (Malloy and Bradley, 2021). Given the insufficient information regarding fertility decision during COVID-19 pandemic in Iran, this study was conducted to evaluate fertility decision and its effective factors among Iranian reproductive-aged women during the COVID-19 pandemic. Frontiers in Psychology frontiersin.org Materials and methods The psychometric properties of the Persian version of this questionnaire were assessed by Baezzat et al. (2017). During the psychometric evaluation of this questionnaire in Iran, the number This was a cross-sectional study conducted on 600 (300 pregnant and 300 non-pregnant women) during the COVID-19 02 Frontiers in Psychology frontiersin.org frontiersin.org Afshari et al. 10.3389/fpsyg.2022.993122 TABLE 1 Demographic characteristics of two groups with positive and negative attitudes toward pregnancy and childbearing. TABLE 1 Demographic characteristics of two groups with positive and negative attitudes toward pregnancy and childbearing. of questions was reduced to 23, and the number of subscales rose to four, namely “children as a pillar of life (questions 1, 2, 3, 4, 6, 8, 21, and 23),” child as an obstacle (questions 13, 14, 15, 16, 17, 18),” “postponing fertility to future (questions 5, 7, 9, 11, 12),” and “fertility after fulfilment of preconditions (for example completing education or saving money, questions 10, 19, 20, and 22).” It is worth mentioning that questions 9–20 are scored negatively. The maximum and minimum scores of this questionnaire are 115 and 23, respectively. Because questionnaire does not have a cutoff point, we classified women according to the mean of the scores. Materials and methods We considered scores more than the mean of AFCS as indicating “positive attitudes toward fertility” and those below the mean indicating “negative attitudes toward fertility.” Variables Negative attitude toward pregnancy n = 331 Positive attitude toward pregnancy n = 269 p Value Mean ± SD or N (%) Age (year) 28.96 ± 5.88 28.39 ± 6.2 0.244 Age of husband (year) 29.96 ± 5.57 29.55 ± 5.82 0.914 Living child 0.91 ± 0.92 0.89 ± 0.99 0.22 Length of marriage (year) 4.51 ± 3.22 4.40 ± 3.2 0.660 Number of children 0.92 ± 0.99 0.87 ± 0.91 0.516 Education High school 80 (24.2) 63 (23.4) 0.977 Diploma 195 (58.9) 160 (59.5) University degree 56 (16.9) 46 (17.1) Occupation Housewife 172 (52) 102 (37.9) <0.0001 Employee 159 (48) 167 (62.1) Education of husband High school 69 (20.8) 57 (21.2) 0.290 Diploma 190 (57.4) 167 (62.1) University degree 72 (21.8) 45 (16.7) Occupation of husband Un-employed 38 (11.5) 31 (11.5) 0.055 Self-employed 115 (34.7) 87 (32.3) Employed 178 (53.8) 151 (56.2) Economic status Good 60 (18.1) 58 (21.6) 0.475 Moderate 179 (54.1) 145 (53.9) Poor 92 (27.8) 66 (24.5) Affected with COVID-19 disease Yes 182 (55) 135 (50.2) 0.138 No 149 (45) 134 (49.8) Admission to hospital Yes 113 (34.1) 63 (23.4) 0.003 No 218 (65.9) 206 (76.6) Do you find COVID-19 dangerous? Yes 176 (53.2) 156 (58) 0.136 No 155 (46.8) 113 (42) Have you had vaccination? Yes (1 dose) 71 (21.5) 54 (20.1) 0.507 Yes (2 doses) 92 (27.8) 85 (31.6) No 168 (50.7) 130 (48.3) Pregnancy during pandemic Yes 183 (55.2) 117 (43.5) 0.003 No 148 (44.7) 152 (56.5) Procedure The phone numbers of eligible women were obtained from the six public health centers. The women subsequently received a phone call, and in case they were willing, they were requested to complete two questionnaires on a web page. The first page before the questionnaires start was dedicated to informed consent. Women were requested to sign this form before answering the questions. One of the researchers (MB) was available in case the participants had any question. Data collection started in September 2021 and was completed in March 2022. Statistics All data were entered into SPSS version 23. Normal distribution of continuous data was evaluated using Shapiro–Wilk test. Independent t-test and Chi-square tests were used for comparing continuous and categorical data, respectively. Logistic regression was used to test the association between attitudes toward fertility and other variables. p < 0.05 was considered statistically significant. Results We invited 750 women to participate in this study, of which 150 of them did not respond, or did not complete questionnaires, and 600 (300 pregnant and 300 non-pregnant women) returned completed questionnaires. Table 1 shows the demographic characteristics of the participants with negative and positive attitudes toward childbearing during the COVID-19 pandemic. As evident from this table, the two groups did not show any significant difference regarding age, age of husband, length of marriage, and number of children. A significantly higher rate of employment and significantly lower rates of pregnancy and hospitalization due to COVID-19 were observed in women with positive attitudes toward childbearing. Apart from these, the two groups did not have any other significant differences regarding other variables. Table 2 shows the views of participants in the two groups of negative and positive attitudes toward fertility according to AFCS questionnaire. As this table shows, women with a positive attitude toward pregnancy and childbearing were more likely to consider a “child as a pillar of life,” and were less likely to consider a “child Table 2 shows the views of participants in the two groups of negative and positive attitudes toward fertility according to AFCS g y g questionnaire. As this table shows, women with a positive attitude toward pregnancy and childbearing were more likely to consider a “child as a pillar of life,” and were less likely to consider a “child Frontiers in Psychology 03 frontiersin.org 10.3389/fpsyg.2022.993122 Afshari et al. of complications and death of the pregnant mother in case of getting infected, reluctance to be vaccinated during pregnancy, no adequate quarantine during pregnancy due to employment, and the risk of pregnancy during the COVID-19 pandemic. as an obstacle” or to be considering “postponing fertility to future” or “after fulfillment of preconditions” (p < 0.0001). The total score of fertility decision in women with positive attitude toward fertility was 95.43 ± 18.51 compared to 46.73 ± 17.2 in women with negative attitude toward fertility (p < 0.0001). For a better understanding of the relationship between attitudes toward childbearing, logistic regression was used, and the results are presented in Table 4. As this table shows, none-employed women had 0.595 times the odds of having a positive attitude toward fertility (95% CI: 0.423–0.836). Women who were not pregnant had 1.5 times the odds of having a positive attitude toward childbearing (95% CI: 1.067–2.112). Results In addition, women who were hospitalized during the pandemic had 0.520 times the odds of having a positive attitude toward fertility (95% CI: 0.342–0.790). Table 3 shows the reasons for lack of interest in childbearing during the COVID-19 pandemic among the two groups with negative and positive attitudes toward pregnancy. The two groups did not show any significant difference regarding factors such as inappropriate social conditions during the pandemic (losing job, or income reduction), the unknown effect of the COVID-19 infection on the fetus and the pregnant mother, higher probability TABLE 2 Comparison of scores of fertility decision among two groups with positive and negative attitudes toward pregnancy and childbearing during the COVID-19 pandemic. Reasons TABLE 4 Logistic regression for assessing the relationship between demographic characteristics and having a positive attitude toward pregnancy Model Odds ratio 95% CI Lower Upper None employed vs. employed 0.595 0.423 0.836 No Pregnancy during pandemic vs. pregnancy during pandemic 1.501 1.067 2.112 Good Economic status vs. Poor 1.294 0.788 2.126 Moderate Economic status vs. Poor 1.030 0.690 1.538 One dose Vaccination vs. no vaccination 0.958 0.617 1.486 Two doses Vaccination vs. no vaccination 1.006 0.678 1.493 No hospitalization vs. hospitalization 0.520 0.342 0.790 Affected with COVID-19 infection vs. not affected with infection 1.009 0.683 1.472 Discussion Components Negative toward pregnancy n = 331 Positive toward pregnancy n = 269 p Value Mean ± SD The child as a pillar of life 11.36 ± 6.53 46.98 ± 9.34 <0.0001 Child as an obstacle 12.69 ± 5.59 23.68 ± 4.66 <0.0001 Postponing fertility to the future 6.68 ± 2.80 16.21 ± 2.40 <0.0001 Fertility after fulfilment of preconditions 15.81 ± 2.28 8.56 ± 2.11 <0.0001 Total scores 46.73 ± 17.2 95.43 ± 18.51 <0.0001 This study was designed to evaluate the fertility decision of Iranian women in the reproductive age during the COVID-19 pandemic. The results of the present study showed that almost half of the studied women postponed their pregnancy to after the pandemic, which is consistent with Chu et al. in China, who found that fertility intention of 47.7% of women was affected by COVID-19 outbreak (Chu et al., 2022). Results of a study by Albeitawi et al. on 814 fertile and infertile women in Jordan showed that 58.7% of fertile and 76.6% of infertile women believed that pregnancy during pandemic is dangerous (Albeitawi et al., TABLE 4 Logistic regression for assessing the relationship between demographic characteristics and having a positive attitude toward pregnancy and childbearing pregnancy. Reasons Negative toward pregnancy n = 331 Positive toward pregnancy n = 269 p Value N (%) Inappropriate social conditions during the pandemic 75 (22.7) 56 (20.8) 0.48 The unknown effect of the COVID-19 infection on the fetus and pregnant mother 33 (10) 21 (7.8) 0.39 More possibility of complications and death of the pregnant mother in case of infection 20 (6) 18 (6.7) 0.43 Reluctance to be vaccinated during pregnancy 40 (12.1) 29 (10.8) 0.70 Being employed does not allow adequate quarantine during pregnancy 149 (45) 127 (47.2) 0.62 Pregnancy is not dangerous during the COVID-19 pandemic 14 (4.2) 18 (6.7) 0.20 Negative toward pregnancy n = 331 Positive toward pregnancy n = 269 p Value Frontiers in Psychology Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Our results indicated that women who were not hospitalized were more positive toward fertility. In line with our results, Akinyemi et al. (2022) in a study on 774 married and non-pregnant women found that COVID-19 pandemic caused women to postpone their pregnancy, and this decision was related to age, higher education, and household food insecurity. Model 04 Afshari et al. 10.3389/fpsyg.2022.993122 10.3389/fpsyg.2022.993122 some limitations. First, we did not recruit women randomly, and this may affect the generalizability of the study. Second, we did not assess the opinion of husbands about fertility decision. Future studies may wish to consider the view-points of husbands in this regard. 2022), which is consistent with our results. Furthermore, Kahn et al. who evaluated 1,179 mothers of young children during COVID-19 pandemic found that around 40% of these women postponed their pregnancy to after the pandemic (Kahn et al., 2021) which is in line with our study. Contrary to our results, however, a study in sub-Saharan, Kenya showed that almost 85% of participants insisted on their fertility decision during the COVID-19 pandemic (Zimmerman et al., 2022). The reason for this discrepancy may be due to the fact that COVID-19 virus in sub-Saharan Africa may not be regarded as dangerous as it is in other countries. For example, a study by Osuagwu et al. (2022) showed that 72.2 and 84.5% of sub-Saharan people were resistant to or hesitant about COVID-19 vaccination, respectively. Acknowledgments This study was derived from a research project supported financially by Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. We would like to thank all women who participated in this study. In conclusion, women who had positive attitudes toward fertility during pandemic were willing to postpone their pregnancy to the future, but they believed that a child is a pillar of life. Ethics statement The studies involving human participants were reviewed and approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences (Ref. ID: IR.AJUMS. REC.1400.334). The patients/participants provided their written informed consent to participate in this study. Evidence shows that females and those who are suspicious of having COVID-19 disease were more likely to be afraid of the disease (Quadros et al., 2021). Studies show that women generally are more fearful and have more negative expectations about the consequences of a pandemic compared with men (Alsharawy et al., 2021). Our results showed that pregnant women have more negative attitudes toward childbearing. A qualitative study by Sahin and Kabakci (2021) in Turkey showed that pregnant women during pandemic experienced more fear and anxiety, which create exerts negative emotional effects on pregnant women. Author contributions All authors equally contributed to the conception of the study. MB collected the data. All authors were involved in data analysis and interpretation. PaA prepared the manuscript. All authors contributed to the article and approved the submitted version. Our results also showed that women with a positive attitude obtained significantly higher scores from the subscales of “considering children as a pillar of life,” having child as an obstacle, “postponing pregnancy to future,” and “fertility after fulfilment of preconditions,” and a higher total score of fertility decision compared to women with a negative attitude toward fertility during the COVID-19 pandemic. Frontiers in Psychology Conclusion The results of this study showed that half of the studied women postponed their pregnancy to after the pandemic. Also, employed women, women who were not pregnant, and women who were not hospitalized during the pandemic were more likely to have positive attitudes toward fertility during COVID-19 pandemic. Policymakers should pursue strategies to clarify the benefits and harms of pregnancy during crises such as the COVID-19 pandemic. According to our results, employed and non-pregnant women had a positive attitude toward fertility. Although employment was not assessed in other studies, a study by Fakehi et al. (2021) showed that nulliparous Iranian women with higher level of education, and better economic status had negative attitude toward fertility during pandemic that is not in line with our results. The reason for this discrepancy may be due to the fact that Fakehi et al. (2021) only recruited nulliparous women, while we recruited nulliparous, multiparous, and pregnant women. Strengths and limitations of the study This is the first scholarly attempt to evaluate the opinion of women (pregnant and non-pregnant) toward fertility during COVID-19 pandemic in Iran. Despite its merits, this study has The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 05 frontiersin.org frontiersin.org 10.3389/fpsyg.2022.993122 10.3389/fpsyg.2022.993122 Afshari et al. References Kahn, L. G., Trasande, L., Liu, M., Lee, S. S. M., Brubaker, S. G., and Jacobson, M. H. (2021). Factors associated with changes in pregnancy intention among women who were mothers of young children in new York City following the COVID-19 outbreak. JAMA Netw. Open 4:e2124273. doi: 10.1001/ jamanetworkopen.2021.24273 Adhikari, R. (2010). Demographic, socio-economic, and cultural factors affecting fertility differentials in Nepal. BMC Preg. Childbirth 10:19. doi: 10.1186/1471-2393-10-19 Akinyemi, J. O., Dipeolu, O. I., Adebayo, A. M., Gbadebo, B. M., Ajuwon, G. A., Olowolafe, T. A., et al. (2022). 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https://openalex.org/W3176380681	https://www.research.unipd.it/bitstream/11577/3449037/1/children-08-00541-v2.pdf	English	null	Monitoring Public Perception of Health Risks in Brazil and Italy: Cross-Cultural Research on the Risk Perception of Choking in Children	Children	2,021	cc-by	9,398	Abstract: One of the most relevant public health issues among pediatric injuries concerns foreign body (FB) aspiration. The risk perception of choking hazards (CH) and risk perception, in general, are complex multifactorial problems that play a signiﬁcant role in deﬁning protective behavior. Risk prevention policies should take this aspect into account. A lack of scientiﬁc knowledge of FB injury risk perception may be evidenced in Brazil and other newly developed countries. This study aims to characterize the differences and peculiarities in risk perception of CH between Italian and Brazilian populations. The risk perception among adults in Italy and Brazil between September and October 2017 was investigated in a survey. A Multiple Correspondence Analysis was carried out to identify the latent components characterizing the risk perception in Italian and Brazilian population samples. The most relevant dimension characterizing risk perception is the “Professional–educational status and the related perception of Risk” (13% of factorial inertia). The Italians identify batteries and magnets as the most dangerous choking risks (20% of responses). On the other hand, Brazilian people, mainly manual laborers (22%) with secondary or primary education (94%), perceive coins as the most dangerous items (30% of responses, p < 0.001). Socio-economic issues characterize the subjective risk perception of Italian and Brazilian survey respondents. In this framework, data-driven prevention strategies could be helpful to tailor intervention strategies to the cultural context to which they are addressed. Received: 15 May 2021 Accepted: 22 June 2021 Published: 24 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: (accidental) foreign body injuries/object injuries; hazard-choking; cross-cultural study Article Monitoring Public Perception of Health Risks in Brazil and Italy: Cross-Cultural Research on the Risk Perception of Choking in Children Alexander Hochdorn 1, Alexia Oliveira 2 , Giulia Lorenzoni 3 , Andrea Francavilla 3, Solidea Baldas 4, Paola Berchialla 5 , Alessandra Oliveira 2 , Vicente Paulo Alves 2 , Dario Gregori 3,* and Danila Azzolina 3,6 1 Department of Social and Work Psychology, University of Brasília, Brasília 72220-275, Brazil; alexander.hochdorn@gmail.com g 2 School of Medicine and Healthcare Sciences, Catholic University of Brasília, Brasília 72220-275, Brazil; alexia_v.o@hotmail.com (A.O.); a.oliveira53@gmail.com (A.O.); vicerap@gmail.com (V.P.A.) g p g 3 Unit of Biostatistics, Epidemiology and Public Health, Department of Cardiac Thoracic Vascular Sciences and Public Health, University of Padua, 35131 Padua, Italy; giulia.lorenzoni@unipd.it (G.L.); andrea.francavilla@studenti.unipd.it (A.F.); danila.azzolina@ubep.unipd.it (D.A.) p ( ); p 4 Prochild ONLUS, 34129 Trieste, Italy; solidea.baldas@prochild.eu y p 5 Department of Clinical and Biological Sciences, University of Torino, 10124 Torino, Italy; paola.berchialla@unito.it 6 Department of Medical Science, University of Ferrara, 44121 Ferrara, Italy ment of Medical Science, University of Ferrara, 44121 Ferrara, * Correspondence: dario.gregori@unipd.it * Correspondence: dario.gregori@unipd.it Citation: Hochdorn, A.; Oliveira, A.; Lorenzoni, G.; Francavilla, A.; Baldas, S.; Berchialla, P.; Oliveira, A.; Alves, V.P.; Gregori, D.; Azzolina, D. Monitoring Public Perception of Health Risks in Brazil and Italy: Cross-Cultural Research on the Risk Perception of Choking in Children. Children 2021, 8, 541. https:// doi.org/10.3390/children8070541 Academic Editor: Jason Gilliland Received: 15 May 2021 Accepted: 22 June 2021 Published: 24 June 2021 Citation: Hochdorn, A.; Oliveira, A.; Lorenzoni, G.; Francavilla, A.; Baldas, S.; Berchialla, P.; Oliveira, A.; Alves, V.P.; Gregori, D.; Azzolina, D. Monitoring Public Perception of Health Risks in Brazil and Italy: Cross-Cultural Research on the Risk Perception of Choking in Children. Children 2021, 8, 541. https:// doi.org/10.3390/children8070541 Academic Editor: Jason Gilliland Received: 15 May 2021 Accepted: 22 June 2021 Published: 24 June 2021 children children children 1. Introduction Risk perception, which deﬁnes how people think and feel about the risks they face, represents a cognitive issue and plays a signiﬁcant role in protective behavior [1,2]. Risk awareness is a multidimensional and complex aspect inﬂuenced by psychological and socio-cultural features [3–6]. The World Health Organization (WHO) speciﬁes that real- ity’s knowledge and experience deeply inﬂuence hazard perception, which is eventually associated with cultural aspects [7,8]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Given the complexity of risk perception, prevention policies should be tailored to the speciﬁc social framework [9]. Conversely, worldwide intervention policies are usually https://www.mdpi.com/journal/children Children 2021, 8, 541. https://doi.org/10.3390/children8070541 Children 2021, 8, 541 2 of 14 managed with general and non-speciﬁc guidelines [7]. In many circumstances, prevention activities are based on education [10]. This is the case of preventing childhood injuries, where policies are still in progress in the so-called Newly Industrialized Countries [11]. One of the most relevant public health issues among pediatric injuries concerns foreign body (FB) aspiration. In most cases, FBs are expelled spontaneously [12], but a not negligible proportion of FBs get stuck in the aero-digestive tract, leading to severe complications [13]. p Referring to the Brazilian context, different studies have been conducted to evaluate the prevalence of unintentional injuries and FB aspiration injuries in preschool children [14]. Some aimed to investigate the prevalence of FB ingestion in Brazilian settings [15]. In contrast, others (i.e., the WHO study) investigated the FB injury prevalence in Brazil and additional newly developed countries, focusing on a global perspective [16]. y p g g p p However, a lack of scientiﬁc knowledge and prevention policies concerning FB in- juries may be evidenced, mainly in Brazil and in newly developed countries [17]. These considerations justify the need to start up a pilot survey in Brazil to compare the results with a similar survey conducted in Italy, representing a developed country. The immediate identiﬁcation of a lack of interventions is crucial for the efﬁcient imple- mentation of culturally tailored prevention strategies. Handy tools offering an immediate picture of the intervention policy optimality have been poorly implemented. Recently, data- mining instruments have been proposed in the literature to provide a multidimensional comparison among countries in the ﬁeld of epidemiology of FB injuries [18]. 1. Introduction Previous research aimed to analyze the characteristics of FB injuries in Bosnia and Herzegovina, a newly industrialized country, by comparing cases from the other European countries via Multiple Correspondence Analysis (MCA) [18]. This paper aims to broaden the application of the existing tools to monitor the risk perception with potential impact on public health policies. The main idea is that once a speciﬁc country has been tested for prevention policies, other nations can be compared with it, and whatever differences are evidenced, if any, can be interpreted as an early signal of lack of implementations. Once the shortcomings in risk prevention policies have been identiﬁed, the intervention to be implemented should be tailored to the socio-economic context to which they refer [8,18]. Italy is at the cutting edge of a prevention strategy. In this direction, recent efforts were made on regulations, disseminating activities (safe food), and targeted initiatives to schools. For example, the Italian Choking Prevention project (CHOP) [19] was established to identify the most effective educational strategy for preventing FB injuries. The prevention policy’s actions should also consider the parent’s risk perception, which is an essential and culture-related element [7]. This work aims to characterize the main aspects of risk perception of choking hazard (CH) through an MCA analysis of Italy and Brazil as performed in similar research contexts for cross-cultural comparability purposes [18]. Italy was considered a benchmark because the country has implemented consolidated prevention strategies in the CH prevention area, while Brazil is a newly developed country where little is known about the practices adopted to prevent choking hazard events. Brazil still does not have enough tools to reduce the rate of aspiration of foreign bodies. The latest FB injury data from the Ministry of Health date back to 2012 [20]; the data revealed that 756 children up to 14 years old died from suffocation. The injuries caused by ingestion or aspiration of foreign bodies remain a serious public health problem in the pediatric population [21]. Data Collection Instrument The questionnaire has been developed in the context of the Susy Safe project [22] and has been used in other research settings as well [23]. It is administered to collect data on the risk perception of CH among adults having no formal health degree. The data collection Children 2021, 8, 541 3 of 14 tool includes the principal sociodemographic information: gender, age, employment type, and educational attainment. tool includes the principal sociodemographic information: gender, age, employment type, and educational attainment. The questionnaire includes ten images of jewelry, popcorn, batteries, hotdogs, toys, stationery, candies, coins, nuts, and seeds. These are the same items of the questionnaire previously administered in Italy [23]; they seem to represent most of the CH ﬁndings, particularly in a child aged 0–3 years [17]. Participants were asked to look at the images and answer by classifying a maximum of two items (per age group) that they considered to be the highest CH for children aged (A) less than 1-year-old; (B) 1–2 years old; and (C) 3–6 years old. According to the literature, the age classiﬁcation has been deﬁned considering that the risk of injury is highest in children between 1 and 2 years of age [24]. Additional details about the data collection tool are reported elsewhere [23]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted following the Declaration of Helsinki and approved by the local ethics committee (“Commissao Nacional de Ètica em Pasquisa” project identiﬁcation code = 17988619.7.0000.5553). No compensation was provided for the survey participants in both the Italian and Brazilian samples. 2.3. Statistical Analysis 2.3. Statistical Analysis 2.2. Adults without Health Education Survey Italian sample. The questionnaire was administered to 742 subjects not having a speciﬁc academic qualiﬁcation in any health ﬁeld between 2014 and 2016. The inclusion criteria were age over 18 years and the ability to read and speak Italian. The questionnaire was self-administered with a pencil and paper. Subjects were enrolled during multiple national events between 2014 and 2016. These public events aimed to sensitize adult supervisors on the issue of food choking injuries in children [23]. Only Italian respondents were selected to perform the analysis. Other details about questionnaire administration in the Italian sample can be found elsewhere [23]. p Brazilian sample. The questionnaire was administered to 172 subjects without a speciﬁc degree in healthcare sciences in 2017. The inclusion criteria were age over 18 years and the ability to understand and speak Portuguese. The questionnaire was not self-administrated since not all respondents could read and write in their native language. p g g The questionnaire was translated into Portuguese with the help of a committee [25]. In this case, two translators worked together to produce a consensus. g p All subjects have been recruited in the University Hospital of Ceilândia, a densely inhabited satellite town of Brazil’s Federal Capital Brasília. 2.3.1. Multiple Correspondence Analysis 2.3.1. Multiple Correspondence Analysis 2.3.1. Multiple Correspondence Analysis An approach based on Multiple Correspondence Analysis (MCA) was chosen to char- acterize respondents. MCA is a statistical method used to create multivariate contingency tables, and thus it does not require the deﬁnition of a dependent variable. MCA aims to summarize associations between variables in large, potentially complex datasets [26]. g p y p Orthogonal factorial axes (latent dimensions) were extracted in descending order, explaining the highest variability of the data matrix. The results were summarized in a two-dimensional subspace reporting the respective coordinates of individuals (respondents) and categories (variable modalities). For this study, individual and variable MCA summaries were reported: For this study, individual and variable MCA summaries were report For individuals, the relative coordinates were graphically reported in a biplot to identify similar groups according to their characteristics recognized by latent dimensions. A comparison between Italy and Brazil was performed in terms of median factor loadings (coordinates) regarding selected axes. Children 2021, 8, 541 4 of 14 For variable modality, the results were summarized considering the relative coordinate and contribution of each variable’s modality to the axis’s deﬁnition to identify the main characteristics contributing to the latent dimension. The standardized coordinates, presented as test values, were used to deﬁne speciﬁc characterization on each side of the latent dimensions. If the test value’s absolute value for one particular variable modality is superior to 2, then the speciﬁed coordinate is signiﬁcantly different from zero [27]. MCA was performed using the R packages FactomineR [28] and Factoextra [29]. 2.3.2. Descriptive Statistics Descriptive statistics were carried out according to the children’s country of origin (Italian vs. Brazil). Categorical data were reported as relative and absolute frequencies, continuous data as median and I and III quartiles. Wilcoxon rank sum tests were per- formed for continuous variables and the Pearson’s Chi-square test, or Fisher’s exact test, whatever appropriate, were used for categorical variables. Analyses were performed using R 3.5.2 [30]. 3. Results Variable N Brazil Italy Combined p-Value (N = 172) (N = 742) (N = 914) Gender: Female 902 159 (92%) 575 (79%) 734 (81%) <0.001 Male 13 (8%) 155 (21%) 168 (19%) Age 904 32.0 (25.0–39.0) 42.0 (37.0–49.0) 41.0 (34.0–48.0) <0.001 Job: Housewife 807 19 (11%) 62 (10%) 81 (10%) <0.001 Unemployed 17 (10%) 31 (5%) 48 (6%) Teacher/Ofﬁce Worker 24 (14%) 221 (35%) 245 (30%) Self-employed 60 (35%) 29 (5%) 89 (11%) Manager 2 (1%) 172 (27%) 174 (22%) Manual laborer 38 (22%) 63 (10%) 101 (13%) Retired 2 (1%) 24 (4%) 26 (3%) Student 10 (6%) 33 (5%) 43 (5%) Number of children: 0 887 1 (1%) 103 (14%) 104 (12%) <0.001 1–2 105 (61%) 529 (74%) 634 (71%) 3–4 56 (33%) 83 (12%) 139 (16%) >4 10 (6%) 0 (0%) 10 (1%) Education: Higher education 895 104 (60%) 371 (51%) 475 (53%) <0.001 Primary education 58 (34%) 56 (8%) 114 (13%) Post-secondary education 10 (6%) 296 (41%) 306 (34%) Hazardous items for children < 1 year (ﬁrst response): Batteries 894 5 (3%) 144 (20%) 149 (17%) <0.001 Candies 4 (2%) 129 (18%) 133 (15%) Coins 51 (30%) 34 (5%) 85 (10%) Hotdog 3 (2%) 31 (4%) 34 (4%) Jewelry 56 (33%) 135 (19%) 191 (21%) Nuts 1 (1%) 9 (1%) 10 (1%) Popcorn 18 (10%) 62 (9%) 80 (9%) Seeds 15 (9%) 4 (1%) 19 (2%) Stationery 4 (2%) 12 (2%) 16 (2%) Toys 15 (9%) 162 (22%) 177 (20%) 5 of 14 Children 2021, 8, 541 Table 1. Cont. 3. Results Overall, 742 Italian and 172 Brazilian questionnaires were administered (Table 1). In Italy, 79% of the respondents were female compared to 92% in the Brazilian survey (p < 0.001). Overall, 742 Italian and 172 Brazilian questionnaires were administered (Table 1). In Italy, 79% of the respondents were female compared to 92% in the Brazilian survey (p < 0.001). Table 1. Characteristics of Italian and Brazilian respondents. Continuous data are reported as median (I, III quartiles); categorical data are reported as percentages and absolute frequencies. Wilcoxon rank sum tests were performed for continuous variables and the Pearson’s Chi-square test, or Fisher’s exact test, whatever appropriate, for categorical variables. Table 1. Characteristics of Italian and Brazilian respondents. Continuous data are reported as median (I, III quartiles); categorical data are reported as percentages and absolute frequencies. Wilcoxon rank sum tests were performed for continuous variables and the Pearson’s Chi-square test, or Fisher’s exact test, whatever appropriate, for categorical variables. 3. Results Variable N Brazil Italy Combined p-Value (N = 172) (N = 742) (N = 914) Hazardous items for children < 1 year (second response): Batteries 677 1 (5%) 33 (5%) 34 (5%) 0.069 Candies 1 (5%) 126 (19%) 127 (19%) Coins 9 (45%) 219 (33%) 228 (34%) Hotdog 0 (0%) 11 (2%) 11 (2%) Jewelry 0 (0%) 0 (0%) 0 (0%) Nuts 0 (0%) 70 (11%) 70 (10%) Popcorn 1 (5%) 20 (3%) 21 (3%) Seeds 7 (35%) 77 (12%) 84 (12%) Stationery 0 (0%) 29 (4%) 29 (4%) Toys 1 (5%) 72 (11%) 73 (11%) Hazardous items for children aged 1–2 years (ﬁrst response): Batteries 888 7 (4%) 167 (23%) 174 (20%) <0.001 Candies 11 (6%) 119 (17%) 130 (15%) Coins 54 (31%) 22 (3%) 76 (9%) Hotdog 2 (1%) 51 (7%) 53 (6%) Jewelry 33 (19%) 68 (9%) 101 (11%) Nuts 3 (2%) 9 (1%) 12 (1%) Popcorn 11 (6%) 74 (10%) 85 (10%) Seeds 4 (2%) 3 (0%) 7 (1%) Stationery 10 (6%) 30 (4%) 40 (5%) Toys 37 (22%) 173 (24%) 210 (24%) Hazardous items for children aged 1–2 years (second response): Batteries 677 2 (10%) 20 (3%) 22 (3%) <0.001 Candies 2 (10%) 190 (29%) 192 (28%) Coins 6 (29%) 193 (29%) 199 (29%) Hotdog 0 (0%) 26 (4%) 26 (4%) Jewelry 0 (0%) 0 (0%) 0 (0%) Nuts 1 (5%) 85 (13%) 86 (13%) Popcorn 3 (14%) 13 (2%) 16 (2%) Seeds 5 (24%) 27 (4%) 32 (5%) Stationery 1 (5%) 34 (5%) 35 (5%) Toys 1 (5%) 68 (10%) 69 (10%) Hazardous items for children aged 3–6 years (ﬁrst response): Batteries 886 15 (9%) 167 (23%) 182 (21%) <0.001 Candies 22 (13%) 119 (17%) 141 (16%) Coins 30 (18%) 22 (3%) 52 (6%) Hotdog 8 (5%) 51 (7%) 59 (7%) Jewelry 12 (7%) 68 (9%) 80 (9%) Nuts 4 (2%) 9 (1%) 13 (1%) Popcorn 9 (5%) 74 (10%) 83 (9%) Seeds 7 (4%) 3 (0%) 10 (1%) Stationery 21 (12%) 30 (4%) 51 (6%) Toys 42 (25%) 173 (24%) 215 (24%) Hazardous items for children aged 3–6 years (second response): Batteries 676 1 (5%) 20 (3%) 21 (3%) 0.01 Candies 2 (10%) 190 (29%) 192 (28%) Coins 8 (40%) 193 (29%) 201 (30%) Hotdog 1 (5%) 26 (4%) 27 (4%) Jewelry 0 (0%) 0 (0%) 0 (0%) Nuts 0 (0%) 85 (13%) 85 (13%) Popcorn 0 (0%) 13 (2%) 13 (2%) Seeds 4 (20%) 27 (4%) 31 (5%) Stationery 3 (15%) 34 (5%) 37 (5%) Toys 1 (5%) 68 (10%) 69 (10%) Table 1. 3. Results Cont. Children 2021, 8, 541 6 of 14 Table 1. Cont. Variable N Brazil Italy Combined p-Value (N = 172) (N = 742) (N = 914) Have any children in your household ever experienced risk of choking?: No 840 134 (78%) 462 (69%) 596 (71%) 0.024 Yes 38 (22%) 206 (31%) 244 (29%) What object caused the accident?: Food 222 10 (26%) 149 (81%) 159 (72%) <0.001 Non-Food 28 (74%) 35 (19%) 63 (28%) Involving risk of death: No 480 26 (68%) 391 (88%) 417 (87%) 0.001 Yes 12 (32%) 51 (12%) 63 (13%) Table 1. Cont. The differences in occupational compositions of the two samples are signiﬁcant (p < 0.001); the majority of Brazilian respondents were manual laborers (35%) or were self-employed (22%). In Italy, instead, the greater part of the sample comprises ofﬁce workers (35%) and managers (27%). In the Brazilian sample, 33% of subjects have at least three children, while, in the Italian sample, this proportion is smaller and equal to 12% (p < 0.001). According to the educational level, 6% of Brazilian respondents hold post-degree educational attainment compared to 41% of Italian respondents (p < 0.001). The Italian sample perceived the batteries and toys as the most hazardous items for their children aged less than one year. Brazilians perceived coins and jewelry instead as more harmful. The second lead choice for Italians was “coins and candies”; while Brazilians chose “coins and seed” (Table 1). Considering the risk perception for 1- and 2-year-old children, the Italians consider the most dangerous FB items to be batteries and toys. Coins and toys were the ﬁrst choices for Brazilians. The second-choice distribution is consistent with the responses provided for the children aged less than 1 year (Table 1). For 3-year-old and older children, the Italians perceive toys and batteries as the most dangerous objects. Brazilians instead think the most dangerous ones are toys and coins. The second choice is candies and coins for Italians, and stationery and coins for Brazilian respondents (Table 1). A higher proportion of Italian respondents (31%) experienced choking accidents among their children compared to Brazilians (22%), p = 0.024 (Table 1). Among Italians, in- jury accidents were mainly due to food ingestion, 81% vs. 26% among Brazilians (p < 0.001), and a smaller proportion of them involved the risk of death (12% vs. 32%, p < 0.001) (Table 1). 3. Results Most Italian adults (86%), having at least one child, experienced the risk of choking at least once in comparison with the Brazilian sample (p = 0.024) (Table S1). Among them, a considerable proportion of adults has more than four children (24%), p < 0.001 (Table S1, Supplementary Materials). The 9% of the adults experiencing a choking hazard for their child perceived batteries as the second hazardous item compared to the 3% in the remaining sample (p = 0.027) (Table S1, Supplementary Materials). Finally, comparing Italian and Brazilian countries, the frequency of adults in manage- rial or teaching positions reporting FB injury episodes in their children is similar (Table S2, Supplementary Materials). MCA Results The ﬁrst two latent dimensions explain 20% of the overall variability. The ﬁrst latent dimension explains most of the overall inertia (13%). The most important contributors to the ﬁrst dimension are the professional status of respondents (self-employment working status) and educational attainment (primary education Level) (Table 2). 7 of 14 Children 2021, 8, 541 Table 2. Contribution (percentage) and test values (standardized coordinates) of variables’ levels for each of the two dimensions. In bold, the most important contributors are deﬁned as variables’ levels with a greater percentage of explained data variance. In bold are also reported the greater test values (absolute values) on both positive and negative sides of the axes. The medians of coordinates of factors contributing to each dimension are for “Professional-educational status and perception of Risk” (Dim 1): 0.83 (Brazil) and −0.24 (Italy); for “Factors affecting Risk perception in children <1 year” (Dim 2): 0.01 (Brazil) and 0.07 (Italy). MCA Results ) ( ) ( y) Categories Professional−Educational Status and Perception of Risk (Dim 1) Factors Affecting Risk Perception in Children <1 Year (Dim 2) Contributions Test Value Contributions Test Value Gender: Female 0.23 2.67 0.23 −2.01 Male 1.03 −2.67 1.02 2.01 Job: Housewife 0.06 0.62 0.4 −1.19 Unemployed 0.48 1.7 0.18 −0.78 Teacher/Ofﬁce Worker 1.64 −3.65 0.03 −0.34 Self-employed 5.4 5.85 0.08 −0.53 Manager 1.98 −3.78 0.28 1.07 Manual laborer 1.36 2.96 0.26 −0.98 Retired 0.08 −0.69 0.83 1.66 Student 0.01 −0.22 1.22 2.04 Number of children: 0 1.13 −2.68 2.93 3.27 1–3 0.24 −2.17 0.28 −1.79 3–4 2.25 3.87 0.04 −0.39 >4 2.99 4.13 0.27 −0.94 Education: Higher education 0 −0.1 0.08 0.75 Primary education 6.18 6.32 0.16 −0.76 Post-secondary education 2.19 −4.33 0.01 −0.26 Hazardous items for children < 1 year (ﬁrst response): Batteries 1.93 −3.62 3.45 −3.65 Candies 0.99 −2.56 2.08 2.8 Coins 3.65 4.77 0.15 −0.72 Hotdog 0.09 −0.74 0.01 0.17 Jewelry 1.45 3.22 0.01 0.17 Nuts 0.07 −0.65 0.53 1.31 Popcorn 0.26 1.26 0.15 −0.72 Seeds 3.53 4.51 0.02 0.28 Stationery 0.15 0.93 0.35 1.07 Toys 1.72 −3.48 0.2 0.9 Hazardous items for children < 1 year (second response): Batteries 3.08 −4.65 8.39 −5.8 Candies 0.11 −0.85 0.52 1.4 Coins 9.61 7.7 0 0.04 Hotdog 0.56 −1.83 16.56 −7.53 Jewelry 1.37 2.95 0.05 0.41 Nuts 0.01 −0.21 2.63 2.93 Popcorn 0.58 −1.9 10.16 6.02 Seeds 1.57 2.99 0.1 −0.58 Stationery 0.8 2.17 0.12 −0.63 Toys 0.31 −1.51 3.03 3.58 Hazardous items for children aged 1–2 years (ﬁrst response): Batteries 2.16 −3.92 8.55 −5.89 Candies 0 0.09 0.47 1.34 Coins 5.36 5.67 0.12 0.64 Hotdog 0.3 −1.35 16.52 −7.55 Jewelry 0.22 1.17 0.11 0.64 Nuts 0 0.04 2.73 2.99 Popcorn 0.7 −2.09 9.75 5.89 Seeds 1.77 3.17 0.09 −0.53 Stationery 2.07 3.51 0.13 −0.68 Toys 0.08 −0.77 3.18 3.68 Have any children in your household ever experienced risk of choking?: no 0 0.18 0.42 2.16 yes 0 −0.18 1.03 −2.16 What object caused the accident?: Food 0.14 −1.68 0 0.07 Non-Food 0.36 1.68 0 −0.07 Involving risk of death: No 0.02 −0.99 0.01 0.61 Yes 0.15 0.99 0.1 −0.61 Children 2021, 8, 541 8 of 14 The contributors to the second factor (Table 2) are more related to choking risk percep- tion in infants aged less than 1 year. 4. Discussion This work shows that some socio-economic peculiarities in the Brazilian context are connected to a different deﬁnition of risk perception of choking injuries compared to the Italian setting. Some studies conducted on injury prevention in Brazil showed that some social groups and individuals are at higher risk for unintentional injuries [31]. The identiﬁed situations at risk are heterogeneous, and they may include aspects related to education, salary, access to health services, unemployment, or the absence of a family network. The latter was shown to be fundamental, particularly in families with a larger number of children [32]. Considering the peculiarities of FB injuries risk perception, the results showed that Ital- ians perceived both batteries and magnets as more harmful for their children. Conversely, the Brazilian respondents identify a higher risk in coins and buttons. Epidemiological surveillance is helpful to deﬁne the conﬁgurations of choking injuries in children [33]. The literature shows that the majority of FB injuries in children are related to coins and marble ingestion [34], but the most dangerous items are magnets [35] and batteries [36], due to the increased chance of severe complications. The prevention policies recently implemented in Italy have raised public awareness about the serious complications arising from the ingestion of batteries and magnets [19]. Concerning the results of this work, the Brazilian respondents perceive coins, which are among the main causes of incidents across children [36], as more dangerous. This risk perception probably derives from personal experience rather than from an actual risk awareness, mainly because coins are much more common in daily life for Brazilian respondents than batteries. In our research, the Italians declare that the FB injury events are mainly due to the ingestion of food items compared to the Brazilian sample who reported a greater number of events due to non-food items. The literature conﬁrmed that, in European countries, most FB injuries are caused by inorganic items [23,37], and food items, in particular nuts and seeds, are the most frequently retrieved from a child’s upper airway tract [34]. Other research has shown that most FB injury events in European countries occur during a meal when the adult is distracted. In newly developed countries, where policies are less focused on the topic, FB injury happens mainly under adult supervision while the child is playing [18]. MCA Results The information provided by the latent factors could be synthesized by considering the most contributing variables on the latent dimensions [18] (Table 2). For example, the ﬁrst factor has been named “Professional–educational status and perception of Risk”, for the reason that the socio-economic features contribute the most to a variable’s modalities. The “Factors affecting Risk perception in children < 1 year” (Table 2) are instead the most contributing variable modalities on the second latent factor. A further interpretation of latent axes may be performed with standardized coor- dinates (test values) to specify respondents’ peculiarities according to their locations on selected axes. Considering the ﬁrst dimension, on the negative side of the factor (negative test values), it is possible to identify two groups of respondents characterized as Italian males (median loading −0.24) with a high education level, in managerial employment without children. This group of respondents identiﬁes batteries as a more hazardous object for children in all age classes (Table 2). On the positive side of these factors (positive test values), Brazilian respondents are reported, where (median loading 0.84) their primary education was classiﬁed as own worker or manual worker, and the number of children was greater than four. Brazilian respondents identify coins as the most dangerous objects (Table 2). According to the second latent dimension, it is possible to distinguish, on the positive side, the subjects identifying a potential FB injury hazard in toys and popcorn. On the negative side instead, hot dogs and batteries are identiﬁed as most hazardous. The differences in geographical and cultural belonging are mainly evident in the ﬁrst latent factor (the most important in terms of explained variance) (Figure 1). Figure 1. MCA individual biplot for dimensions 1 (x-axis) and 2 (y-axis), according to the children’s country of origin (Italy vs. Brazil). Figure 1. MCA individual biplot for dimensions 1 (x-axis) and 2 (y-axis), according to the children’s country of origin (Italy vs. Brazil). 9 of 14 Children 2021, 8, 541 4. Discussion On the one hand is the greater awareness of the problem in Italy, shaped by the recent FB-related prevention policies, which probably facilitated the respondent’s recall of events that involved less serious outcomes. On the other hand, the Brazilian respondents, less aware of the problem, could be prone to remember only the events involving more severe outcomes. The literature shows that the majority of FB injuries in children are related to coins and marble ingestion [34], but the most dangerous items are magnets [35] and batteries [36], due to the increased chance of severe complications. The hazard related to these latter items is underestimated, in our data, by the Brazilian respondents. Mortality due to foreign body injury is in fact a serious problem in Brazil; FB airway obstruction is the leading cause of death from external causes in infants up to 1 year of age. Most of the cases treated were children around the ﬁrst 3 years of life, with a prevalence of boys, in a ratio of 2:1 [21]. In Italy, instead, the number of children hospitalized for FB injuries [33] has decreased in the last decade, but at the same time, the awareness of the problem has improved [23]. Our data demonstrated that by performing a comparison between Italian and Brazilian countries, the frequency of adults in managerial or teaching positions reporting FB injury episodes in their children is similar between Italy and Brazil. This could illustrate the fact that a demographic–occupational position could be an issue that deﬁnes attitudes and perceptions of risk in a similar way in Italy and Brazil. These results can be viewed as a starting point of some preliminary consideration; preventive actions and targeted policies should be necessary for Brazil to increase awareness and education about the real risk concerning FB injuries. Compared to European countries, Brazil is characterized by higher levels of socio- cultural differences (economic, educational, and ethnic). In this context, the application of prevention policies can be of crucial importance, especially if the attempt is to reach the economically more vulnerable population groups [42,43]. The application of prevention policies, especially if oriented towards awareness and education about the problem, can reach, with focused interventions, even the most disadvantaged segments of the population. However, the policies do not act alone in this direction. 4. Discussion This highlights a lack of awareness of the problem in the Brazilian sample, which over-represents the non-food items among the objects involved in FB injuries and underestimates the perception of risk associated with ingestion of batteries and magnets. Research conducted in an emergency ward of a general hospital in Brazil underlined that most FB injuries among children occur in the home environment where many items and situations can establish risk scenarios [38]. In some situations, inadequate child supervision is related to the fact that the adult responsible for the caregiving activities is simultaneously involved in other activities at the same time. Adult presence does not prevent FB injuries; research demonstrated that 4.3% of the children presenting in the emergency room were alone when the incident occurred [38]. This could happen when, even though the adult caregiver is available for supervision, he lacks the understanding of what constitutes a threat to the child. In these situations, the caregiver could inadvertently allow the child to play with dangerous items, such as batteries and magnets. p y g g A considerable part of the Brazilian sample belongs to low-income classes [39,40]. The people originating from underdeveloped urban areas, mostly Black or Brown people, with poor educational levels, have limited access to healthcare and social policies. As pointed out by different authors [39,40], the levels of awareness regarding situations representing a considerable risk for one’s physical and/or psychological well-being are extremely precarious. Indeed, a relevant percentage of all Brazilian mothers, who took part in the survey, were very young at the moment of data collection, sometimes having more than one child. Children 2021, 8, 541 10 of 14 10 of 14 Inexperience, precarious socio-economic backgrounds and low educational levels are often co-responsible for everyday injuries, which could be avoided in more socially advan- taged situations and conditions. Considering a certain problem or phenomenon concerning its socio-cultural background, an intersectional view is indispensable for healthcare, social, and psychological research carried out in countries with a post-colonial past, such as the Americas. Despite the greater number of Italian respondents, compared to Brazilians, declaring to have experienced FB injuries in their children, a greater percentage of events with a fatal outcome is reported in the Brazilian sample. The literature shows that recall of events can be inﬂuenced by actual risk awareness [41]. In this regard we could highlight two aspects. 4. Discussion Preventive actions would be easier to implement if the educational substrate of the population is homogeneous and if the population is already oriented to inform itself about public health issues [44]. Concerning FB injury prevention, a few simple measures that could be implemented by any population group, disadvantaged or not, would be helpful. For example, the FB injury events in several cases involve objects present in the children’s homes; it can be assumed that modiﬁcation of the domestic environment could be an important preventive issue, as well as the adoption of safe behaviors and adequate surveillance habits by parents and caregivers [45]. Within this general framework, guidelines and prevention strategies oriented to the child’s developmental stage could be essential. Certainly, a dual intervention including prevention policies but also the promotion of equal access to education and employment can make the difference [46]. Our questionnaire focused on the socio-demographic peculiarities of the analyzed sample. However, culture is a broader concept that deﬁnes the customary beliefs, social forms, and material traits of a racial, religious, or social group [47]. Brazil is characterized Children 2021, 8, 541 11 of 14 11 of 14 by a wider heterogeneity of ethnic–religious groups in comparison with Italy. The country is a cultural patchwork; it was ofﬁcially colonized by the Portuguese and, at the same time, it had distinctive African and indigenous inﬂuences [48]. Italians, French, Dutch, Arabs, Japanese, and other ethnic groups moved to Brazil in the course of its history [49]. This cultural heterogeneity impacts risk perception [50]. Furthermore, it is difﬁcult to discuss Brazil without mentioning the social exclusion connected to the cultural differences; many ethnic groups still face severe limitations in accessing education, social integration, labor market, and healthcare facilities [51]. We have highlighted in our research that the occupational status (manual laborer status) with a lower level of education and a higher number of children for Brazilian respondents deﬁnes a different perception of risk compared to the Italians who tend to have a more homogeneous cultural connotation. The literature demonstrates that [52] the general lack of attention during parental caregiving might cause more FB injury cases, especially among disadvantaged popula- tions [53]. Therefore, special attention should be paid not only to improve the overall awareness within the closer family context but also to enhance the professional caregiving experience [54]. Study Limitations The ﬁrst study limitation consists of a possible incomparability between a relatively small national European context, i.e., Italy, and a macro-national Brazilian context. A single state—the Federal District—was considered to resolve this geopolitical inequality. Most educated people and college students are in the Brazilian Federal District (Brasília—17.6%), exactly where most participants are sampled for many psychological studies [63]. From a statistical standpoint, the current survey concerns the asymmetric distribution of the sample sizes. Such a difference is due to the exploratory character of this type of research. However, MCA allows accounting for both small sample sizes [64] and unbalanced data [65]. Our study ﬁndings warrant future research to compare risk perception at the popula- tion level between different European countries and the Brazilian population. 4. Discussion Moreover, Brazilian parents are younger than European parents and only a few of them were engaged in caring for one child. A signiﬁcant proportion of them carry out extra-domestic and physically demanding full-time jobs. In this setting, families may have difﬁculties in appropriately supervising children; this is a relevant issue because the literature conﬁrmed that a momentary lack of attention is one of the most common causes of FB injury in children [13,37,55–57]. In that regard, a relevant target of prevention is related to parental awareness and generally to a collectively shared recognition of a health problems in the pediatric age [58]. However, the literature from newly industrialized countries [17,59] or Eastern Euro- pean nations [60,61], demonstrates that extensive and comprehensive prevention policies on the topic are still in progress [60]. The injury prevention policies should be tailored to the socio-economic peculiarities of the targeted population [8,18]. The principal contribution of this survey is to underline the socio-cultural differences between Italy (a developed country) and Brazil (a newly developed country). Cultural peculiarities are considered factors inﬂuencing the risk perception and, subsequently, the epidemiology of FB injuries in the pediatric age. Such intersectional and transnational perspectives could help in tailoring the edu- cational interventions to a speciﬁc cultural setting [62]. Therefore, this study could be considered one of the ﬁrst attempts to analyze an epidemiologically relevant problem through an intersectional, post-modern, and intercultural perspective. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions Socio-economic factors are important issues concerning subjective risk perception of FB injuries. Different socio-cultural characteristics of Italian and Brazilian survey re- Children 2021, 8, 541 12 of 14 12 of 14 spondents were evidenced by our data. A data-driven and cross-cultural approach may be helpful or is recommended in designing injury prevention policies. The intervention policies should address the heterogeneity and complexity of risk perception. spondents were evidenced by our data. A data-driven and cross-cultural approach may be helpful or is recommended in designing injury prevention policies. The intervention policies should address the heterogeneity and complexity of risk perception. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/children8070541/s1, Table S1: Characteristics and item choices for adults having and not having children ever experienced risk of choking, Table S2: Adults having any children in the household ever experienced risk of choking in managerial position/ofﬁce and position/teachers category in Italy and Brazil. Author Contributions: Conceptualization, D.G. and A.H.; methodology, D.A., P.B.; formal analysis, D.A.; investigation, A.O. (Alessandra Oliveira), A.O. 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https://openalex.org/W4386330098	https://www.frontiersin.org/articles/10.3389/fcell.2023.1197109/pdf	English	null	Sin3a associated protein 130 kDa, sap130, plays an evolutionary conserved role in zebrafish heart development	Frontiers in cell and developmental biology	2,023	cc-by	13,388	OPEN ACCESS OPEN ACCESS EDITED BY Diego Franco, University of Jaén, Spain REVIEWED BY Jiandong Liu, University of North Carolina at Chapel Hill, United States Marina Campione, National Research Council (CNR), Italy CORRESPONDENCE Michael Tsang, tsang@pitt.edu RECEIVED 30 March 2023 ACCEPTED 17 August 2023 PUBLISHED 30 August 2023 CITATION DeMoya RA, Forman-Rubinsky RE, Fontaine D Jr, Shin J, Watkins SC, Lo CW and Tsang M (2023), Sin3a associated protein 130 kDa, sap130, plays an evolutionary conserved role in zebraﬁsh heart development. Front. Cell Dev. Biol. 11:1197109. doi: 10.3389/fcell.2023.1197109 EDITED BY Diego Franco, University of Jaén, Spain REVIEWED BY Jiandong Liu, University of North Carolina at Chapel Hill, United States Marina Campione, National Research Council (CNR), Italy Ricardo A. DeMoya1, Rachel E. Forman-Rubinsky1, Deon Fontaine Jr1, Joseph Shin1, Simon C. Watkins 2, Cecilia W. Lo1 and Michael Tsang1 Ricardo A. DeMoya1, Rachel E. Forman-Rubinsky1, Deon Fontaine Jr1, Joseph Shin1, Simon C. Watkins 2, Cecilia W. Lo1 and Michael Tsang1* Ricardo A. DeMoya1, Rachel E. Forman-Rubinsky1, Deon Fontaine Jr1, Joseph Shin1, Simon C. Watkins 2, Cecilia W. Lo1 and Michael Tsang1* 1Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States, 2Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States CITATION DeMoya RA, Forman-Rubinsky RE, Fontaine D Jr, Shin J, Watkins SC, Lo CW and Tsang M (2023), Sin3a associated protein 130 kDa, sap130, plays an evolutionary conserved role in zebraﬁsh heart development. Hypoplastic left heart syndrome (HLHS) is a congenital heart disease where the left ventricle is reduced in size. A forward genetic screen in mice identiﬁed SIN3A associated protein 130 kDa (Sap130), part of the chromatin modifying SIN3A/ HDAC complex, as a gene contributing to the etiology of HLHS. Here, we report the role of zebraﬁsh sap130 genes in heart development. Loss of sap130a, one of two Sap130 orthologs, resulted in smaller ventricle size, a phenotype reminiscent to the hypoplastic left ventricle in mice. While cardiac progenitors were normal during somitogenesis, diminution of the ventricle size suggest the Second Heart Field (SHF) was the source of the defect. To explore the role of sap130a in gene regulation, transcriptome proﬁling was performed after the heart tube formation to identify candidate pathways and genes responsible for the small ventricle phenotype. Genes involved in cardiac differentiation and cardiac function were dysregulated in sap130a, but not in sap130b mutants. TYPE Original Research PUBLISHED 30 August 2023 DOI 10.3389/fcell.2023.1197109 TYPE Original Research PUBLISHED 30 August 2023 DOI 10.3389/fcell.2023.1197109 TYPE Original Research PUBLISHED 30 August 2023 DOI 10.3389/fcell.2023.1197109 cardiac development, second heart ﬁeld, SIN3A/HDAC complex, congenital heart disease, zebraﬁsh frontiersin.org OPEN ACCESS Confocal light sheet analysis measured deﬁcits in cardiac output in MZsap130a supporting the notion that cardiomyocyte maturation was disrupted. Lineage tracing experiments revealed a signiﬁcant reduction of SHF cells in the ventricle that resulted in increased outﬂow tract size. These data suggest that sap130a is involved in cardiogenesis via regulating the accretion of SHF cells to the growing ventricle and in their subsequent maturation for cardiac function. Further, genetic studies revealed an interaction between hdac1 and sap130a, in the incidence of small ventricles. These studies highlight the conserved role of Sap130a and Hdac1 in zebraﬁsh cardiogenesis. Front. Cell Dev. Biol. 11:1197109. doi: 10.3389/fcell.2023.1197109 COPYRIGHT © 2023 DeMoya, Forman-Rubinsky, Fontaine, Shin, Watkins, Lo and Tsang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Introduction SET and MYND domain-containing lysine methyltransferase 4 (smyd4) mutants also result in reduced ventricle size in zebraﬁsh and mouse, suggesting there is a common requirement of gene regulation for specifying heart organ size in vertebrates (Trotter and Archer, 2008). RNA sequencing (RNA-seq) analysis of smyd4 zebraﬁsh mutants revealed dysregulation of cardiac muscle contraction and metabolism genes. Moreover, cell culture studies revealed human SMYD4 and HDAC1 interact, further supporting a central requirement for hdac1 in zebraﬁsh cardiogenesis (Xiao et al., 2018). Taken together these suggest a potential epigenetic role for sap130a during development as part of the Sin3a complex. complex and genetically heterogenous. Mouse models of HLHS were recovered from a large-scale mutagenesis screen (Liu et al., 2017), and among 8 lines, the Ohia mutant line was identiﬁed to have a digenic etiology for HLHS. This is comprised of mutations in SIN3A associated protein 130 kDa (SAP130) and protocadherin 9 (PCDHA9) that together causes HLHS comprising hypoplasia of all left-sided heart structures including the ventricle, aorta/aortic valve, and mitral valve. In pigs a CRISPR generated SAP130 allele caused embryonic lethality and tricuspid dysplasia and atresia, indicating SAP130 involvement in cardiac development in higher vertebrates (Gabriel et al., 2021). In zebraﬁsh, maternal zygotic sap130a (MZsap130a) mutants resulted in a diminutive ventricle by 72 h post fertilization (hpf), conﬁrming that SAP130 retains a conserved function among vertebrates during heart development (Liu et al., 2017; Gabriel et al., 2021). SAP130 was identiﬁed as an interacting protein in the SIN3A complex, binding both SIN3A and Histone Deacetylase 1 (HDAC1), thought to stabilize the complex. It was theorized that the SAP130 C-terminus functioned as a transcriptional repressor in association with the SIN3A complex, while the N-terminus paradoxically could function as an activator (Fleischer et al., 2003). A knock-out allele of SAP130 in mice is peri-implantation lethal, unlike global knockouts of HDAC1 and SIN3A which die at later stages of development (Lagger et al., 2002; Dannenberg et al., 2005; Liu et al., 2017). These suggest multiple roles and stages of development where SAP130/SIN3A/HDAC1 are critical for life. SIN3A and HDACs epigenetically regulate transcription through histone and non-histone deacetylation events and are classically associated with gene repression. However, some studies have shown this complex to be a transcriptional activator in other contexts (Han et al., 2011; Kadamb et al., 2013; Adams et al., 2018). Zebraﬁsh husbandry All zebraﬁsh experiments and protocols were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh in agreement with NIH guidelines. Wild-type AB, Tg(myl7:GFP)twu34 (Huang et al., 2003), Tg(nkx2.5:kaeda)fb9 (Guner-Ataman et al., 2013), sap130apt32a (Liu et al., 2017), hdac1b382 (Ignatius et al., 2013). Adult tail ﬁn clips or whole embryos for genotyping assays was performed as previously described (Jing, 2012). Restriction fragment length polymorphism (RFLP) genotyping for sap130apt32a, sap130bpt35b, sin3abpt36a and hdac1b382 used the primers and enzymes listed in Supplementary Table S1. All zebraﬁsh experiments and protocols were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh in agreement with NIH guidelines. Wild-type AB, Tg(myl7:GFP)twu34 (Huang et al., 2003), Tg(nkx2.5:kaeda)fb9 (Guner-Ataman et al., 2013), sap130apt32a (Liu et al., 2017), hdac1b382 (Ignatius et al., 2013). Adult tail ﬁn clips or whole embryos for genotyping assays was performed as previously described (Jing, 2012). Restriction fragment length polymorphism (RFLP) genotyping for sap130apt32a, sap130bpt35b, sin3abpt36a and hdac1b382 used the primers and enzymes listed in Supplementary Table S1. In addition to the Sin3a/Hdac1 complex, related chromatin modifying complexes like the BAF complex, have been shown to be involved in cardiogenesis (Lickert et al., 2004; Wang et al., 2004; Stankunas et al., 2008; Hang et al., 2010; Hargreaves and Crabtree, 2011; Takeuchi et al., 2011; Lei et al., 2012; Singh and Archer, 2014; Nakamura et al., 2016; Xiao et al., 2016; Sun et al., 2018; Alfert et al., 2019; Hota et al., 2019; Lei et al., 2019; Chen et al., 2022; Auman et al., 2023). A study describing the loss of smarcc1a, a BAF chromatin remodeling complex protein, in zebraﬁsh resulted in dysmorphic cardiac chambers further highlighting the importance of chromatin remodeling in proper heart formation (Auman et al., 2023). Another part of the BAF complex in zebraﬁsh brg1, when mutated reveals a Introduction HDACs have been reported to regulate many aspects of development, including cardiac development in zebraﬁsh, mouse, and chick models, as evidenced by treatment with a pan HDAC small molecule inhibitor, Trichostatin A (Hargreaves and Crabtree, 2011; McKinsey, 2011; Martinez et al., 2015). Zebraﬁsh studies have revealed that hdac1 is involved in Second Heart Field (SHF) development and in adult cardiac regeneration (Song et al., 2019; Buhler et al., 2021). In zebraﬁsh, hdac1 mutants have less cardiomyocytes (CMs) in the ventricle while inhibition of hdac1 (and other class I HDACs) reveal reduced proliferation during regenerative events (Montgomery et al., 2007; Nambiar et al., 2007; Song et al., 2019; Buhler et al., 2021). Zebraﬁsh hdac1 mutants are embryonic lethal, similar to the mouse models, but MZsap130a mutants are viable as adults suggesting that hdac1 and sap130a may have distinct functions in zebraﬁsh cardiogenesis. Here we investigate the role of sap130 genes in zebraﬁsh by studying mutations in both sap130a and sap130b. Transcriptome proﬁling of 36hpf MZsap130a mutants revealed over 5,000 genes to be differentially expressed, including genes involved in the cardiac development and function. In genetic studies, an increase in embryos with small ventricles (SVs) were noted in MZsap130a embryos that were also heterozygous for hdac1. Furthermore, MZsin3ab mutants exhibit a SV phenotype at 48hpf. Collectively, these studies suggest a role for sin3ab/hdac1/sap130a in the SHF during zebraﬁsh cardiogenesis. Frontiers in Cell and Developmental Biology Introduction Congenital heart diseases (CHDs) affect approximately 1% of live births per year and causes have been attributed to environmental and genetic factors (Nora, 1968; Fahed et al., 2013; Costain et al., 2016). Hypoplastic left heart syndrome (HLHS) is a critical CHD characterized by a reduced volume in the left ventricle and aortic and valve malformations (Connor and Thiagarajan, 2007; Barron et al., 2009). The genetic etiology of HLHS is 01 Frontiers in Cell and Developmental Biology frontiersin.org frontiersin.org DeMoya et al. 10.3389/fcell.2023.1197109 10.3389/fcell.2023.1197109 reduction in CM proliferation leading to a smaller ventricle after 28hpf. The brg1 mutants reveal changes in a working myocardium marker nppa, similar to mouse Brg1 mutants (Takeuchi et al., 2011). Other types of epigenetic regulation such as methylation are shown to be paired with chromatin remodeling events and are involved in cardiogenic processes (Carrozza et al., 2005; Joshi and Struhl, 2005; Keogh et al., 2005; Brown et al., 2006; Donlin et al., 2012; Voelkel et al., 2013; Singh and Archer, 2014; Xiao et al., 2018; Zhu et al., 2018; Bisserier et al., 2021). SET and MYND domain-containing lysine methyltransferase 4 (smyd4) mutants also result in reduced ventricle size in zebraﬁsh and mouse, suggesting there is a common requirement of gene regulation for specifying heart organ size in vertebrates (Trotter and Archer, 2008). RNA sequencing (RNA-seq) analysis of smyd4 zebraﬁsh mutants revealed dysregulation of cardiac muscle contraction and metabolism genes. Moreover, cell culture studies revealed human SMYD4 and HDAC1 interact, further supporting a central requirement for hdac1 in zebraﬁsh cardiogenesis (Xiao et al., 2018). Taken together these suggest a potential epigenetic role for sap130a during development as part of the Sin3a complex. reduction in CM proliferation leading to a smaller ventricle after 28hpf. The brg1 mutants reveal changes in a working myocardium marker nppa, similar to mouse Brg1 mutants (Takeuchi et al., 2011). Other types of epigenetic regulation such as methylation are shown to be paired with chromatin remodeling events and are involved in cardiogenic processes (Carrozza et al., 2005; Joshi and Struhl, 2005; Keogh et al., 2005; Brown et al., 2006; Donlin et al., 2012; Voelkel et al., 2013; Singh and Archer, 2014; Xiao et al., 2018; Zhu et al., 2018; Bisserier et al., 2021). frontiersin.org Lineage tracing Lineage tracing of cardiac progenitors at 24hpf was performed on Tg(nkx2.5:kaede) and Tg(nkx2.5:kaede);sap130apt32a/pt32a embryos and was described by Guner-Ataman et al. (Guner-Ataman et al., 2013). Using the Zeiss Imager M2 confocal microscope at 40x, the ROI (Region of Interest) was selected to photoconvert the peristaltic heart tube at 24hpf. Embryos were mounted in low melting temperature agarose droplets on 35 mm dishes. The embryos were then freed from the agarose and raised in darkness until 48hpf, when the looped heart was imaged at 40x. Cardiac functional analysis To measure cardiac function in embryonic zebraﬁsh, we used a custom-built light sheet microscope which followed a design based on the openSPIM platform (Pitrone et al., 2013; Girstmair et al., 2016). This ‘T’ design illuminates the sample bilaterally and uses a four-channel laser launch for maximum versatility. Tg(myl7:EGFP) and Tg(myl7:EGFP);sap130am/m embryos at 48hpf embryos were placed into E3 and Tricaine (307 nmol concentration) to anesthetize them before mounting for imaging. Low melting point agarose was heated and cooled to 42°C. 100 µL agarose placed onto a dish and after 45 s of cooling, 48hpf embryo was added to the agarose and drawn into a custom cut 1 mL straight-barreled syringe. The agarose is allowed to solidify, and the syringe is placed into a sample manipulator capable of 3D movement + rotation (Picard Technologies, Inc.). The agarose-embedded embryos were extruded from the syringe and positioned in a lateral view, with anterior to the left and posterior to the right, before recording 100 frames at 50–75 frames per second using a Prime 95B sCMOS camera (Photometrics, Inc.). Fiji ImageJ software was used to identify end-diastole and end-systole frames to calculate ventricle area, length (distance between ventricular apex and out-ﬂow tract opening), and diameter for each embryo (distance between the walls of the chamber, taken from the middle of length measurement). These data were used to estimate chamber volumes and calculate end-diastole and systole volumes, ejection fraction (%), fractional Imaging A Leica M205 FA stereomicroscope was used to take images of the hearts from Tg(myl7:EGFP) WT and mutant embryos at 36 and 48hpf. For imaging the Tg(myl7:memGFP) OFT, a Nikon A1 inverted confocal microscope was used at 72hpf. Tg(myl7:memGFP) embryos were anesthetized in 7x MS-222/10 mM BDM (2,3-butanedione monoxime) and mounted in low melting agarose on MaTek glass bottom petri dish (MaTek, Part No: P35G-1.5–14-C) and imaged at a 40x water immersion. For counting cardiomyocytes at 72hpf, Tg (myl7: memGFP) and MZsap130a;Tg(myl7:EGFP) were injected with 50 pg of H2b-mCherry mRNA at the 1-2 cell stage. Injected embryos were incubated at 28°C and mounted on an inverted confocal microscope at 72hpf on a Nikon A1 microscope. Ventricular cardiomyocytes were designated as positive for both mCherry nuclei and membrane GFP expression using Fiji ImageJ and the orthogonal views tool. In situ probe synthesis, whole mount in situ hybridization RNA probe generation and whole mount in situ hybridization for nkx2.5, ltbp3, myh7 and myh6 was performed as previously described with DIG RNA labeling kit (Millipore Sigma cat# 11175025910) (Znosko et al., 2010). CRISPR/Cas9 mutant allele generation The CRISPR/Cas9 protocol (Gagnon et al., 2014) was used to establish mutant lines. This protocol used Sp6 in vitro transcribed sgRNAs targeting the sequence ccgTGGGAGGGAAAACAATGCTG for sap130b and cctGCTCCTCTTCAGCCATACAG for sin3ab, where lower case letters represent the protospacer motif sequence. sgRNA was incubated at room temperature with Cas9 protein (NEB, Cat# M0646T). AB* embryos were injected at the one-cell stage with the sgRNA and Cas9 cocktail in a 1 nL volume at 25 pg sgRNA/nL. RFLP 02 frontiersin.org 10.3389/fcell.2023.1197109 DeMoya et al. puriﬁed with the RNeasy Micro Kit (Qiagen#74004). A minimum 50 embryos or 180 hearts were pooled together for each condition. The RNA-seq used was 0.5–1 μg RNA for each condition and was sent to the Genomics Research Core at the University of Pittsburgh. The raw sequence reads were processed and mapped to the Zebraﬁsh Reference Genome GRCz11 using CLC Genomics Workbench 20 RNAseq analysis tool. A count matrix was exported and the bioinformatic analysis was carried out in R (R Core Team, 2021) using the edgeR package for 36hpf whole embryo and 48hpf heart tissue data. Results for DEGs in Supplementary Tables, S2–S6 (Robinson et al., 2010). To identify cardiac changes with whole embryo resolution we deﬁned DEGs as those with an FDR ≤ 0.05 and log2FC > ±0.4. After determining differentially expressed genes they were entered into DAVID (https://david. ncifcrf.gov/summary.jsp) for functional annotation clustering. Results for DAVID clustering in Supplementary Tables, S2–S6 (Sherman et al., 2022). was performed to determine protected mutated bands present 24hrs after injection to determine gRNA efﬁciency and injected embryos were raised to adults outcrossed to AB. DNA mutations in sap130b and sin3ab were veriﬁed by PCR TOPO-TA cloning (ThermoFisher, #K4575J10) from adult heterozygous animals and Sanger sequenced. gRNA sequence information Supplementary Table S1. ConSurf and R generated phylogenetic trees and protein diagram ConSurf (https://consurf.tau.ac.il/consurf_index.php) was used to align multiple Sap130 protein sequences across many species (Berezin et al., 2004). The sap130a amino acid sequence from zebraﬁsh was input to ConSurf and the output was collected and plotted in R, with ggtree, ggplot2 and phytools (Revell and Graham Reynolds, 2012; Wickham, 2016; Yu et al., 2018; Yu, 2020). A multiple sequence alignment (MSA) was performed on Sap130 protein sequences from UniProt and distance calculations to plot simple phylogeny trees using R CRAN packages seqinr, msa, Biostrings, ggtree, ggplot2 (Charif et al., 2005; Bodenhofer et al., 2015; Lifschitz et al., 2022). For plotting the protein sequences and conserved domains reported by UniProt, the R packages ggplot and drawProteins were used (Brennan, 2018). frontiersin.org Frontiers in Cell and Developmental Biology Frontiers in Cell and Developmental Biology Adult heart measurements At 48hpf MZsap130a mutant embryos were scored for ventricle size and raised in separate tanks. MZsap130a mutants and aged matched AB controls were measured for length and weight before hearts were extracted for DIC imaging at 4-6mpf. Fiji-ImageJ was used to measure the ventricle surface area and bulbus arteriosus surface area. These data were plotted using Graphpad Prism 9.3. FIGURE 1 sap130a and sap130b have non-overlapping functions in the zebraﬁsh heart (A, B) A simple distance matrix phylogeny tree of Sap130a and Sap130b in broad or teleost speciﬁc contexts (C) Schematic of Sap130a and Sap130b protein sequences from the UniProt database highlighting the conserved regions and predicted mutant proteins. Unorganized sequence in pink, C-terminal conserved domain in blue, which contains the binding domain for SIN3A and HDAC1 (D, E) Representative images of Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP) and MZsap130b;Tg(myl7:EGFP) mutant hearts at 48hpf. V and A are ventricle and atria, respectively. Scale bar 100 μm. FIGURE 1 sap130a and sap130b have non-overlapping functions in the zebraﬁsh heart (A, B) A simple distance matrix phylogeny tree of Sap130a and Sap130b in broad or teleost speciﬁc contexts (C) Schematic of Sap130a and Sap130b protein sequences from the UniProt database highlighting the conserved regions and predicted mutant proteins. Unorganized sequence in pink, C-terminal conserved domain in blue, which contains the binding domain for SIN3A and HDAC1 (D, E) Representative images of Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP) and MZsap130b;Tg(myl7:EGFP) mutant hearts at 48hpf. V and A are ventricle and atria, respectively. Scale bar 100 μm. FIGURE 1 sap130a and sap130b have non-overlapping functions in the zebraﬁsh heart (A, B) A simple distance matrix phylogeny tree of Sap130a and Sap130b in broad or teleost speciﬁc contexts (C) Schematic of Sap130a and Sap130b protein sequences from the UniProt database highlighting the conserved regions and predicted mutant proteins. Unorganized sequence in pink, C-terminal conserved domain in blue, which contains the binding domain for SIN3A and HDAC1 (D, E) Representative images of Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP) and MZsap130b;Tg(myl7:EGFP) mutant hearts at 48hpf. V and A are ventricle and atria, respectively. Scale bar 100 μm. RNAseq sample preparation and data analysis Total RNA was extracted from whole embryos or isolated hearts (36hpf and 48hpf, respectively) using Trizol (Invitrogen) and was Frontiers in Cell and Developmental Biology 03 frontiersin.org DeMoya et al. DeMoya et al. 10.3389/fcell.2023.1197109 shortening (µm), Total stroke volume, cardiac output, and heart rate as an average of all cycles captured for each ﬁsh. The volumes calculated are under the assumption of a prolate sphere shape (pi/6). The equations used are as follows (Yalcin et al., 2017); FIGURE 1 sap130a and sap130b have non-overlapping functions in the zebraﬁsh heart (A, B) A simple distance matrix phylogeny tree of Sap130a and Sap130b in broad or teleost speciﬁc contexts (C) Schematic of Sap130a and Sap130b protein sequences from the UniProt database highlighting the conserved regions and predicted mutant proteins. Unorganized sequence in pink, C-terminal conserved domain in blue, which contains the binding domain for SIN3A and HDAC1 (D, E) Representative images of Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP) and MZsap130b;Tg(myl7:EGFP) mutant hearts at 48hpf. V and A are ventricle and atria, respectively. Scale bar 100 μm. Ejection Fraction % ( ) SV EDV100 Stoke Volume SV ( ) End Diastole Volume −End Systole Volume Fractional Shortening Diastole diameter −Systole diameter Diastole diameter Fractional Area Change End Diastole Area −End Systole Area End Diastole Area 100 Heart Rate # of Cycles Aquisition time s ( ) These were implemented using R scripting and RStudio to automate the calculations, and then data were plotted using Graphpad PRISM 9.3. Each data point represents an average of 3 or more contraction cycles per ﬁsh (Yalcin et al., 2017). FIGURE 1 sap130a and sap130b have non-overlapping functions in the zebraﬁsh heart (A, B) A simple distance matrix phylogeny tree of Sap130a and Sap130b in broad or teleost speciﬁc contexts (C) Schematic of Sap130a and Sap130b protein sequences from the UniProt database highlighting the conserved regions and predicted mutant proteins. Unorganized sequence in pink, C-terminal conserved domain in blue, which contains the binding domain for SIN3A and HDAC1 (D, E) Representative images of Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP) and MZsap130b;Tg(myl7:EGFP) mutant hearts at 48hpf. V and A are ventricle and atria, respectively. Scale bar 100 μm. Statistics However, given that the C- terminal domains are most conserved, Sap130a and Sap130b can potentially compensate for one another in zebraﬁsh (Figure 1C). MZsap130a mutants develop SVs in 36% of the population by 72hpf (Liu et al., 2017). The incomplete Statistics For analysis of RNA-seq data we used the edgeR package, utilizing a quasi-likelihood negative binomial generalized log-linear model to our count data comparing AB* control to MZsap130a or MZsap130b mutant embryos at 36hpf. For heart tissue RNA-seq, edgeR’s likelihood ratio test was used to interpret up or downregulation of genes. For all other statistical analysis, signiﬁcance was calculated using two-tailed, unpaired Student’s t-test, one-way ANOVA or Fisher’s exact text using GraphPad Prism version 9.3. (Fleischer et al., 2003). Determining protein sequence similarities can predict functional structures across species and offer insight into the potential for functional redundancy between Sap130a and Sap130b. ConSurf was used for a multispecies comparison of 145 unique SAP130 protein sequences to determine their similarity and conserved domains (Berezin et al., 2004). In general, Sap130a and Sap130b are dissimilar, but they both contained conserved N- and C-terminus domains represented by repetitive predicted structural and functional residues (Supplementary Figure S1). Comparing SAP130 proteins to a small group of common species Sap130a and Sap130b are most like one another, suggesting they could serve similar functions (Figure 1A). Narrowing the comparison to a smaller set of protein sequences among other teleost, Sap130a and Sap130b are distinct suggesting in teleost these genes could have evolved distinct functions (Figure 1B). However, given that the C- terminal domains are most conserved, Sap130a and Sap130b can potentially compensate for one another in zebraﬁsh (Figure 1C). MZsap130a mutants develop SVs in 36% of the population by 72hpf (Liu et al., 2017). The incomplete (Fleischer et al., 2003). Determining protein sequence similarities can predict functional structures across species and offer insight into the potential for functional redundancy between Sap130a and Sap130b. ConSurf was used for a multispecies comparison of 145 unique SAP130 protein sequences to determine their similarity and conserved domains (Berezin et al., 2004). In general, Sap130a and Sap130b are dissimilar, but they both contained conserved N- and C-terminus domains represented by repetitive predicted structural and functional residues (Supplementary Figure S1). Comparing SAP130 proteins to a small group of common species Sap130a and Sap130b are most like one another, suggesting they could serve similar functions (Figure 1A). Narrowing the comparison to a smaller set of protein sequences among other teleost, Sap130a and Sap130b are distinct suggesting in teleost these genes could have evolved distinct functions (Figure 1B). sap130b is not required for heart development Sap130a AUG start codon antisense-morpholino (MO) studies suggested the SVs arise from decreased ventricular CMs (Liu et al., 2017), but where or when CMs are lost was not explored. To determine if the SVs are due to decreased cardiac progenitors, we performed Whole Mount In Situ Hybridization (WISH) at 10 somite stage with nkx2.5, an early cardiac progenitor marker. We discovered no differences between MZsap130a and controls (Figure 3A). This suggests that the early cardiac progenitors were present in the MZsap130a embryos. To proﬁle a later stage of the First Heart Field (FHF) and the chambers of the heart we performed WISH at 24hpf with myosin heavy chain 7 (myh7, ventricle) and myosin heavy chain 6 (myh6, atria). No difference between WT and mutant embryos were observed, suggesting the FHF is intact (Figure 3B). At 36hpf and 48hpf the atrial chamber showed no change, but the ventricle was smaller (Figure 3C, Supplementary Figure S2). This phenotype was observed again when imaging the MZsap130a;Tg(myl7:EGFP) at 36hpf (Figure 4A). Many studies have detailed the second heart ﬁeld accretion between 24 and 48hpf in zebraﬁsh (Grimes et al., 2008; de Pater et al., 2009; Hami et al., 2011; Lazic and Scott, 2011). These SHF cells trail behind the heart tube and add to the ventricle continuously. There is speculation as to how many SHF cells are ventricular CMs, between 30%–40% of the total ventricular CMs by 48hpf has been proposed (Felker et al., 2018). The SV heart phenotype arising at 36hpf and the lack of changes seen in FHF markers suggest the SHF might be an inﬂuenced cell population where CMs are lost in MZsap130a mutants. sap130b is not required for heart development The offspring produced the expected number of double mutants (7/120 (5.8%)) from the expected (1/16 (6.25%)) from a double heterozygous in-cross. However, the adult double sap130a/b mutants are much smaller than their double heterozygous siblings and failed to produce offspring when bred (Figure 2C). MZsap130a;sap130bpt35b/+ mutant in-crosses, resulted in 39% of the embryos with SVs at 48hpf, which is in the same range as MZsap130a mutants indicating the zygotic loss of sap130b did not contribute to increased cardiac defects (Figure 2D). These observations suggest sap130b is not required for zebraﬁsh cardiogenesis. Sap130a AUG start codon antisense-morpholino (MO) studies suggested the SVs arise from decreased ventricular CMs (Liu et al., 2017), but where or when CMs are lost was not explored. To determine if the SVs are due to decreased cardiac progenitors, we performed Whole Mount In Situ Hybridization (WISH) at 10 somite stage with nkx2.5, an early cardiac progenitor marker. We discovered no differences between MZsap130a and controls penetrance of the SV phenotype was hypothesized to be the result of sap130b compensating for the loss of sap130a. To address this, we generated a mutation in sap130b using CRISPR/Cas9 technology. This produced an allele (7bp del, 1bp sub (G>C))sap130bpt35b/pt35b that introduced a premature stop codon in exon 6 of sap130b disrupting the N-terminus and eliminating the C-terminal region (Figure 1C, Supplementary Tables S1). Using the Tg(myl7:EGFP) line, which labels the heart with green ﬂuorescent protein, we found that 48% of the MZsap130a;Tg(myl7:EGFP) mutant embryos had the SV heart phenotype at 48hpf (Figure 1D). In contrast, only 17% of the MZsap130b;Tg(myl7:EGFP) mutant embryos had SVs by 48hpf (Figure 1E). We generated double mutants to further explore if sap130a and sap130b have any redundant functions (Figures 2A, B). The offspring produced the expected number of double mutants (7/120 (5.8%)) from the expected (1/16 (6.25%)) from a double heterozygous in-cross. However, the adult double sap130a/b mutants are much smaller than their double heterozygous siblings and failed to produce offspring when bred (Figure 2C). MZsap130a;sap130bpt35b/+ mutant in-crosses, resulted in 39% of the embryos with SVs at 48hpf, which is in the same range as MZsap130a mutants indicating the zygotic loss of sap130b did not contribute to increased cardiac defects (Figure 2D). These observations suggest sap130b is not required for zebraﬁsh cardiogenesis. Frontiers in Cell and Developmental Biology sap130b is not required for heart development Zebraﬁsh were part of the teleost-speciﬁc genome duplication event 350 million years ago (Alsop and Vijayan, 2009), resulting in two sap130 genes, sap130a and sap130b. Deﬁning the SAP130 protein domains based on homology with other model organisms will provide insight into the potential conserved functional domains. In mammals, both SIN3A and HDAC1 proteins were shown to interact with SAP130 at the C-terminus between amino acids 836–1,047, suggesting that SAP130 may act as a stabilizing scaffold between these proteins Frontiers in Cell and Developmental Biology 04 frontiersin.org DeMoya et al. 10.3389/fcell.2023.1197109 FIGURE 2 MZsap130a;sap130bpt35b/pt35b mutants are not healthy (A) sap130a;sap130b double heterozygous adults, male (top) and female (bottom). (B) sap130a;sap130b double homozygous adults, male (top) and female (bottom). (C) Graph quantifying weight to length ratio for adults from a sap130a; sap130b double heterozygous in-cross, pvals are for one-way ANOVA, error bars are standard error mean (SEM). Red points represent females and males in black. (D) Graph quantifying the heart phenotype proportions for Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP), MZsap130b;Tg(myl7:EGFP), and MZsap130a;sap130bpt35b/+;Tg(myl7:EGFP), pvals are for ﬁsher’s exact test. Scale bar 5 mm. FIGURE 2 MZsap130a;sap130bpt35b/pt35b mutants are not healthy (A) sap130a;sap130b double heterozygous adults, male (top) and female (bottom). (B) sap130a;sap130b double homozygous adults, male (top) and female (bottom). (C) Graph quantifying weight to length ratio for adults from a sap130a; sap130b double heterozygous in-cross, pvals are for one-way ANOVA, error bars are standard error mean (SEM). Red points represent females and males in black. (D) Graph quantifying the heart phenotype proportions for Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP), MZsap130b;Tg(myl7:EGFP), and MZsap130a;sap130bpt35b/+;Tg(myl7:EGFP), pvals are for ﬁsher’s exact test. Scale bar 5 mm. penetrance of the SV phenotype was hypothesized to be the result of sap130b compensating for the loss of sap130a. To address this, we generated a mutation in sap130b using CRISPR/Cas9 technology. This produced an allele (7bp del, 1bp sub (G>C))sap130bpt35b/pt35b that introduced a premature stop codon in exon 6 of sap130b disrupting the N-terminus and eliminating the C-terminal region (Figure 1C, Supplementary Tables S1). Using the Tg(myl7:EGFP) line, which labels the heart with green ﬂuorescent protein, we found that 48% of the MZsap130a;Tg(myl7:EGFP) mutant embryos had the SV heart phenotype at 48hpf (Figure 1D). In contrast, only 17% of the MZsap130b;Tg(myl7:EGFP) mutant embryos had SVs by 48hpf (Figure 1E). We generated double mutants to further explore if sap130a and sap130b have any redundant functions (Figures 2A, B). FIGURE 4 Since SAP130 has been shown to be part of the SIN3A complex, we reasoned that the phenotype may be caused by altered regulation of cardiac gene expression during development. A whole embryo RNA sequencing (RNAseq) experiment, separating the SV and “normal” (NV) siblings in the MZsap130a mutants, was performed at 36hpf. We ﬁrst performed our analysis looking for differences in the wildtype, compared to NV and SV separately ﬁnding 2,826 differentially expressed genes (DEGs) in common, with 812 unique DEGs for NV and 1979 for SV. Functional annotation of these gene groups revealed that NV and SV embryos are similar when compared to the wildtype transcriptome (Supplementary Table S2). Comparing the controls to all MZsap130a samples (both NV and SV), we observed 5,002 DEGs that included many cardiac speciﬁc transcripts. Among the DEGs we found sarcomere and cardiac conduction genes were dysregulated, suggesting CM biology has changed in the MZsap130a embryos (Figures 4B, C, Supplementary Tables S3, S4). To identify potential pathways involved in heart function and development, we used the Database for Annotation, Visualization, and Integrated Discovery (DAVID) functional annotation of downregulated genes. This showed enrichment for cardiac contraction and adrenergic signaling in CMs, further suggesting a role for sap130a in CM function (Figure 4D, Supplementary Table S6). To conﬁrm cardiac speciﬁc changes in these same transcripts, MZsap130a mutant hearts and controls were harvested at 48hpf and the transcriptome was proﬁled, showing similar cardiac gene expression changes (Figure 4E, Supplementary Table S5). MZsap130b whole embryo transcriptome was also proﬁled at 36hpf and less gene expression changes (617 DEGs) were noted (Supplementary Figure S3, Supplementary Table S6). Moreover, the sarcomere gene expression changes seen in MZsap130a was not detected in the MZsap130b transcriptome. DAVID functional annotation of the 278 DEGs common between MZsap130a and MZsap130b mutants, belonged to heme binding and biosynthesis, oxygen binding, and iron binding KEGG pathways, suggesting involvement in hematopoiesis (Supplementary Table S6). The expression proﬁle for these hematopoietic related genes was opposite in MZsap130a and MZsap130b, suggesting distinct functions during hematopoiesis (Supplementary Figure S3). These data suggest that sap130a and sap130b could be involved in hematopoiesis that correlates with sin3aa/ab gene knockdown studies showing strong hematopoietic defects (Huang et al., 2013). S6). To conﬁrm cardiac speciﬁc changes in these same transcripts, MZsap130a mutant hearts and controls were harvested at 48hpf and the transcriptome was proﬁled, showing similar cardiac gene expression changes (Figure 4E, Supplementary Table S5). FIGURE 4 FIGURE 4 FIGURE 3 Cardiac gene expression in MZsap130a and MZsap130b (A) WISH of nkx2.5 at 10 somite stage for AB, MZsap130a and MZsap130b. (B) WISH of myh6 and myh7 at 24hpf for AB, MZsap130a and MZsap130b. (C) WISH of myh7 at 36hpf in AB* and MZsap130a. Scale bar 100 μm. RNAseq reveals cardiac contraction and conduction is altered in MZsap130a (A) Representative images of Tg(myl7:EGFP) and MZsap130a;Tg(myl7:EGFP) embryos collected for whole embryo RNAseq at 36hpf. (B) Volcano plot of 36hpf whole embryo RNAseq data. (C) Heatmap of sarcomere and conduction genes (Supplementary Figure S6) at 36hpf from whole embryos. (D) DAVID functional annotation cluster 8 from downregulated genes in MZsap130a, showing DAVID calculated p-values. (E) Heatmap of 48hpf heart tissue RNAseq data for the same genes found in panel C. V and A are ventricle and atria, respectively. Scale bar 100 μm. complex has been shown to regulate sarcomere speciﬁc genes like titins, troponins, and actins important for cardiac contraction (van Oevelen et al., 2010). Since SAP130 has been shown to be part of the SIN3A complex, we reasoned that the phenotype may be caused by altered regulation of cardiac gene expression during development. A whole embryo RNA sequencing (RNAseq) experiment, separating the SV and “normal” (NV) siblings in the MZsap130a mutants, was performed at 36hpf. We ﬁrst performed our analysis looking for differences in the wildtype, compared to NV and SV separately ﬁnding 2,826 differentially expressed genes (DEGs) in common, with 812 unique DEGs for NV and 1979 for SV. Functional annotation of these gene groups revealed that NV and SV embryos are similar when compared to the wildtype transcriptome (Supplementary Table S2). Comparing the controls to all MZsap130a samples (both NV and SV), we observed 5,002 DEGs that included many cardiac speciﬁc transcripts. Among the DEGs we found sarcomere and cardiac conduction genes were dysregulated, suggesting CM biology has changed in the MZsap130a embryos (Figures 4B, C, Supplementary Tables S3, S4). To identify potential pathways involved in heart function and development, we used the Database for Annotation, Visualization, and Integrated Discovery (DAVID) functional annotation of downregulated genes. This showed enrichment for cardiac contraction and adrenergic signaling in CMs, further suggesting a role for sap130a in CM function (Figure 4D, Supplementary Table complex has been shown to regulate sarcomere speciﬁc genes like titins, troponins, and actins important for cardiac contraction (van Oevelen et al., 2010). RNAseq reveals sap130a is involved in regulating cardiac sarcomere and conduction genes Sap130a AUG start codon antisense-morpholino (MO) studies suggested the SVs arise from decreased ventricular CMs (Liu et al., 2017), but where or when CMs are lost was not explored. To determine if the SVs are due to decreased cardiac progenitors, we performed Whole Mount In Situ Hybridization (WISH) at 10 somite stage with nkx2.5, an early cardiac progenitor marker. We discovered no differences between MZsap130a and controls Variants in genes encoding sarcomere proteins have been linked to CHDs (Jones et al., 1996; Morano et al., 2000; Ching et al., 2005; Zhu et al., 2006; Monserrat et al., 2007; Granados-Riveron et al., 2010; Postma et al., 2011; Fahed et al., 2013; van Engelen et al., 2013). The Sin3A Frontiers in Cell and Developmental Biology 05 frontiersin.org frontiersin.org DeMoya et al. 10.3389/fcell.2023.1197109 FIGURE 4 RNAseq reveals cardiac contraction and conduction is altered in MZsap130a (A) Representative images of Tg(myl7:EGFP) and MZsap130a;Tg(myl7:EGFP) embryos collected for whole embryo RNAseq at 36hpf. (B) Volcano plot of 36hpf whole embryo RNAseq data. (C) Heatmap of sarcomere and conduction genes (Supplementary Figure S6) at 36hpf from whole embryos. (D) DAVID functional annotation cluster 8 from downregulated genes in MZsap130a, showing DAVID calculated p-values. (E) Heatmap of 48hpf heart tissue RNAseq data for the same genes found in panel C. V and A are ventricle and atria, respectively. Scale bar 100 μm. FIGURE 3 Cardiac gene expression in MZsap130a and MZsap130b (A) WISH of nkx2.5 at 10 somite stage for AB, MZsap130a and MZsap130b. (B) WISH of myh6 and myh7 at 24hpf for AB, MZsap130a and MZsap130b. (C) WISH of myh7 at 36hpf in AB* and MZsap130a. Scale bar 100 μm. FIGURE 4 MZsap130b whole embryo transcriptome was also proﬁled at 36hpf and less gene expression changes (617 DEGs) were noted (Supplementary Figure S3, Supplementary Table S6). Moreover, the sarcomere gene expression changes seen in MZsap130a was not detected in the MZsap130b transcriptome. DAVID functional annotation of the 278 DEGs common between MZsap130a and MZsap130b mutants, belonged to heme binding and biosynthesis, oxygen binding, and iron binding KEGG pathways, suggesting involvement in hematopoiesis (Supplementary Table S6). The expression proﬁle for these hematopoietic related genes was opposite in MZsap130a and MZsap130b, suggesting distinct functions during hematopoiesis (Supplementary Figure S3). These data suggest that sap130a and sap130b could be involved in hematopoiesis that correlates with sin3aa/ab gene knockdown studies showing strong hematopoietic defects (Huang et al., 2013). Frontiers in Cell and Developmental Biology 06 frontiersin.org DeMoya et al. DeMoya et al. 10.3389/fcell.2023.1197109 FIGURE 5 MZsap130a show cardiac functional deﬁcits (A–C) Shows systole and diastole frames from recordings of live ventricular contractions in Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP) and MZsap130b;Tg(myl7: EGFP) at 48hpf. (D, E) Quantiﬁed cardiac parameters total stroke volume (TSV) and cardiac output (CO), pvals from one-way ANOVA, error bars are SEM. Each point represents individual ventricle and color coded for 3+ experiments. For Tg(myl7:EGFP), n = 115; MZsap130a NV, n = 36; MZsap130a SV, n = 30; MZsap130b NV, n = 57; MZsap130b SV, n = 13. Scale bar 20 μm. Whole embryo and heart tissue MZsap130a RNA-seq data revealed sarcomere genes such as actins and myosins were dysregulated, indicating that sarcomere dysfunction could be part for the MZsap130a mutant phenotype. These data also showed downregulation of CM conduction genes such as cxcr4b and gja3, resulting in changes in cardiogenesis (Severs et al., 2004; Stankunas et al., 2008; Chi et al., 2010; Itou et al., 2012; Mortensen et al., 2017; Jiang et al., 2019). dococs226 (gja3) mutants report having changes in cardiac conduction that lead to CM morphological changes in the ventricle (Chi et al., 2010). Rat studies have shown Cxcr4 involvement in cardiac conduction (Pyo et al., 2006). While MZcxcr4b mutants are reported to have abnormal organ morphogenesis, including heart looping defects (Jiang et al., 2019). Changes were found in calcium channel (cacna1sb, cacng7a, cacnb1, cacna1bb) and sodium channel (scn4aa/ab, scn2b) genes, known to be important to CM biology (Haverinen et al., 2018; Papa et al., 2022; Shah et al., 2022). Sap130a regulates cardiac function Global loss of sap130a showed downregulation of sarcomere genes such as actc1, ttn.1, and ttn.2 (Figures 4B–E). This suggested that cardiac function could be diminished in MZsap130a mutants. The DAVID functional annotation tool revealed enrichment for cardiac muscle contraction genes that were decreased in the MZsap130a mutant embryos (Figure 4D). To determine ventricle chamber function in mutants, confocal light sheet microscopy was used to record live cardiac contractions at 48hpf. These recordings provided us with multiple frames of diastole and systole for chamber volume estimation (Figures 5A–C, and Supplementary Movie S1–S5). Volume estimations were used to calculate the cardiac parameters Total Stroke Volume (TSV), and Cardiac Output (CO) (Yalcin et al., 2017). The light sheet data revealed that all MZsap130a mutants had deﬁcits in CO, TSV, fractional shortening, and ejection fraction (Figures 5D, E, and Supplementary Figure S5). The MZsap130b mutant hearts revealed no signiﬁcant difference from WT function, both in TSV and CO, but showed an increase in End- systolic volume which could explain the increase in CO through increased contraction force (Figures 5D, E and Supplementary Figure S5). The heart tissue RNA-seq identiﬁed cardiac contraction genes myh7, actc1, ttn.1, ttn.2, scn4ab, and cacna1sb were dysregulated in MZsap130a mutants, supporting the contraction deﬁcits measured at 48hpf (Supplementary Figure S6). These data show that sap130a has a role in zebraﬁsh cardiac sarcomere regulation. FIGURE 4 Furthermore, transcriptome analysis revealed that MZsap130a mutants showed dysregulation of a wide range of genes critical for cardiac maturation and function. These include genes associated with fatty acid metabolism (ppt2), glycogen metabolism (ugp2a, phka2), and mitochondria (slc25a44a, slc25a42, mtrf1, mrpl58) found downregulated in MZsap130a mutants in whole embryos at 36hpf and speciﬁcally in the heart at 48hpf (Supplementary Figure S3). Deﬁcits in mitochondrial function have been shown in HLHS patients and other HLHS models including Sap130 mouse mutants and rbfox mutant zebraﬁsh (Liu et al., 2017; Huang et al., 2022). Collectively, MZsap130a mutants show changes in sarcomere, conduction and metabolism associated genes, all integral parts of CM maturation and function. FIGURE 5 MZsap130a show cardiac functional deﬁcits (A–C) Shows systole and diastole frames from recordings of live ventricular contractions in Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP) and MZsap130b;Tg(myl7: EGFP) at 48hpf. (D, E) Quantiﬁed cardiac parameters total stroke volume (TSV) and cardiac output (CO), pvals from one-way ANOVA, error bars are SEM. Each point represents individual ventricle and color coded for 3+ experiments. For Tg(myl7:EGFP), n = 115; MZsap130a NV, n = 36; MZsap130a SV, n = 30; MZsap130b NV, n = 57; MZsap130b SV, n = 13. Scale bar 20 μm. Frontiers in Cell and Developmental Biology frontiersin.org MZsap130a mutants have longer outﬂow tract FIGURE 7 FIGURE 7 Tg(myl7:memGFP) reveals longer OFT by 72hpf in MZsap130a (A) Quantiﬁed OFT lengths at 72hpf, pval is from a one ANOVA, SEM error bars. Each point represents a single embryo, Tg(myl7:memGFP), n = 18; MZsap130a;Tg(myl7:memGFP) NV, n = 20; MZsap130a; Tg(myl7:memGFP) SV, n = 9 (B) Representative images of Tg(myl7: memGFP) and MZsap130a;Tg(myl7:memGFP), white lines demarcate OFT length, pvals from one way ANOVA, SEM error bars. (C) Tg(myl7:memGFP)+;H2b:mCherry + ventricular CM counts for three WT or MZsap130a SV heart, pval is from a t-test, SEM error bars (D) Tg(myl7:memGFP)+;H2b:mCherry + out-ﬂow tract cell counts for three WT or MZsap130a SV heart, pval is from a t-test, SEM error bars. V and A are ventricle and atria, respectively. Scale bar 100 μm. FIGURE 7 Tg(myl7:memGFP) reveals longer OFT by 72hpf in MZsap130a (A) Quantiﬁed OFT lengths at 72hpf, pval is from a one ANOVA, SEM error bars. Each point represents a single embryo, Tg(myl7:memGFP), n = 18; MZsap130a;Tg(myl7:memGFP) NV, n = 20; MZsap130a; Tg(myl7:memGFP) SV, n = 9 (B) Representative images of Tg(myl7: memGFP) and MZsap130a;Tg(myl7:memGFP), white lines demarcate OFT length, pvals from one way ANOVA, SEM error bars. (C) Tg(myl7:memGFP)+;H2b:mCherry + ventricular CM counts for three WT or MZsap130a SV heart, pval is from a t-test, SEM error bars (D) Tg(myl7:memGFP)+;H2b:mCherry + out-ﬂow tract cell counts for three WT or MZsap130a SV heart, pval is from a t-test, SEM error bars. V and A are ventricle and atria, respectively. Scale bar 100 μm. Supplementary Figure S9. Lineage tracing the SHF with MZsap130a;Tg(nkx2.5:kaede) embryos revealed that the SVs acquire less SHF (green area) compared to the wildtype and MZsap130a mutant siblings that develop normal ventricles (Figures 6B–D). Moreover, the OFTs in the MZsap130a mutants were longer at 48hpf in some embryos with SVs (Supplementary Figure S10). The longer OFTs were much more pronounced at 72hpf, and every SV heart had a longer OFT (Figures 7A, B, and Supplementary Figure S10). To count the CMs in the ventricle and OFT of the MZsap130a mutants, we injected Tg(myl7:memGFP) and MZsap130a;Tg(myl7:memGFP) embryos with H2b:mCherry mRNA. We detected an increase in OFT cells that was concomitant with a decrease in ventricular CMs (Figures 7C, D and Supplementary Figure S11). This suggested that the lost ventricular CMs contributed to OFT cells instead and was further evidenced at adult stages. MZsap130a;Tg(myl7:EGFP) embryos were scored at 48hpf for ventricle size and reared separately into adulthood. Frontiers in Cell and Developmental Biology MZsap130a mutants have longer outﬂow tract The earliest observation of smaller ventricles in MZsap130a mutants was at 36hpf, a stage when SHF cells are migrating into the ventricle. Extensive studies have reported the contribution of SHF cells to the ventricle during this time (Grimes et al., 2008; de Pater et al., 2009; Lazic and Scott, 2011; Knight and Yelon, 2016; Felker et al., 2018; Song et al., 2019). RNA-seq data showed that SHF progenitor markers ltbp3, mef2cb and isl1, isl2a/b were decreased (Supplementary Figure S7). These genes are known to label SHF progenitors at the arterial and venous poles. WISH at 30hpf revealed a decrease in ltbp3 expression in MZsap130a mutants (Supplementary Figure S8). Together these data suggest that the SHF in the MZsap130a mutants was affected such that insufﬁcient CMs contribute to the ventricle by 48hpf. To determine if this occurs, we performed lineage tracing Frontiers in Cell and Developmental Biology 07 frontiersin.org 10.3389/fcell.2023.1197109 DeMoya et al. FIGURE 6 Lineage tracing reveal changes to SHF in MZsap130a at 48hpf (A) Diagram showing how the FHF heart tube at 24hpf was photoconverted to red, leaving SHF progenitors unlabeled in green and imaging at 48hpf. (B, C) Confocal imagines of Tg(nkx2.5: kedge) and MZsap130a;Tg(nkx2.5:kedge) at 48hpf with the heart tube being photoconverted at 24hpf, the red outlined region represents area measurements collected. (D) Quantiﬁed SHF (green area) accreted by 48hpf, pval is from a one ANOVA, error bars are SEM. Each point represents a single embryo, Tg(nkx2.5:kedge), n = 15; MZsap130a;Tg(nkx2.5:kedge) NV, n = 14; MZsap130a;Tg(nkx2.5: kedge) SV, n = 10. V and A are ventricle and atria, respectively. Scale bar 100 μm. FIGURE 7 Tg(myl7:memGFP) reveals longer OFT by 72hpf in MZsap130a (A) Quantiﬁed OFT lengths at 72hpf, pval is from a one ANOVA, SEM error bars. Each point represents a single embryo, Tg(myl7:memGFP), n = 18; MZsap130a;Tg(myl7:memGFP) NV, n = 20; MZsap130a; Tg(myl7:memGFP) SV, n = 9 (B) Representative images of Tg(myl7: memGFP) and MZsap130a;Tg(myl7:memGFP), white lines demarcate OFT length, pvals from one way ANOVA, SEM error bars. (C) Tg(myl7:memGFP)+;H2b:mCherry + ventricular CM counts for three WT or MZsap130a SV heart, pval is from a t-test, SEM error bars (D) Tg(myl7:memGFP)+;H2b:mCherry + out-ﬂow tract cell counts for three WT or MZsap130a SV heart, pval is from a t-test, SEM error bars. V and A are ventricle and atria, respectively. Scale bar 100 μm. MZsap130a mutants have longer outﬂow tract Images of heart revealed a larger bulbus arteriosus (BA) area, the adult structure derived from the OFT, and decreased ventricular area (Figure 8). The observations in the MZsap130a adults from small ventricle embryos correlates with the notion that sap130a is involved in SHF cell fate decisions between ventricular CMs and OFT cells. Lineage tracing reveal changes to SHF in MZsap130a at 48hpf (A) Diagram showing how the FHF heart tube at 24hpf was photoconverted to red, leaving SHF progenitors unlabeled in green and imaging at 48hpf. (B, C) Confocal imagines of Tg(nkx2.5: kedge) and MZsap130a;Tg(nkx2.5:kedge) at 48hpf with the heart tube being photoconverted at 24hpf, the red outlined region represents area measurements collected. (D) Quantiﬁed SHF (green area) accreted by 48hpf, pval is from a one ANOVA, error bars are SEM. Each point represents a single embryo, Tg(nkx2.5:kedge), n = 15; MZsap130a;Tg(nkx2.5:kedge) NV, n = 14; MZsap130a;Tg(nkx2.5: kedge) SV, n = 10. V and A are ventricle and atria, respectively. Scale bar 100 μm. experiments using Tg(nkx2.5:kaede) embryos (Guner-Ataman et al., 2013). In this transgenic line, the FHF cells can be permanently labeled at 24hpf, photo-converting only the heart tube. Next, we imaged at 48hpf to determine the addition of green cells to the ventricle (Figure 6A, and 08 frontiersin.org DeMoya et al. 10.3389/fcell.2023.1197109 FIGURE 8 Adult MZsap130a hearts have large bulbus arteriosus (A, B) AB* and MZsap130a adult SV hearts extracted at approximately 4–6 months post fertilization. (C) Quantiﬁcation of ventricle area of unﬁxed hearts, pval is from a one ANOVA solid black bars are mean, dotted lines represent up and lower 25th percentiles. Each point represents a single heart, AB, n = 14; MZsap130a NV, n = 7; MZsap130a SV, n = 12. (D) Quantiﬁcation of BA area. of unﬁxed hearts, pval is from a one ANOVA solid black bars are mean, dotted lines represent up and lower 25th percentiles. Each point represents a single heart, n = 10 for all groups. V, A, and BA are ventricle, atria, and bulbus arteriosus respectively, Scale bar 200 μm. FIGURE 9 sap130a shows an association with hdac1 and MZsin3ab mutants have SVs (A) Representative image of Tg(myl7:EGFP) and MZsap130a; hdac1b382/+;Tg(myl7:EGFP) NV and SVs at 48hpf. (B) Representative image of Tg(myl7:EGFP) and MZsin3ab;Tg(myl7:EGFP) NV and SVs at 48hpf. (C) Quantiﬁcation of heart phenotype proportions in Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP), hdac1b382/+;Tg(myl7: EGFP) and MZsap130a;hdac1b382/+;Tg(myl7:EGFP). The p-values are ﬁsher’s exact test. MZsap130a mutants have longer outﬂow tract V, A, and BA are ventricle, atria, and bulbus arteriosus respectively, Scale bar 200 μm. FIGURE 9 sap130a shows an association with hdac1 and MZsin3ab mutants have SVs (A) Representative image of Tg(myl7:EGFP) and MZsap130a; hdac1b382/+;Tg(myl7:EGFP) NV and SVs at 48hpf. (B) Representative image of Tg(myl7:EGFP) and MZsin3ab;Tg(myl7:EGFP) NV and SVs at 48hpf. (C) Quantiﬁcation of heart phenotype proportions in Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP), hdac1b382/+;Tg(myl7: EGFP) and MZsap130a;hdac1b382/+;Tg(myl7:EGFP). The p-values are ﬁsher’s exact test. (D) Quantiﬁcation of heart phenotype proportions in Tg(myl7:EGFP) and MZsin3ab;Tg(myl7:EGFP). The p-values are ﬁsher’s exact test. V and A are ventricle and atria, respectively. Scale bar 100 μm. sap130a genetically interacts with hdac1 during SHF accretion Zebraﬁsh hdac1 is required for ventricle formation (Song et al., 2019; Buhler et al., 2021). We explored the potential interaction of Sap130a and Hdac1 by analyzing heart development in MZsap130a; hdac1+/b382 embryos. While hdac1 homozygous mutants develop cardiac defects, heterozygous mutants are viable and show a similar proportion of SVs like in the MZsap130a mutants. An increase in SV phenotype was noted in MZsap130a;hdac1+/b382 suggesting MZsap130a mutants are sensitized to hdac1 gene dosage (Figures 9A, C). These data revealed an association between hdac1 heterozygous status and ventricle size and this increase in a MZsap130a background (Figure 9C). This suggests that sap130a and hdac1 genetically interact in zebraﬁsh and function in the same complex like in mammals. mutants using CRISPR/Cas9. The sin3ab pt36a allele generated disrupted amino acids 862–867. In MZsin3ab mutants an in complete penetrant SV phenotype was observed in 44% (Figures 9B, D). WISH data at 30hpf, revealed that ltbp3 expression in MZsin3ab mutants was reduced, similar to the MZsap130a mutants in Supplementary Figure S8. It is not surprising that the penetrance of the phenotype in MZsin3ab was also incomplete since both sin3aa and sin3b could compensate for the disruption of sin3ab. These data suggest that sin3ab is involved in ventricular development in zebraﬁsh, a phenotype that is reminiscent of sap130a and hdac1 mutants. Frontiers in Cell and Developmental Biology MZsap130a mutants have longer outﬂow tract (D) Quantiﬁcation of heart phenotype proportions in Tg(myl7:EGFP) and MZsin3ab;Tg(myl7:EGFP). The p-values are ﬁsher’s exact test. V and A are ventricle and atria, respectively. Scale bar 100 μm. FIGURE 8 Adult MZsap130a hearts have large bulbus arteriosus (A, B) AB and MZsap130a adult SV hearts extracted at approximately 4–6 months post fertilization. (C) Quantiﬁcation of ventricle area of unﬁxed hearts, pval is from a one ANOVA solid black bars are mean, dotted lines represent up and lower 25th percentiles. Each point represents a single heart, AB, n = 14; MZsap130a NV, n = 7; MZsap130a SV, n = 12. (D) Quantiﬁcation of BA area. of unﬁxed hearts, pval is from a one ANOVA solid black bars are mean, dotted lines represent up and lower 25th percentiles. Each point represents a single heart, n = 10 for all groups. V, A, and BA are ventricle, atria, and bulbus arteriosus respectively, Scale bar 200 μm. FIGURE 8 Adult MZsap130a hearts have large bulbus arteriosus (A, B) AB and MZsap130a adult SV hearts extracted at approximately 4–6 months post fertilization. (C) Quantiﬁcation of ventricle area of unﬁxed hearts, pval is from a one ANOVA solid black bars are mean, dotted lines represent up and lower 25th percentiles. Each point represents a single heart, AB, n = 14; MZsap130a NV, n = 7; MZsap130a SV, n = 12. (D) Quantiﬁcation of BA area. of unﬁxed hearts, pval is from a one ANOVA solid black bars are mean, dotted lines represent up and lower 25th percentiles. Each point represents a single heart, n = 10 for all groups. V, A, and BA are ventricle, atria, and bulbus arteriosus respectively, Scale bar 200 μm. FIGURE 8 Adult MZsap130a hearts have large bulbus arteriosus (A, B) AB and MZsap130a adult SV hearts extracted at approximately 4–6 months post fertilization. (C) Quantiﬁcation of ventricle area of unﬁxed hearts, pval is from a one ANOVA solid black bars are mean, dotted lines represent up and lower 25th percentiles. Each point represents a single heart, AB*, n = 14; MZsap130a NV, n = 7; MZsap130a SV, n = 12. (D) Quantiﬁcation of BA area. of unﬁxed hearts, pval is from a one ANOVA solid black bars are mean, dotted lines represent up and lower 25th percentiles. Each point represents a single heart, n = 10 for all groups. Data availability statement All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI GEO under GSE228451. Acknowledgments We are grateful to members of the MT lab, Donghun JS, Elizabeth Rochon, and Neil Hukriede for reading the manuscript and experimental suggestions. Tim Feinstein, who built the light sheet scope and mounting system. Manush Saydmohammed and Shoulin Li for initial work on MZsap130a mutant and the Tg(myl7: memGFP) line. Conﬂict of interest Taken together this study support the importance of Sap130a/ Sin3a/Hdac complex in zebraﬁsh cardiogenesis. These observations build upon previous studies in zebraﬁsh hdac1 and reiterates the importance of context speciﬁc components for the Sin3a complex during cardiogenesis. The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Funding This research was supported by funding from the National Institutes of Health R01HL142788 to MT and CL, and R01HL156398 to MT This research was supported in part by the University of Pittsburgh Center for Research Computing through the resources provided. Open Access funding provided by University of Pittsburgh. This research was supported in part by the University of Pittsburgh Center for Research Computing, RRID: SCR_022735, through the resources provided. Speciﬁcally, this work used the HTC cluster, which is supported by NIH award number S10OD028483. This project used the University of Pittsburgh HSCRF Genomics Research Core, RRID: SCR_018301, for RNA-seq experiments. RNA-seq data were aligned with CLC genomics Workbench Version 20 (QIAGEN), licensed through the Molecular Biology Information Service of the Health Sciences Library System, University of Pittsburgh. The catalytic unit of the SIN3A complex is comprised of class I HDACs, which deacetylate lysine residues to alter gene expression or protein function. The hdac1 mutant, cardiac really gone (crg) exhibit decreased ventricular CMs by 36hpf (Song et al., 2019). This is also the timepoint MZsap130a mutants show decreased ventricular size. This suggests sap130a and hdac1 could have overlapping functions during SHF development. Although SAP130 was shown to have HDAC-independent functions from in vitro studies (Fleischer et al., 2003), genetic studies suggest sap130a and hdac1 interact for proper ventricular cardiomyocyte development. This supports a model in which Sap130a associates with the Sin3a/Hdac1 complex and/or an Hdac1-independent X-factor to regulate transcription (Supplementary Figure S13). Author contributions Our study reveals the consequence of disrupting members of the SIN3A complex, resulting in improper heart development. In the MZsap130a mutants, the main phenotype is a small ventricle leading to larger OFT and bulbus arteriosus in adulthood. Developmentally this arises from the failure of SHF progenitors to migrate into the growing ventricle. We come to this conclusion because the WISH data for nkx2.5 and myh7 showed no changes prior to the 24 hpf, indicating the FHF is intact. The phenotype arising at 36hpf is in line with observations showing the addition of SHF cells between 24 and 48hpf and with our lineage tracing experiments (Grimes et al., 2008; de Pater et al., 2009; Hami et al., 2011; Lazic and Scott, 2011; Felker et al., 2018). In the Ohia mouse mutant, the combination of PCDHA9 and a SAP130 mutations caused an HLHS etiology inﬂuencing the FHF structures. The prominent phenotype included a hypoplastic left ventricle and valve abnormalities in 11% of mouse embryos. In the zebraﬁsh, the sap130a mutation is predicted to be a null mutant producing a hypoplastic ventricle in 48% of embryos. The difference seen between the mouse and zebraﬁsh can be explained by the difference in the number of ventricle chambers, the specialized development of the mammalian OFT, and the changes seen during the evolution of this specialized pump from sea to land (Jensen et al., 2013). A recent study of SAP130 pig CRISPR mutants show tricuspid dysplasia and atresia, highlighting the complex role of Sap130 in heart development across different species (Gabriel et al., 2021). RD, CL, and MT designed research; RD, RF-R, DF, and JS performed research; RD and MT analyzed data CL and MT responsible for funding. RD wrote the manuscript. All authors contributed to the article and approved the submitted version. Discussion Both MZsap130a whole embryo and heart speciﬁc RNA-seq datasets revealed sarcomere genes to be down and cell cycle genes to be upregulated, similar to SIN3A knock-out and knock-down studies (Supplementary Figure S12) (van Oevelen et al., 2008; van Oevelen et al., 2010; Dobi et al., 2014). For example, the cell cycle genes vrk1, e2f7, and haus6 are increased in MZsap130a mutants, while we did not ﬁnd evidence of expanded cardiac progenitors. These similarities in up and down DEGs point to the possibility that sap130a associates with sin3aa or sin3ab in zebraﬁsh, similar to mammals. To further explore the importance of SIN3A in heart development, we generated MZsin3ab In this study, we have revealed a role for sap130a in zebraﬁsh cardiogenesis. We describe a null allele of sap130a, resulting in small ventricles through the delay and failure of SHF cells to migrate into the ventricle. Without sap130a, some of the SHF progenitors permanently become OFT cells. Transcriptome proﬁling of the MZsap130a embryos at 36hpf and hearts at 48hpf revealed that expression of sarcomere, conduction, and metabolism genes were dysregulated. This suggest that the CMs fail to terminally differentiate and properly function. Frontiers in Cell and Developmental Biology 09 frontiersin.org DeMoya et al. DeMoya et al. 10.3389/fcell.2023.1197109 10.3389/fcell.2023.1197109 Frontiers in Cell and Developmental Biology References Adams, G. 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https://openalex.org/W3216915900	https://epub.ub.uni-muenchen.de/100034/1/fpsyg-12-769206.pdf	English	null	Early and Later Perceptions and Reactions to the COVID-19 Pandemic in Germany: On Predictors of Behavioral Responses and Guideline Adherence During the Restrictions	Frontiers in psychology	2,021	cc-by	18,501	ORIGINAL RESEARCH doi: 10.3389/fpsyg.2021.769206 published: 26 November 2021 Keywords: COVID-19, trust, behavior change, guideline adherence, social distancing, political orientation, acceptance, health anxiety Early and Later Perceptions and Reactions to the COVID-19 Pandemic in Germany: On Predictors of Behavioral Responses and Guideline Adherence During the Restrictions Eva Lermer 1,2,3†, Matthias F. C. Hudecek 1,2,4†, Susanne Gaube 1,5, Martina Raue 6 and 1 LMU Center for Leadership and People Management, LMU Munich, Munich, Germany, 2 Institute of Business Psychology, FOM University of Applied Sciences, Munich, Germany, 3 Applied Business and Media Psychology program, Ansbach University of Applied Sciences, Ansbach, Germany, 4 Department of Experimental Psychology, University of Regensburg, Regensburg, Germany, 5 Department of Infection Prevention and Infectious Diseases, University Hospital of Regensburg, Regensburg, Germany, 6 MIT AgeLab, Massachusetts Institute of Technology, Cambridge, MA, United States, 7 Department of Obstetrics and Gynecology and Center for Gynecological Endocrinology and Reproductive Medicine, University Hospital, LMU Munich, Munich, Germany Edited by: Bojana M. Dinic, University of Novi Sad, Serbia In March 2020, the German government enacted measures on movement restrictions and social distancing due to the COVID-19 pandemic. As this situation was previously unknown, it raised numerous questions about people’s perceptions of and behavioral responses to these new policies. In this context, we were specifically interested in people’s trust in official information, predictors for self-prepping behavior and health behavior to protect oneself and others, and determinants for adherence to social distancing guidelines. To explore these questions, we conducted three studies in which a total of 1,368 participants were surveyed (Study 1 N = 377, March 2020; Study 2 N = 461, April 2020; Study 3 N = 530, April 2021) across Germany between March 2020 and April 2021. Results showed striking differences in the level of trust in official statistics (depending on the source). Furthermore, all three studies showed congruent findings regarding the influence of different factors on the respective behavioral responses. Trust in official statistics predicted behavioral responses in all three studies. However, it did not influence adherence to social distancing guidelines in 2020, but in 2021. Furthermore, adherence to social distancing guidelines was associated with higher acceptance rates of the measures and being older. Being female and less right-wing orientated were positively associated with guidelines adherence only in the studies from 2020. This year, political orientation moderated the association between acceptance of the measures and guideline adherence. This investigation is one of the first to examine perceptions and reactions during the COVID-19 pandemic in Germany across 1 year and provides insights into important dimensions that need to be considered when communicating with the public. In March 2020, the German government enacted measures on movement restrictions and social distancing due to the COVID-19 pandemic. As this situation was previously unknown, it raised numerous questions about people’s perceptions of and behavioral responses to these new policies. In this context, we were specifically interested in people’s trust in official information, predictors for self-prepping behavior and health behavior to protect oneself and others, and determinants for adherence to social distancing guidelines. To explore these questions, we conducted three studies in which a total of 1,368 participants were surveyed (Study 1 N = 377, March 2020; Study 2 N = 461, April 2020; Study 3 N = 530, April 2021) across Germany between March 2020 and April 2021. Edited by: Bojana M. Dinic, University of Novi Sad, Serbia Results showed striking differences in the level of trust in official statistics (depending on the source). Furthermore, all three studies showed congruent findings regarding the influence of different factors on the respective behavioral responses. Trust in official statistics predicted behavioral responses in all three studies. However, it did not influence adherence to social distancing guidelines in 2020, but in 2021. Furthermore, adherence to social distancing guidelines was associated with higher acceptance rates of the measures and being older. Being female and less right-wing orientated were positively associated with guidelines adherence only in the studies from 2020. This year, political orientation moderated the association between acceptance of the measures and guideline adherence. This investigation is one of the first to examine perceptions and reactions during the COVID-19 pandemic in Germany across 1 year and provides insights into important dimensions that need to be considered when communicating with the public. Reviewed by: Valerio Capraro, Middlesex University, United Kingdom Yossi Maaravi, Interdisciplinary Center Herzliya, Israel Reviewed by: Valerio Capraro, Middlesex University, United Kingdom Yossi Maaravi, Interdisciplinary Center Herzliya, Israel Correspondence: Eva Lermer eva.lermer@hs-ansbach.de †These authors share first authorship Specialty section: This article was submitted to Personality and Social Psychology, a section of the journal Frontiers in Psychology Received: 01 September 2021 Accepted: 28 October 2021 Published: 26 November 2021 Edited by: Edited by: Bojana M. Dinic, University of Novi Sad, Serbia INTRODUCTION rates (Oksanen et al., 2020). Since health authorities used infection and death statistics to justify their strict regulations and encouraged everyone to help “flatten the curve” (of new infections), we expected trust in these official statistics to be an important predictor of compliance with the protective measures. Therefore, we aimed at investigating trust in official information from different sources and formulated the following research question (RQ): On Sunday March 22, 2020, Angela Merkel, the German Chancellor, announced that in the fight against the spread of the novel Coronavirus, she and the prime ministers of Germany agreed that public gatherings of more than two people would be prohibited temporarily for 14 days (Frankfurter Allgemeine Zeitung, 2020). Movement restrictions and social/physical distancing provisions have never existed before in the Federal Republic of Germany, and so it was unclear how people would react to them. Obviously, the COVID-19 pandemic has raised many questions in many scientific disciplines. Social sciences offered an abundance of theories to predict and explain human behavior in extreme conditions – such as a pandemic. One of the first researchers recommending the application of relevant knowledge from the social and behavioral sciences to the context of the COVID-19 pandemic was Bavel et al. (2020). The extent to which they meet the research zeitgeist is reflected in the number of citations: In October 2021, only about 1.5 years after the publication of their article, it has already been cited over 2,400 times. We also wanted to contribute to a better understanding of how people behave in this new situation. Therefore, it was important for us to examine which variables are central for acceptance of the measure and behavioral responses in this context. Based on previous studies in the areas of pandemics (e.g., Ebola: Vinck et al., 2019), prevention measures (e.g., Rykkja et al., 2011), and risk communication (e.g., Baumgartner and Hartmann, 2011), we selected a set of potentially relevant variables. These include trust, political orientation, health anxiety, and uncertainty tolerance.f RQ 1: How much do people trust in statistics on COVID-19 from official authorities? RQ 1: How much do people trust in statistics on COVID-19 from official authorities? In the course of the COVID-19 pandemic, the media constantly reported about people’s reactions to the new circumstances. INTRODUCTION This included increased purchasing or even hoarding of products such as disinfectants, face masks, food and toilet paper (Statista, 2020a; Statistisches Bundesamt, 2020), as well as differences in people’s compliance with social distancing measures (Lehrer et al., 2020; Statista, 2020b). Uncertainty about the virus itself, its origin, or the appropriate measures to combat it, coupled with a growing group of people who challenge established facts set the stage for the rise of conspiracy theories. In such an environment, merely trying to convince people of the severity of the disease and the effectiveness of the prevention measures may not be sufficient to encourage protective behavior such as social distancing. Therefore, it is important to not only understand how much people trust in official infection statistics, but also to explore further in the pandemic relevant variables. First, it must be understood which variables are central to behavioral responses to subsequently develop appropriate communication strategies. As behavioral responses, we considered three types of behavior: (A) Self-centered prepping behavior (e.g., stocking up on face masks, food, or other essential goods; the term is also used by Imhoff and Lamberty, 2020, for hoarding everyday goods in the COVID-19 pandemic), and protective behavior to not infect (B) oneself and (C) others. We differentiate here between protective behavior for oneself and for others for different reasons. For example, risk research shows that risk assessments differ depending on who the target person is (i.e., self vs. other, see Lermer et al., 2013, 2019). Furthermore, people differ in prosocial behavior (e.g., Eagly, 2009). While this is more pronounced in some than in others, it need not be related to their self-protective behavior. Like some previous studies (e.g., Longstaff and Yang, 2008; van der Weerd et al., 2011), we consider trust to be an important variable for human behavior in the context of a pandemic. The APA Dictionary of Psychology (2020) defines trust as “reliance on or confidence in the dependability of someone or something.” However, trust is a broad concept and can refer to different aspects, depending on the perspective. The relevant perspective for us at the time of the first study was trust in infection statistics from official authorities, that is, the figures communicated by official institutions and governments. Citation: Lermer E, Hudecek MFC, Gaube S, Raue M and Batz F (2021) Early and Later Perceptions and Reactions to the COVID-19 Pandemic in Germany: On Predictors of Behavioral Responses and Guideline Adherence During the Restrictions. Front. Psychol. 12:769206. doi: 10.3389/fpsyg.2021.769206 November 2021 \| Volume 12 \| Article 769206 1 Frontiers in Psychology \| www.frontiersin.org Reactions to COVID-19 in Germany Lermer et al. Frontiers in Psychology \| www.frontiersin.org Participants and Procedure p To explore our RQs, we conducted an online survey end of March, 2020, when the restrictions in Germany started. At that time (March 23), 24,774 people had been infected and 94 had died from or with COVID-19 in Germany (WHO, 2020b). A total of 571 people clicked on the questionnaire. Data from participants who failed to answer the attention check item (i.e., If you add two and three, you get four) correctly or did not finish the questionnaire were not included. The final sample encompassed data from 377 participants (78% female) who worked and studied part-time at a university with locations in 32 cities across Germany (Mage = 26.54, SDage = 4.97, and Rangeage = 19–53). There is no information about the individual characteristics of those participants who were excluded. However, all participants originate from the same pool of people (employees or self-employed persons who were enrolled at the FOM University of Applied Sciences as part-time students). One of the participants reported being infected with COVID-19. Forty participants reported that they felt sick, and 10 of those assumed they were infected. All other participants reported that they felt healthy. None of the (possibly) infected participants were excluded. Participants were rewarded with course credits for their participation. p Anxiety is associated with high uncertainty and often motivates people to take action which should reduce uncertainty (Raghunathan and Pham, 1999), such as increased information seeking (Valentino et al., 2009). The COVID-19 pandemic is a threat that is both dreadful and highly uncertain. Research has shown that these affective states strongly influence people’s perceptions of risk (Fischhoff et al., 1978). Perceived risk is influenced by uncertainty (Vives and FeldmanHall, 2018). Uncertainty during the current pandemic is high because SARS- CoV-2 is a novel virus that has until recently not been known to scientists. As a result, it is unclear how the pandemic will develop and difficult to accurately assess one’s personal risk. Uncertainty is a state that is perceived as discomforting and people generally strive to avoid it (Schneider et al., 2017). However, people differ in their tolerance for uncertainty (Grenier et al., 2005). Research on the tolerance of uncertainty goes back to Frenkel-Brunswik (1949) who observed that people systematically differ in dealing with ambiguous situations (Dalbert, 1999). People with a low level of uncertainty tolerance employ vigilant coping strategies such as intensified information seeking about the threatening event. INTRODUCTION Previous research has shown that trust in political systems may influence people’s reactions to restrictions, that is, trust is positively correlated with acceptance of prevention measures in a society (e.g., anti-terror measures, Rykkja et al., 2011) and linked to law compliance (Marien and Hooghe, 2011). Also, Rowe and Calnan (2006) have shown that trust in public systems and authorities positively influences the way people follow instructions. Greater trust in policy makers is associated with greater compliance in health policies such as testing or quarantining. These relationships could also be demonstrated in past pandemics (e.g., Ebola: Morse et al., 2016; Blair et al., 2017; Asian influenza and H1N1 pandemic: Siegrist and Zingg, 2014). There are some good summaries of the relevance of trust in the context of Coronavirus pandemic (e.g., Balog-Way and McComas, 2020; Devine et al., 2021). Only recently, in the context of the COVID-19 pandemic, it has been shown that trust in institutions is associated with lower mortality p Complex and alarming world events are often accompanied by the emergence of conspiracy theories (McCauley and Jacques, 1979; Leman and Cinnirella, 2007; Jolley and Douglas, 2014). These theories assume that the event in question is the result of a secret plot of a powerful group (Imhoff and Bruder, 2014). Previous research suggests that political orientation may be associated with conspiracy beliefs. For instance, van Prooijen et al. (2015) found a positive association between extreme political ideologies (at both sides the right and the left) and the tendency to believe in conspiracy theories. The authors conclude that “political extremism and conspiracy beliefs are strongly associated due to a highly structured thinking style that is aimed at making sense of societal events” (p. 570). A study in Italy has shown that believing in conspiracies is linked to right-wing political orientation (Mancosu et al., 2017). In November 2021 \| Volume 12 \| Article 769206 Frontiers in Psychology \| www.frontiersin.org 2 Reactions to COVID-19 in Germany Lermer et al. their recent study in the context of the COVID-19 pandemic, Imhoff and Lamberty (2020) showed that conservative political orientation was positively associated with self-centered prepping behavior (e.g., stocking up on face masks, food, or other essential goods; the term is also used by Imhoff and Lamberty, 2020, for hoarding everyday goods in the COVID-19 pandemic). Due to these findings, we included political orientation in this research. INTRODUCTION Furthermore, at least two variables seem to be central to behavioral responses during health threatening events: health anxiety and uncertainty tolerance. Today, numerous studies can be found showing that the COVID-19 pandemic increased levels of anxiety (e.g., Baloran, 2020; Choi et al., 2020; Petzold et al., 2020; Roy et al., 2020; Buspavanich et al., 2021). Fewer studies, however, specifically examine health anxiety and its links to reactions to the COVID-19 pandemic. Research shows that anxiety is linked to safety-seeking behavior (Abramowitz et al., 2007; Tang et al., 2007; Helbig-Lang and Petermann, 2010). For example, health anxiety has been linked to an increase in health information searching (Baumgartner and Hartmann, 2011). Sometimes, however, health anxiety can lead people to avoid relevant information that creates discomfort (Kőszegi, 2003). Avoiding information about a diagnosis, for example, seems to help reduce stress and anxiety, while delaying beneficial action (Golman et al., 2017). In a recent article, Asmundson and Taylor (2020) report that people with high health anxiety also tend to engage in maladaptive behaviors such as panic purchasing. Thus, we were interested in the impact of health anxiety on people’s behavioral responses in the COVID-19 pandemic.t and age seemed important to us to be considered as well. Especially because results of recent studies in the COVID-19 context suggest that these are relevant characteristics regarding behavioral responses. For example, it was shown that women and older participants tended to be more willing to wear face masks (e.g., Capraro and Barcelo, 2020). Also, the results of a study conducted by Li and Liu (2020) suggest that women tend to be engaged in more protective behaviors during the COVID-19 pandemic than men. Furthermore, this also seems to be true for being of older age (Li and Liu, 2020). In sum, we aimed at understanding how trust, political orientation, health anxiety, and uncertainty tolerance, in addition to gender and age, influence people’s self-centered prepping behavior and protective behavior to avoid infection of oneself or others. RQ 2: How does trust in official statistics, political orientation, health anxiety, uncertainty tolerance, age and gender influence people’s prepping, and health behaviors during the COVID-19 pandemic? Participants and Procedure In the context of the COVID-19 pandemic, this could result in reading the news more often than usual. At the same time, people with a low level of uncertainty tolerance tend to show avoidance strategies such as turning away from dreadful information about the threat (Grenier et al., 2005). Thus, we were interested in the impact of uncertainty tolerance on people’s behavioral responses in the COVID-19 pandemic. Furthermore, the variables gender Frontiers in Psychology \| www.frontiersin.org Self-Centered Prepping Behavior Self Centered Prepping Behavior Self-centered prepping behavior in the context of COVID-19 was measured using three items: “I bought face masks;” “I stocked up on food;” and “I stocked up on disinfectant.” The answer format was yes or no. Yes answers were summed up to a self-centered prepping behavior sum value. At the time of the study, it was not yet clear (at least to the public) that wearing a mask was more protective for others than for oneself. In addition, masks were a scarce commodity at the time. At the beginning of the Corona pandemic, not even system-relevant institutions (e.g., hospitals) were supplied with sufficient amounts of masks (Biermann et al., 2020; WHO, 2020a). Thus, masks were difficult to obtain at that time. Also, an official requirement to wear masks in public (e.g., while shopping and public transportation) was not introduced throughout Germany until April 29, 2020 (Mitze et al., 2020; The Federal Government Germany, 2020). We therefore understand buying face masks as a behavior to hoard a certain good to build up a stock on them for a certain period of time. With this understanding, we follow the conceptualization of self-prepping behavior described by Imhoff and Lamberty (2020). Measuresi Trust in Official Infection Statistics At the time of the survey, media and reporting in Germany mainly covered figures on the incidence of infection from official sources of the governments of China, the EU, Germany, and the Robert Koch Institute (RKI; the RKI is the government’s central scientific institution in the field of biomedicine. It is one of the most important bodies for the safeguarding of public health in Germany.). Trust in statistics from governments and the RKI was measured with four items on a seven-point November 2021 \| Volume 12 \| Article 769206 Frontiers in Psychology \| www.frontiersin.org 3 Reactions to COVID-19 in Germany Lermer et al. TABLE 1 \| Participants’ trust in official information in Study 1, Study 2, and Study 3. I trust the statistics from Study 1 (N = 377) March 2020 M (SD) Study 2 (N = 461) April 2020 M (SD) Study 3 (N = 530) April 2021 M (SD) China 2.89 (1.58) 2.23 (1.36) 2.28 (1.38) European Countries 4.28 (1.67) 3.83 (1.66) 3.95 (1.73) German Government 4.85 (1.72) 4.63 (1.76) 4.53 (1.88) Robert Koch Institute 5.22 (1.64) 4.94 (1.76) 4.89 (1.90) The scale ranged from 1 = strongly disagree to 7 = strongly agree. TABLE 1 \| Participants’ trust in official information in Study 1, Study 2, and Study 3. Likert scale (1 = strongly disagree to 7 = strongly agree). The items were all designed in the same way: “I trust the statistics from … (1) China, (2) European Countries, (3) German government, (4) RKI on the Corona virus.” These items were averaged to an index of trust (Cronbach’s α = 0.86), which was used as an independent variable to investigate RQ 2. Protective Behavior for Self-Protection Protective behavior to avoid infection was measured using four items. Individuals were asked to indicate change in behavior or new behavior on a five-point Likert scale (1 = strongly disagree to 5 = strongly agree). Items were: “I avoid public transport (contact with other people/visiting cafés and restaurants/meetings with friends) in order not to get infected.” These items were averaged to an index of behavior change for self-protection (Cronbach’s α = 0.81). Health Anxiety y Health anxiety was measured using the German version of the health anxiety inventory (MK-HAI) developed by Bailer and Witthöft (2014). This scale assesses the trend toward health-related concerns with 14 items on a five-point Likert scale (1 = strongly disagree to 5 = strongly agree); sample item: “I spend a lot of time worrying about my health.” These items were averaged to an index of health anxiety (Cronbach’s α = 0.93). Uncertainty Tolerance We measured uncertainty tolerance with the Uncertainty Tolerance (UT) Scale developed by Dalbert (1999). This questionnaire captures the tendency to assess uncertain situations as threats or challenges with eight items on a six-point Likert scale (1 = strongly disagree to 6 = strongly agree); sample item: “I like to know what to expect.” These items were averaged to an index of uncertainty tolerance (Cronbach’s α = 0.70). Frontiers in Psychology \| www.frontiersin.org Political Orientation Participants’ political orientation was measured using the Left– Right Self-Placement scale developed by Breyer (2015). This scale measures political attitudes on a left-right dimension with a single item asking participants to locate themselves on a 10-point Likert scale with the poles left and right. Protective Behavior for Othersh The protective behavior to not infect others was measured using the same four items as to measure behavior change for self-protection. However, these items were related to other people; a sample item reads, “I avoid public transport to protect others.” These items were averaged to an index of behavior change for others (Cronbach’s α = 0.88). STUDY 1 RESULTS To answer RQ 1, participants’ trust in statistics on COVID-19 infections from different official authorities was analyzed, and the results are shown in Table 1. Trust in statistics from China was by far the lowest, whereas trust in statistics from the RKI was highest. To answer RQ 2, we analyzed associations with behavioral responses by using correlation analyses. Results can be found in Table 2.i All variables, except uncertainty tolerance, showed significant associations with one of the three behavioral responses. To investigate the role of these variables to predict behavior change, we conducted linear multiple regression analyses. Findings are shown in Table 3. Results show that lower levels of trust and higher levels of health anxiety are associated with more prepping behavior. Higher levels of trust in official statistics, being female and being of younger age within our sample were shown to be significant predictors for self-protecting behavior. There was also a tendency of higher levels of health anxiety to predict November 2021 \| Volume 12 \| Article 769206 4 Reactions to COVID-19 in Germany Lermer et al. TABLE 2 \| Means, SDs, and correlations for gender, age, trust in official information, political orientation, health anxiety and uncertainty tolerance with self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others in Study 1. TABLE 2 \| Means, SDs, and correlations for gender, age, trust in official information, political orientation, health anxiety and uncertainty tolerance with self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others in Study 1. behavior during the COVID-19 pandemic (Oosterhoff and Palmer, 2020). (c) Health anxiety predicted both self-centered prepping behavior and behavior change to protect oneself. Research has shown that anxiety leads to actions to reduce uncertainty (Raghunathan and Pham, 1999), and both self- centered prepping behavior and recommended behavior changes (e.g., hygiene behavior) may serve this purpose among individuals with high health anxiety. Furthermore, anxiety has been repeatedly linked to general hoarding behavior (Coles et al., 2003; Timpano et al., 2009), and trait anxiety has also been positively linked to preventative behavior during the COVID-19 pandemic (e.g., avoiding going out and avoiding physical contact; Erceg et al., 2020). (d) Women were more likely to change their behavior to protect both themselves and others. STUDY 1 RESULTS UT 0.001 −0.048 −0.091 Total: M (SD) 0.58 (0.74) 4.38 (0.75) 4.35 (0.88) Men: M (SD) 0.55 (0.82) 4.13 (0.93) 4.05 (1.01) Women: M (SD) 0.58 (0.82) 4.45 (0.69) 4.43 (0.81) t-Test 0.42 2.76 3.10 Cohen’s d 0.03 0.39 0.41 In sum, these findings not only show differences in people’s trust in official statistics depending on their source but also that trust influences their behavior. These study results demonstrate that trust gained through clear and transparent information and communication of public authorities is a key to decrease uncertainty, limit the spread of false beliefs, and encourage behavior change to protect everyone’s health. A limitation of Study 1 is that we used a dichotomous answer format to assess participants prepping behavior. Furthermore, we did not measure explicitly the trust in government, acceptance of social distancing measures, and guideline adherence. Therefore, a follow-up study was planned where we would assess self-centered prepping behavior in a more detailed way. The aim was to reinvestigate the found correlations and to additionally include the variables trust in government, acceptance of social distancing measures, and guideline adherence and by this expanding the insights gained from Study 1. Herewith, we wanted to follow the call for replication-extension studies (Bonett, 2012; Wingen et al., 2020). behavior change to avoid infections, which did not reach the significance threshold of p < 0.05 (p = 0.09). In the third model, being female was significantly associated with behavior change to not infect others. Furthermore, this latter model also indicated a tendency of higher levels of trust in official statistics and more right-oriented participants to be less likely to change their behavior to protect others. However, these results did not reach the significance threshold of 0.05 (trust: p = 0.08; political orientation: p = 0.07). STUDY 1 DISCUSSION Six major findings arise from Study 1: (a) Trust in official statistics from different authorities depended on the source of the statistics: Data from China were believed much less than data from Europe or Germany. Data from the RKI were most trusted. (b) Trust in official statistics was negatively correlated with self-centered prepping behavior, but positively correlated with behavior to protect oneself and others. This is also in line with other studies showing that trust in institutions of the political system is positively linked to law compliance (e.g., Marien and Hooghe, 2011). Moreover, the public health recommendations mostly focused on hygiene behavior to avoid infections rather than self-centered prepping behavior. In other words, by showing less self-prepping behavior and more of the recommended protective behavior, participants complied with the official recommendations, which may explain why trust decreased self-prepping behavior. Furthermore, these results are in line with a recently conducted study in which social trust (trust in others) was negatively linked to self-prepping Frontiers in Psychology \| www.frontiersin.org STUDY 1 RESULTS Women not only tend to judge risks as higher than men (e.g., Slovic, 1999) but also engage more in caring behavior (e.g., Archer, 1996) and show more safety-seeking than men (Byrnes et al., 1999; Lermer et al., 2016a; Raue et al., 2018). However, it is important to note that safety behavior may also increase health anxiety (Olatunji et al., 2011), which suggest a potential bidirectional effect. (e) Participants with right-wing political orientations were less likely to change their behavior to protect others.if information, political orientation, health anxiety and uncertainty tolerance with self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others in Study 1. Prepp. beh. BC for self BC for others 1. Gender 0.008 0.149 0.197* 2. Age 0.101 0.112* −0.031 3. Trust Info. −0.114* 0.146** 0.100# 4. Left–Right 0.027 −0.116* −0.120* 5. MK-HAI 0.127* 0.092# 0.059 6. UT 0.001 −0.048 −0.091 Total: M (SD) 0.58 (0.74) 4.38 (0.75) 4.35 (0.88) Men: M (SD) 0.55 (0.82) 4.13 (0.93) 4.05 (1.01) Women: M (SD) 0.58 (0.82) 4.45 (0.69) 4.43 (0.81) t-Test 0.42 2.76 3.10 Cohen’s d 0.03 0.39 0.41 Gender: 1 = male, 2 = female; Trust Info. = Trust in official information (Range = 1–7), higher values indicate more trust; Left–Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = Health anxiety (Range = 1–5), higher values indicate more anxiety; UT = Uncertainty tolerance (Range = 1–6), higher values indicate more uncertainty tolerance; Prepp. beh. = Self-centered prepping behavior (Study 1: Range = 0–3), higher values indicate more self-centered prepping behavior; BC for self = behavior change for self-protection/to avoid infection (Range = 1–5), higher values indicate more behavior change; and BC for others = Behavior change in order to not infect others (Range = 1–5), higher values indicate more behavior change. #indicates p < 0.10. indicates p < 0.05; indicates p < 0.01; indicates p < 0.001. Bold highlighted values are at least p < 0.10. Prepp. beh. BC for self BC for others 1. Gender 0.008 0.149 0.197*** 2. Age 0.101 0.112* −0.031 3. Trust Info. −0.114* 0.146** 0.100# 4. Left–Right 0.027 −0.116* −0.120* 5. MK-HAI 0.127* 0.092# 0.059 6. STUDY 2 METHOD As the COVID-19 pandemic progressed, the duration of the government’s restrictions was extended. To underpin our findings from Study 1, and to further explore the development of perceptions and reactions to the pandemic related restrictions, we replicated and extended Study 1. In addition to reinvestigating our three research questions, we addressed trust in the government as well as acceptance of and adherence to social distancing guidelines. Trust in authorities is an important factor for the acceptance of many measures and is therefore particularly worth protecting November 2021 \| Volume 12 \| Article 769206 5 Reactions to COVID-19 in Germany Lermer et al. TABLE 3 \| Multiple linear regressions on self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others in Study 1. Study 1 Unstandardized coefficients Standardized coefficients B SE(B) β t p Prepp. beh. (Constant) 0.09 0.34 0.28 0.778 Gender 0.04 0.09 0.02 0.44 0.657 Age 0.01 0.00 0.07 1.48 0.137 Trust Info. −0.05 0.02 −0.10* 2.02 0.044 Left–Right 0.00 0.02 0.02 0.40 0.688 MK-HAI 0.12 0.04 0.13* 2.52 0.012 adj. R2 0.024 F 2.80* 0.017 BC for self (Constant) 2.97 0.35 8.51 0.000 Gender 0.29 0.09 0.15** 3.10 0.002 Age 0.01 0.00 −0.11* 2.32 0.021 Trust Info. 0.08 0.02 0.14 2.91 0.004 Left–Right −0.03 0.02 −0.07 1.45 0.136 MK-HAI 0.08 0.05 0.08# 1.70 0.089 adj. R2 0.064 F 6.04* 0.000 BC for others (Constant) 3.55 0.40 8.72 0.000 Gender 0.35 0.11 0.16 3.23 0.001 Age 0.00 0.00 0.00 0.12 0.904 Trust Info. 0.05 0.32 0.09# 1.77 0.077 Left–Right −0.05 0.02 −0.09# 1.80 0.073 MK-HAI 0.43 0.05 0.03 0.73 0.463 adj. R2 0.039 F 3.94 0.002 Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left–Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Prepp. Beh. = self-centered prepping behavior (Study 1: Range = 0–3; Study 2: Range = 1–7), higher values indicate more self-centered prepping behavior; BC for self = behavior change for self-protection/to avoid infection (Range = 1–5), higher values indicate more behavior change; and BC for others = behavior change in order to not infect others (Range = 1–5), higher values indicate more behavior change. #indicates p < 0.10. STUDY 2 METHOD indicates p < 0.05; indicates p < 0.01; *indicates p < 0.001. Bold highlighted values are at least p < 0.10. TABLE 3 \| Multiple linear regressions on self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others in S d β Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left–Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Prepp. Beh. = self-centered prepping behavior (Study 1: Range = 0–3; Study 2: Range = 1–7), higher values indicate more self-centered prepping behavior; BC for self = behavior change for self-protection/to avoid infection (Range = 1–5), higher values indicate more behavior change; and BC for others = behavior change in order to not infect others (Range = 1–5), higher values indicate more behavior change. #indicates p < 0.10. indicates p < 0.05; indicates p < 0.01; indicates p < 0.001. Bold highlighted values are at least p < 0.10. RQ 3: Which factors influence adherence to social distancing guidelines? and enhancing (Betsch et al., 2020d). As mentioned above, Rykkja et al. (2011) found that trust in political systems influences citizens’ attitude toward prevention measures. Research from previous epidemics showed that people who had less trust in the government took fewer precautions against the Ebola virus disease during the 2014–2016 outbreak in Liberia and Congo (Vinck et al., 2019; Oksanen et al., 2020). Furthermore, the social development at that time showed that acceptance of government measures per se is a particularly relevant variable. To explore this RQ, we analyzed the impact of the variables relevant for behavior change from Study 1, as well as trust in government and acceptance of measures on adherence to social distancing guidelines. Frontiers in Psychology \| www.frontiersin.org Self-Centered Prepping Behavior Self Centered Prepping Behavior In Study 2, we assessed self-centered prepping behavior in a more detailed way. In order address some limitations of the first study, a Likert scale was used instead of a dichotomous response format and a symmetrical formulation of the items (“purchased” instead of “stocked up” and “bought”). In addition, three more items were developed to examine a wider range of behaviors (e.g., buying hygiene products or disposable gloves). In total, we used six items where participants were asked to indicate on a seven-point Likert scale (1 = strongly disagree to 7 = strongly agree), how much each statement applied to them: “Purchased face masks” and “Purchased larger quantities of food [disinfectants/toilet paper/hygiene products/disposable gloves] than usual.” These items were averaged to an index of self-centered prepping behavior (Cronbach’s α = 0.79). Participants and Procedure Our second online survey was conducted between April 8 and April 23, 2020. For the recruitment of participants, we used the same sampling strategy as in Study 1 – only the attention check item was changed. Again, data from participants who failed to answer the attention check item (i.e., If you would like to continue with this study then select “agree”; which was the fourth of five response options) correctly or did not finish the questionnaire were not included. We changed the attention check item in comparison to Study 1 because it seemed more valid. In the first study, the attention check was passed by clicking on the rightmost answer option. Here, however, participants could also have passed the check by showing a pattern when answering, as always by clicking on the rightmost During the pandemic, the media increasingly reported violations of the health protective measures, and the closure of businesses, which led to high rates of unemployment. Around mid-April, people started demonstrating against the measures (Kölner Stadt-Anzeiger, 2020). The behavior of participants in demonstrations against the current measures showed that acceptance of the measures has a strong influence on adherence to social distancing guidelines. Thus, we assessed participants’ trust in the government and acceptance of measures and raised the following research question: November 2021 \| Volume 12 \| Article 769206 6 Reactions to COVID-19 in Germany Lermer et al. answer option. A total of 495 people clicked on the questionnaire. As in the previous study, we only included data from participants who did complete the questionnaire and who passed the attention check. Again, no information about the individual characteristics of the excluded persons is available, but all participants originate from the same pool of people. The remaining sample consisted of 461 (75% female) employed part-time students from the same university with locations in 32 cities across Germany as in Study 1 (Mage = 26.45, SDage = 4.05, Rangeage = 20–49). In Study 2, participants also entered the first digit of their postal code. Data showed that the participants came from all nine postal code areas in Germany. Acceptance of the Measures Acceptance of the Measures To assess participants’ acceptance of safety measures, four items with a seven-point Likert scale (1 = strongly disagree to 7 = strongly agree) answer format were developed: “I think the current measures taken by the German government to combat the COVID-19 pandemic are good,” “I think the German government’s communication of current measures to combat the COVID-19 pandemic is good,” “I think the current measures taken by the federal government to combat the COVID-19 pandemic are appropriate,” and “I think that the people responsible for planning and implementing the current measures have the necessary competence.” These items were averaged to an index of acceptance (Cronbach’s α = 0.89). STUDY 2 RESULTS As in Study 1, to answer RQ 1, people’s level of trust in statistics on COVID-19 from official authorities was compared and displayed in Table 1. Again, findings show that trust in statistics from China was by far lowest, whereas trust in statistics from the RKI was highest. g To reinvestigate RQ 2, correlation analyses from Study 1 were replicated and presented in Table 4. Whereas in Study 1, all variables except uncertainty tolerance showed significant correlations with behavior change, and in Study 2, all variables showed significant links with at least one behavior variable. For comparison reasons, the same variables as in Study 1 were included in multiple regressions on the dependent variables of self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others (see Table 5). Again, results showed that health anxiety was To reinvestigate RQ 2, correlation analyses from Study 1 were replicated and presented in Table 4. Whereas in Study 1, all variables except uncertainty tolerance showed significant correlations with behavior change, and in Study 2, all variables showed significant links with at least one behavior variable. For comparison reasons, the same variables as in Study 1 were included in multiple regressions on the dependent variables of self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others (see Table 5). Again, results showed that health anxiety was Participants and Procedure However, the distribution was not equally distributed with most participants living in postal code area four and five where most of the locations (17 out of 32) of the university are (number of participants from area 0 = 6; 1 = 21; 2 = 57; 3 = 17; 4 = 177; 5 = 83; 6 = 46; 7 = 9; 8 = 79; 9 = 26). Three of the participants reported being infected with COVID-19. Fifteen participants reported that they were sick, and four of those assumed that they were infected. All other participants reported that they were healthy. As in Study 1, all participants remained in the data set independent of their COVID-19 status. Participants were rewarded with course credits for their participation. great trust in the state government” with a seven-point Likert scale (1 = strongly disagree to 7 = strongly agree) answer format. These items were averaged to an index of trust in government (Cronbach’s α = 0.91). Due to Germany’s federal structure, we surveyed trust in the federal government and state government separately – as did the COVID-19 Snapshot Monitoring (COSMO) project, which is a well-known repeated cross-sectional monitoring project during the COVID-19 outbreak in Germany (see for instance COSMO COVID-19 Snapshot Monitoring, 2020). Measures Adherence to the Social Distancing Guidelines To assess participants’ adherence to the social distancing guidelines, five items were adapted from a measure of behavior during the COVID-19 pandemic by Rossmann et al. (2020): Participants were asked to indicate on a five-point Likert scale (1 = never to 5 = very often) how often in the last 10 days the following applied to them: “I met with friends who live outside my household;” “I met with family members who live outside my household;” “I met with older people;” “I violated the 1.5 meters distance rule;” and “I disregarded regulations on social distancing or movement restrictions.” These items were averaged to an index of guideline adherence (Cronbach’s α = 0.64) and recoded so that higher values indicate more adherence. We applied the same measures for trust in official statistics (Cronbach’s α = 0.85), political orientation, health anxiety (Cronbach’s α = 0.93), and uncertainty tolerance (Cronbach’s α = 0.70) as in Study 1. This also applies to the indices behavior change to avoid infection (Cronbach’s α = 0.79) and behavior change to not infect others (Cronbach’s α = 0.80). However, the item “I avoid visiting cafés and restaurants in order not to get infected [/not infect others]” was changed to “I pay more attention to the recommended hygiene rules than before the Coronavirus became known, in order not to get infected [/ not infect others]” due to the lockdown. Trust in the Government Participants’ trust in the government was assessed using two items “I have great trust in the federal government” and “I have November 2021 \| Volume 12 \| Article 769206 Frontiers in Psychology \| www.frontiersin.org 7 Reactions to COVID-19 in Germany Lermer et al. TABLE 4 \| Means, SDs, and correlations for gender, age, trust in official information, political orientation, health anxiety and uncertainty tolerance with self-centered prepping behavior, behavior change to avoid infection and behavior change in order to not infect others in Study 2. TABLE 4 \| Means, SDs, and correlations for gender, age, trust in official information, political orientation, health anxiety and uncertainty tolerance with self-centered prepping behavior, behavior change to avoid infection and behavior change in order to not infect others in Study 2. 95%-CI = [−0.05; 0.07]) for less right-wing orientated participants (1 SD below the mean). For participants with average values (mean centered; B = 0.08, SE = 0.02, t = 3.25, 95%-CI = [0.02; 0.11]) and for those with more right-wing orientation (1 SD above the mean; B = 0.11, SE = 0.03, t = 3.77, 95%-CI = [0.05; 0.17]), results showed a significant relationship between acceptance of the measures and guideline adherence. Political orientation was non-normally distributed, with skewness of 0.08 (SE = 0.11) and kurtosis of 0.09 (SE = 0.22), indicating a right-skewed left-leaning distribution. The average value was M = 4.53 (SD = 0.08) slightly below the mean of the scale, the median = 5. Moreover, while analyses for gender showed no moderation effect (p = 0.869), we found that age was a moderator for the effect of acceptance of measures on guideline adherence (B = 0.02, SE = 0.01, t = 2.35, CI-95% = [0.00; 03]). Analyses of conditional effects revealed no relationship between measure acceptance and guideline adherence (B = 0.03, SE = 0.03, t = 0.83, 95%-CI = [−0.03; 0.09]) for participants aged around 23 years. For participants aged around 25 years (B = 0.06, SE = 0.02, t = 2.30, 95%-CI = [0.01; 0.10]) and for those aged around 29 years (B = 0.11, SE = 0.03, t = 3.95, 95%-CI = [0.06; 0.17]), results showed a significant relationship between acceptance of the measures and guideline adherence. self-centered prepping behavior, behavior change to avoid infection and behavior change in order to not infect others in Study 2. Prepp. beh. BC for self BC for others 1. STUDY 2 DISCUSSION positively associated with self-centered prepping behavior and behavior change to avoid infection of one self. Further, the results again showed that trust in official statistics was positively associated with behavior change to avoid infections of oneself and others. Additionally, behavior change to not infect others was further predicted by being female and less right-oriented, which mirrors the pattern of Study 1. Study 2 successfully replicated the findings from Study 1: (a) In the further course of the pandemic, there were still differences in trust in official statistics from different authorities: Again, data from China were believed much less than data from Europe or Germany, whereas data from the RKI were most trusted. (b) As in Study 1, results showed that health anxiety increases self-centered prepping behavior and behavior change to avoid infections. Also, trust in official statistics increased behavior change to avoid infections. Replicating the findings from Study 1, results from Study 2 indicate that being female, being less politically right-oriented, and having trust in official statistics increases behavior change in order to not infect others.i To investigate who shows more adherence to social distancing guidelines and answer RQ 3, correlations of guideline adherence with relevant variables from Study 1 (gender, age, trust in official statistics, political orientation, and health anxiety) as well as acceptance of the measures and trust in government were analyzed in a first step. The correlation matrix can be found in Table 6. In addition to replicating findings from Study 1, Study 2 aimed at investigating influences on adherence to social distancing guidelines. Results show that guideline adherence was positively associated with older age, being female, less right-wing political orientation, and higher acceptance of the measures. A recently conducted study on guideline adherence during the pandemic in the United States also reports a small positive relationship with age (Bogg and Milad, 2020). However, the authors did not show a significant association with gender. Findings from previous research do however support the assumption that women tend to show more precautionary behaviors to avoid infections. For instance, studies show women generally practice more frequent hand-washing than men (Liao et al., 2010; Park et al., 2010). Furthermore, findings from a meta-analysis (Moran and Del Valle, 2016) indicate inherent differences in how women and men respond to pandemic diseases: women are more likely to practice preventative behavior (e.g., face mask wearing) and avoidance behavior (e.g., avoiding public transit) than men. Trust in the Government Gender 0.029 0.064 0.180 2. Age −0.080# −0.018 0.016 3. Trust Info. −0.055 0.136 0.148 4. Left–Right 0.024 −0.003 −0.130 5. MK-HAI 0.168* 0.133 0.031 6. UT 0.000 −0.105* −0.046 Total: M (SD) 2.05 (1.11) 4.05 (0.85) 4.13 (0.88) Men: M (SD) 1.99 (1.23) 3.97 (0.85) 3.86 (0.95) Women: M (SD) 2.07 (1.07) 4.08 (0.83) 4.22 (0.83) t-Test 0.61 1.10 3.84*** Cohen’s d 0.06 0.13 0.40 Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left–Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; UT = uncertainty tolerance (Range = 1–6), higher values indicate more uncertainty tolerance; Prepp. beh. = self-centered prepping behavior (Study 1: Range = 0–3; Study 2: Range = 1–7), higher values indicate more self-centered prepping behavior; BC for self = behavior change for self-protection/to avoid infection (Range = 1–5), higher values indicate more behavior change; BC for others = behavior change in order to not infect others (Range = 1–5), higher values indicate more behavior change. #indicates p < 0.10. indicates p < 0.05; indicates p < 0.01; indicates p < 0.001. Bold highlighted values are at least p < 0.10. Prepp. beh. BC for self BC for others 1. Gender 0.029 0.064 0.180* 2. Age −0.080# −0.018 0.016 3. Trust Info. −0.055 0.136 0.148 4. Left–Right 0.024 −0.003 −0.130 5. MK-HAI 0.168* 0.133** 0.031 6. UT 0.000 −0.105* −0.046 Total: M (SD) 2.05 (1.11) 4.05 (0.85) 4.13 (0.88) Men: M (SD) 1.99 (1.23) 3.97 (0.85) 3.86 (0.95) Women: M (SD) 2.07 (1.07) 4.08 (0.83) 4.22 (0.83) t-Test 0.61 1.10 3.84* Cohen’s d 0.06 0.13 0.40 Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left–Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; UT uncertainty tolerance (Range 1 6) higher values Frontiers in Psychology \| www.frontiersin.org STUDY 2 DISCUSSION All variables except health anxiety and trust in government showed significant correlations with guideline adherence. Thus, all variables showing significant links were included as predictors in a multiple linear regression with guideline adherence as the dependent variable. Findings are presented in Table 7. Results show that adherence to the social distancing guidelines was positively associated with higher levels of acceptance of the measures, being female and being of older age. There was also a tendency of more right-oriented participants to adhere less to social distancing guidelines (p < 0.10). We also conducted a moderation analysis to test whether political orientation is a moderator of the relationship between acceptance of the measures and guideline adherence. We used Hayes’ PROCESS tool (model 1). Results showed a significant interaction effect of measure acceptance and political orientation (B = 0.03, SE = 0.01, t = 2.46, 95%-CI = [0.01; 05]). Analyses of conditional effects revealed no relationship between measure acceptance and guideline adherence (B = 0.02, SE = 0.03, t = 0.53, November 2021 \| Volume 12 \| Article 769206 Frontiers in Psychology \| www.frontiersin.org 8 Reactions to COVID-19 in Germany Lermer et al. TABLE 5 \| Multiple linear regressions on self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others in Study 2. Study 2 Unstandardized coefficients Standardized coefficients B SE(B) β t p Prepp. beh. (Constant) 0.97 0.50 1.93 0.054 Gender 0.04 0.12 0.01 0.34 0.730 Age 0.02 0.01 0.08# 1.85 0.064 Trust Info. −0.05 0.06 −0.06 1.42 0.156 Left–Right 0.00 0.03 0.00 0.14 0.887 MK-HAI 0.25 0.06 0.17* 3.77 0.000 adj. R2 0.030 F 3.76** 0.002 BC for self (Constant) 3.34 0.38 8.78 0.000 Gender 0.09 0.09 0.05 1.05 0.291 Age −0.01 0.01 −0.00 −0.08 0.929 Trust Info. 0.06 0.02 0.10* 2.29 0.022 Left–Right 0.00 0.02 0.00 1.59 0.874 MK-HAI 0.12 0.05 0.11* 2.46 0.014 adj. R2 0.020 F 2.88* 0.014 BC for others (Constant) 3.19 0.39 8.17 0.000 Gender 0.35 0.09 0.17* 3.67 0.000 Age 0.07 0.01 0.03 0.72 0.472 Trust Info. 0.08 0.03 0.13 2.93 0.004 Left–Right −0.05 0.02 −0.09* 2.01 0.044 MK-HAI 0.01 0.05 0.00 0.19 0.845 adj. R2 0.050 F 5.82*** 0.000 Gender: 1 = male, 2 = female; Trust Info. STUDY 2 DISCUSSION = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Prepp. Beh. = self-centered prepping behavior (Study 1: Range = 0–3; Study 2: Range = 1–7), higher values indicate more self-centered prepping behavior; BC for self = behavior change for self-protection/to avoid infection (Range = 1–5), higher values indicate more behavior change; and BC for others = behavior change in order to not infect others (Range = 1–5), higher values indicate more behavior change. #indicates p < 0.10. indicates p < 0.05; indicates p < 0.01; *indicates p < 0.001. Bold highlighted values are at least p < 0.10. on self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others in TABLE 5 \| Multiple linear regressions on self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others in Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Prepp. Beh. = self-centered prepping behavior (Study 1: Range = 0–3; Study 2: Range = 1–7), higher values indicate more self-centered prepping behavior; BC for self = behavior change for self-protection/to avoid infection (Range = 1–5), higher values indicate more behavior change; and BC for others = behavior change in order to not infect others (Range = 1–5), higher values indicate more behavior change. #indicates p < 0.10. indicates p < 0.05; indicates p < 0.01; indicates p < 0.001. Bold highlighted values are at least p < 0.10. The finding that adherence to social distancing guidelines was positively associated with being less politically right- oriented fits to findings from studies recently conducted in the COVID-19 pandemic in the United States. Conway et al. (2020) argue that although much research suggests that conservatives are more sensitive to disease threats, they seem to be less concerned about the COVID-19 pandemic than liberals. However, the authors add that this ideological effect diminishes as experiences with, and the impact of the COVID-19 pandemic grows. STUDY 2 DISCUSSION Furthermore, our findings are supported by another recently study conducted during the COVID-19 pandemic. In this study, liberals and politically moderates show more guideline adherence than conservatives (van Holm et al., 2020). It is intuitively plausible that guideline adherence increases with acceptance of the measures. However, the moderation analysis revealed that political orientation influences the relationship between acceptance of measures and guideline adherence. This interaction effect showed that for less right- wing-orientated participants adherence to social distancing guidelines was not linked to acceptance of the measures. This link was only found in people with moderate political orientation (average values) and in people with more right-wing orientation. These findings are in line with findings from other studies in the COVID-19 context. For instance, also Capraro and Barcelo (2020) report in a recent preprint that demographic variables and political orientation are relevant characteristics in the context of protective behavior. According to their findings, being female, being older, and being left- leaning are correlated with greater intentions to wearing a face covering. Also, studies from Gollwitzer et al. (2020) and Van Bavel et al. (2020) show that supporters of right-wing political parties were less likely to adhere to protective behavior compared to liberal or left-leaning individuals.t t One year after Study 1 and Study 2, the COVID-19 pandemic was still having major impact on our daily lives and causing restrictions on social contact in Germany. However, since many may also have become accustomed to these circumstances, we aimed at reinvestigating our research questions. Frontiers in Psychology \| www.frontiersin.org STUDY 3 METHOD As the COVID-19 pandemic progressed, restrictive measures in Germany also continued. Therefore, another goal of this November 2021 \| Volume 12 \| Article 769206 9 Reactions to COVID-19 in Germany Lermer et al. in Study 2. A total of 555 people clicked on the questionnaire. As in the previous two studies, data were excluded from participants who did not complete the questionnaire or who failed the attention check. As in the previous studies, no information about the individual characteristics of the excluded persons is available, but all participants originated from the same pool of people. The remaining sample consisted of 530 (69.4% female) employed part-time students from the same university with locations in 32 cities across Germany as in Study 1 and Study 2 (Mage = 26.26, SDage = 4.63, Rangeage = 19–50). As in Study 2, participants also entered the first digit of their postal code. Again, data showed that the participants came from all nine postal code areas in Germany. However again, the distribution was not equally distributed with most participants living in postal code area four where many of the locations of the university are (number of participants from area 0 = 8; 1 = 61; 2 = 54; 3 = 18; 4 = 165; 5 = 73; 6 = 41; 7 = 70; 8 = 77; 9 = 8). Fifteen of the participants reported being infected with COVID-19. Ten participants reported that they were sick, and one of those assumed that she/he was infected. All other participants reported that they were healthy. As in Study 1 and Study 2, all participants remained in the data set independent of their COVID-19 status. Participants were rewarded with course credits for their participation. research project was to investigate the research questions of the two preceding studies 1 year later. For this purpose, we conducted Study 3, a replication of Study 2. Since we had no assumptions regarding changes in perception and behavioral responses to the consequences of the COVID-19 pandemic, we did not formulate explicit hypotheses and instead reexamined our research questions. Participants and Procedure Bold highlighted values are at least p < 0.10. TABLE 7 \| Multiple linear regressions on adherence to the social distancing guidelin Unstandardized coefficients S B SE(B) Guid. Adh. (Constant) 3.04 0.28 Gender 0.20 0.06 Age 0.02 0.00 T t I f 0 01 0 02 Measures We applied the same measures for trust in official statistics (Cronbach’s α = 0.85), political orientation, health anxiety (Cronbach’s α = 0.92), and uncertainty tolerance (Cronbach’s α = 0.66) as in Study 2. This also applies to the indices self- centered prepping behavior (Cronbach’s α = 0.76), behavior change to avoid infection (Cronbach’s α = 0.78), behavior change to not infect others (Cronbach’s α = 0.80), trust in the government (Cronbach’s α = 0.91), acceptance of the measures (Cronbach’s α = 0.89), and adherence to the social distancing guidelines (Cronbach’s α = 0.64). Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Acceptance (Range = 1–7), higher values indicate more acceptance of the measures; Trust Gov. = trust in government (Range = 1–7), higher values indicate more trust in the government; and Guideline adherence = adherence to the social distancing guidelines (Range = 0–5), higher values indicate more adherence.indicates p < 0.05; indicates p < 0.01. Bold highlighted values are at least p < 0.10. TABLE 7 \| Multiple linear regressions on adherence to the social distancing guidelines. Study 2 Unstandardized coefficients Standardized coefficients B SE(B) β t p Guid. Adh. (Constant) 3.04 0.28 10.68 0.000 Gender 0.20 0.06 0.13 2.97 0.003 Age 0.02 0.00 0.13** 2.94 0.003 Trust Info. 0.01 0.02 0.02 0.44 0.656 Left–Right −0.03 0.01 −0.07# 1.68 0.094 Acceptance 0.06 0.02 0.12* 2.31 0.021 adj. R2 0.053 F 6.07*** 0.000 Guid. Adh. = adherence to the social distancing guidelines (Range = 0–5), higher values indicate more adherence, Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation, Acceptance, acceptance of the measures (Range = 1–7), higher values indicate more acceptance. #indicates p < 0.10. indicates p < 0.05; indicates p < 0.01; indicates p < 0.001. Bold highlighted values are at least p < 0.10. TABLE 7 \| Multiple linear regressions on adherence to the social distancing guidelines. Guid. Adh. = adherence to the social distancing guidelines (Range = 0–5), higher values indicate more adherence, Gender: 1 = male, 2 = female; Trust Info. Participants and Procedure Our third online survey was conducted between March 15 and April 28, 2021. The recruitment of participants was as TABLE 6 \| Means, SDs, and correlations for gender, age, trust in official information, political orientation, health anxiety, acceptance of the measures, and trust in the government with adherence to the social distancing guidelines in Study 2. Guideline adherence 1. Gender 0.134 2. Age 0.120** 3. Trust Info. 0.092* 4. Left–Right −0.110* 5. MK-HAI −0.054 6. Acceptance 0.143 7. Trust Gov. 0.120 Total: M (SD) 4.08 (0.62) Men: M (SD) 3.93 (0.61) Women: M (SD) 4.41 (0.62) t-Test 2.93 Cohen’s d 0.39 Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Acceptance (Range = 1–7), higher values indicate more acceptance of the measures; Trust Gov. = trust in government (Range = 1–7), higher values indicate more trust in the government; and Guideline adherence = adherence to the social distancing guidelines (Range = 0–5), higher values indicate more adherence.indicates p < 0.05; indicates p < 0.01. Bold highlighted values are at least p < 0.10. TABLE 6 \| Means, SDs, and correlations for gender, age, trust in official information, political orientation, health anxiety, acceptance of the measures, and trust in the government with adherence to the social distancing guidelines in Study 2. Guideline adherence 1. Gender 0.134* 2. Age 0.120** 3. Trust Info. 0.092* 4. Left–Right −0.110* 5. MK-HAI −0.054 6. Acceptance 0.143 7. Trust Gov. 0.120 Total: M (SD) 4.08 (0.62) Men: M (SD) 3.93 (0.61) Women: M (SD) 4.41 (0.62) t-Test 2.93 Cohen’s d 0.39 Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Acceptance (Range = 1–7), higher values indicate more acceptance of the measures; Trust Gov. = trust in government (Range = 1–7), higher values indicate more trust in the government; and Guideline adherence = adherence to the social distancing guidelines (Range = 0–5), higher values indicate more adherence.indicates p < 0.05; indicates p < 0.01. Measures = trust in official information (Range = 1–7), higher values indicate more trust; Left–Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; UT = uncertainty tolerance (Range = 1–6), higher values indicate more uncertainty tolerance; Prepp. beh. = self-centered prepping behavior (Study 1: Range = 0–3; Study 2: Range = 1–7), higher values indicate more self-centered prepping behavior; BC for self = behavior change for self-protection/to avoid infection (Range = 1–5), higher values indicate more behavior change; and BC for others = behavior change in order to not infect others (Range = 1–5), higher values indicate more behavior change. indicates p < 0.05; indicates p < 0.01; indicates p < 0.001. Bold highlighted values are at least p < 0.10. Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left–Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; UT = uncertainty tolerance (Range = 1–6), higher values indicate more uncertainty tolerance; Prepp. beh. = self-centered prepping behavior (Study 1: Range = 0–3; Study 2: Range = 1–7), higher values indicate more self-centered prepping behavior; BC for self = behavior change for self-protection/to avoid infection (Range = 1–5), higher values indicate more behavior change; and BC for others = behavior change in order to not infect others (Range = 1–5), higher values indicate more behavior change. indicates p < 0.05; indicates p < 0.01; indicates p < 0.001. Bold highlighted values are at least p < 0.10. fi As in Study 2, we conducted moderation analyses to test whether political orientation, gender and age are moderators of the relationship between acceptance of the measures and guideline adherence. We used Hayes’ PROCESS tool (model 1). The distribution of political orientation in Study 3 was like that in Study 2. Also, here political orientation was non-normally distributed, with a skewness of 0.06 (SE = 0.11) and kurtosis of −0.11 (SE = 0.21), indicating a right-skewed left-leaning distribution. The average value was M = 4.40 (SD = 0.07) slightly below the mean of the scale, the median = 5. However, results showed no moderation effect for political orientation (p = 0.954). STUDY 3 DISCUSSION To reinvestigate RQ 2, correlation analyses from Study 1 and Study 2 were replicated and shown in Table 8. Correlations between gender, trust in official statistics, and health anxiety with behavior variables were stronger than in the studies from 2020, whereas the link between age and political orientation with behavior change was weaker. Study 3 successfully replicated findings from Study 1 and Study 2: (a) Also 1 year after the surveys in March and April 2020, there were still differences in trust in official statistics from different authorities: Again, data from China were believed much less than data from Europe or Germany, whereas data from the RKI were most trusted. (b) Results from all three studies showed that health anxiety increases self-centered prepping behavior and behavior change to avoid infections. Also, trust in official statistics increased behavior change to avoid infections. Regarding behavior change in order not to infect others, results in Study 3 slightly differ compared to studies 1 and 2. Whereas in the first two studies, being female, being less politically right-oriented and having trust in official statistics were positively associated with behavior change to protect others, Study 3 indicates that political orientation is no longer a relevant predictor for behavior change in order not to infect others. Moreover, neither political orientation, gender nor age showed up as moderators in Study 3. Instead, health anxiety turned out to predict behavior change in order not to infect others. This leads to the assumption that the Corona pandemic has become less an issue of political orientation than of individual characteristics related to health- related behaviors. For comparison reasons, the same variables as in Study 1 and Study 2 were included in multiple regressions on the dependent variables of self-centered prepping behavior, behavior change to avoid infection, and behavior change in order not to infect others (see Table 9). As in the studies from 2020, results showed that health anxiety was positively associated with self-centered prepping behavior and behavior change to avoid infection of oneself. Furthermore, in 2021, health anxiety was positively associated with behavior change in order not to infect others. Further in line with the previous studies, the results showed that trust in official statistics was positively associated with behavior change to avoid infections of oneself and others. However, in 2021, these associations were much stronger. STUDY 3 RESULTS As in Study 1 and Study 2, to answer RQ 1, people’s level of trust in statistics on COVID-19 from official authorities was compared and displayed in Table 1. The results show, as in the two previous studies, that trust in statistics from China was by far lowest, whereas trust in statistics from the RKI was highest. Measures Moreover, neither gender (p = 0.988), nor age (p = 0.837) moderated the effect. Measures = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation, Acceptance, acceptance of the measures (Range = 1–7), higher values indicate more acceptance. #indicates p < 0.10. indicates p < 0.05; indicates p < 0.01; indicates p < 0.001. Bold highlighted values are at least p < 0.10. November 2021 \| Volume 12 \| Article 769206 10 Reactions to COVID-19 in Germany Lermer et al. TABLE 8 \| Means, SDs, and correlations for gender, age, trust in official information, political orientation, and health anxiety with self-centered prepping behavior, behavior change to avoid infection and behavior change in order to not infect others in Study 3. TABLE 8 \| Means, SDs, and correlations for gender, age, trust in official information, political orientation, and health anxiety with self-centered prepping behavior, behavior change to avoid infection and behavior change in order to not infect others in Study 3. To investigate RQ 3, asking who shows more adherences to social distancing guidelines, correlations of guideline adherence with variables used in Study 2 (gender, age, trust in official statistics, political orientation, health anxiety, acceptance of the measures, and trust in government) were analyzed in a first step. The correlation matrix can be found in Table 10.fi Prepp. beh. BC for self BC for others 1. Gender 0.169** 0.110* 0.164* 2. Age 0.072 0.043 0.018 3. Trust Info. 0.017 0.355* 0.278* 4. Left–Right 0.006 0.016 −0.03 5. MK-HAI 0.261* 0.173*** 0.103* 6. UT −0.115 −0.059 −0.008 Total: M (SD) 3.20 (1.26) 3.62 (0.97) 3.62 (0.98) Men: M (SD) 2.87 (1.28) 3.46 (0.98) 3.38 (1.01) Women: M (SD) 3.34 (1.22) 3.69 (0.97) 3.73 (0.95) t-Test 3.92* 2.54* 3.79*** Cohen’s d 0.38 0.23 0.36 As in Study 2 age, trust in official statistics and acceptance of the measures showed significant correlations with guideline adherence (the variable guidelines adherence was only collected from Study 2 onwards). For comparison reasons, the same variables as in Study 2 were included in a multiple regression on the dependent variable guideline adherence. Findings are presented in Table 11. As the correlational findings already indicated, results showed that adherence to the social distancing guidelines was positively associated with higher levels of acceptance of the measures, being of older age, and having more trust in official statistics. Gender: 1 = male, 2 = female; Trust Info. STUDY 3 DISCUSSION TABLE 9 \| Multiple linear regressions on self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others in Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Prepp. Beh. = self-centered prepping behavior (Study 1: Range = 0–3; Study 2: Range = 1–7), higher values indicate more self-centered prepping behavior; BC for self = behavior change for self-protection/to avoid infection (Range = 1–5), higher values indicate more behavior change; and BC for others = behavior change in order to not infect others (Range = 1–5), higher values indicate more behavior change. indicates p < 0.05; indicates p < 0.01; indicates p < 0.001. Bold highlighted values are at least p < 0.10. As Study 2, Study 3 aimed at investigating influences on adherence to social distancing guidelines. Again, results show that guideline adherence was positively associated with older age and higher acceptance of the measures. In addition, and contrary to Study 2, higher levels of trust in official information turned out to be a relevant predictor for guideline adherence, too. However, no associations were found with gender and political orientation. These findings indicate that the importance of the various predictors for guideline adherence changed as the global pandemic progressed. A relevant factor in this context may be that the levels of general acceptance with preventive measures declined substantially between the time points of studies 2 and 3. Thus, the importance of political orientation might have decreased because support for social distancing guidelines has declined in all population groups. This trend has already been suggested by Conway et al. (2020) who argue that ideological effects diminish as experiences with, and the impact of the COVID-19 pandemic grows. In contrast, trust in official information has become more relevant. This is consistent with recent findings from other studies. Bargain and Aminjonov (2020) found that higher trust was associated with decreased mobility related to non-necessary activities. Fridman et al. (2020) report that higher levels of trust in government information sources are positively related to adherence to social distancing. STUDY 3 DISCUSSION Additionally, being female was positively associated with all behavior variables, which mirrors the pattern of Study 1 and Study 2. However, political orientation was not associated with any behavior variable in Study 3. November 2021 \| Volume 12 \| Article 769206 Frontiers in Psychology \| www.frontiersin.org 11 Reactions to COVID-19 in Germany Lermer et al. on self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others in TABLE 9 \| Multiple linear regressions on self-centered prepping behavior, behavior change to avoid infection, and behavior change in order to not infect others in Study 3. Study 3 Unstandardized coefficients Standardized coefficients B SE(B) β t p Prepp. beh. (Constant) 0.38 0.47 0.80 0.421 Gender 0.48 0.11 0.17* 4.19 0.000 Age 0.03 0.01 0.11 2.76 0.007 Trust Info. 0.01 0.03 0.02 0.51 0.609 Left–Right 0.02 0.03 0.02 0.67 0.499 MK-HAI 0.43 0.06 0.26* 6.44 0.000 adj. R2 0.101 F 12.81* 0.000 BC for self (Constant) 1.06 0.35 3.00 0.003 Gender 0.27 0.08 0.12 3.15 0.002 Age 0.02 0.00 0.09* 2.36 0.018 Trust Info. 0.24 0.02 0.36* 9.12 0.000 Left–Right 0.03 0.02 0.05 1.27 0.202 MK-HAI 0.20 0.05 0.16* 4.05 0.000 adj. R2 0.171 F 22.68* 0.014 BC for others (Constant) 1.54 0.36 4.18 0.000 Gender 0.37 0.09 0.13* 4.12 0.000 Age 0.14 0.00 0.06 1.59 0.111 Trust Info. 0.19 0.02 0.28*** 6.97 0.000 Left-Right 0.00 0.02 0.01 0.32 0.743 MK-HAI 0.11 0.05 0.09* 2.23 0.026 adj. R2 0.113 F 14.34*** 0.000 Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Prepp. Beh. = self-centered prepping behavior (Study 1: Range = 0–3; Study 2: Range = 1–7), higher values indicate more self-centered prepping behavior; BC for self = behavior change for self-protection/to avoid infection (Range = 1–5), higher values indicate more behavior change; and BC for others = behavior change in order to not infect others (Range = 1–5), higher values indicate more behavior change. indicates p < 0.05; indicates p < 0.01; indicates p < 0.001. Bold highlighted values are at least p < 0.10. Frontiers in Psychology \| www.frontiersin.org GENERAL DISCUSSION Today, there are numerous psychological studies on the COVID-19 pandemic context. However, many of these studies focus on screening for negative (mental health) effects of the COVID-19 pandemic. The aim of our studies was to capture early and later perceptions and behavioral reactions to the COVID-19 pandemic. Our three studies give insights into three important dimensions in the context of the COVID-19 pandemic: (a) trust in official statistics, (b) behavior change, and (c) adherence to social distancing guidelines. There are striking differences in participants’ trust in official statistics depending on whether the source is China or the RKI. The high level of trust in statistics from the RKI in our three samples corresponds with results from COSMO - Covid-19 Snapshot Monitoring a German Corona Monitor (COSMO COVID-19 Snapshot Monitoring, 2020). The weekly monitoring survey November 2021 \| Volume 12 \| Article 769206 Frontiers in Psychology \| www.frontiersin.org 12 Reactions to COVID-19 in Germany Lermer et al. (e.g., Erceg et al., 2020). Our analyses also revealed bidirectional effects regarding health anxiety and prepping behavior (in Study 1–3) and between health anxiety and behavior change to avoid own infection (Study 1–3). Behavior change in order not to infect others was only associated with health anxiety in study 3. This is in line with research from Olatunji et al. (2011) and emphasizes the importance of further research in the context health anxiety. Age was not or only negligibly associated with self-centered prepping behavior. This is in line with findings from the German Corona Monitor regarding panic buying (waves 1, 2, and 3: Betsch et al., 2020a,b,c). However, gender seems to be relevant when it comes to behavior change to avoid personal and other person’s risks. In all three studies, women reported higher values on the behavior change variables (both to avoid own infection and to protect others) then men. Previous research has shown that women are more safety- oriented (Lermer et al., 2016b), especially in the health domain (Thom, 2003; Lermer et al., 2016a). Women also tend to behave generally more pro-socially (Archer, 1996) than men. Our findings imply that these observations also apply during the COVID-19 pandemic. results from March 3, 2020, to April 21, 2020, show that trust in the RKI was consistently very high, even higher than trust in the German Federal Ministry of Health, the Federal Government and the WHO. GENERAL DISCUSSION However for 2021, results from Betsch (2021) show that trust in general (in government and in authorities) has declined somewhat. Furthermore, the present findings show that trust in the official statistics is a predictor of behavior change and guideline adherence. Therefore, effort should be made to ensure that trust in the data is maintained, especially in contexts where long-term measures are required, like the COVID-19 pandemic. Health anxiety was linked to self-centered prepping behavior and behavior change to reduce personal risk in all three studies. These findings are not only intuitively plausible but also supported by other studies showing that anxiety is linked to safety behavior Frontiers in Psychology \| www.frontiersin.org 1 TABLE 10 \| Means, SDs, and correlations for gender, age, trust in official information, political orientation, health anxiety, acceptance of the measures, and trust in the government with adherence to the social distancing guidelines in Study 3. Guideline adherence 1. Gender 0.056 2. Age 0.169* 3. Trust Info. 0.179* 4. Left-Right −0.049 5. MK-HAI −0.002 6. Acceptance 0.178* 7. Trust Gov. 0.136 Total: M (SD) 3.51 (0.82) Men: M (SD) 3.45 (0.77) Women: M (SD) 3.55 (0.84) t-Test 1.29 Cohen’s d 0.12 Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Acceptance (Range = 1–7) = higher values indicate more acceptance of the measures; Trust Gov. = trust in government (Range = 1–7), higher values indicate more trust in the government; and Guideline Adherence = adherence to the social distancing guidelines (Range = 0–5), higher values indicate more adherence. indicates p < 0.01; *indicates p < 0.001. Bold highlighted values are at least p < 0.10. TABLE 11 \| Multiple linear regressions on adherence to the social distancing guidel Unstandardized coefficients S B SE(B) Guid. Adh. (Constant) 2.01 0.29 Gender 0.10 0.07 Age 0.03 0.00 Trust Info. 0.08 0.02 Left–Right −0.02 0.02 Acceptance 0.06 0.02 adj. R2 0.075 F 9.49* Guid. Adh. = adherence to the social distancing guidelines (Range = 0–5), higher values indicate (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), (Range = 1–7), higher values indicate more acceptance. GENERAL DISCUSSION indicates p < 0.05; indicates p < 0.01 TABLE 10 \| Means, SDs, and correlations for gender, age, trust in official information, political orientation, health anxiety, acceptance of the measures, and trust in the government with adherence to the social distancing guidelines in Study 3. Results from the present study (samples 2 and 3) indicate a positive effect of acceptance of measures and trust in the government, a moderate positive effect of trust in official statistics and a small negative effect of being more politically right-wing oriented (Study 2) on adherence to social distancing guidelines. Betsch et al. (2020d) report in their Corona Monitor that German acceptance of the measures had risen sharply since mid-March 2020 and then decreased somewhat, with some fluctuations, until April 2021 (Betsch, 2021). However, overall acceptance of most of the measures was still at a high level. Our study is line with these findings. Our results reveal that approximately 1 year after the outbreak of the Coronavirus pandemic, the adherence to official guidelines regarding social distancing declined somewhat. Research has shown that trust in authorities is an important factor for the acceptance of environmental measures (Zannakis et al., 2015) and adherence to health guidelines (Gilles et al., 2011; Prati et al., 2011; Quinn et al., 2013; Sibley, 2020). Political decision-makers and Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Acceptance (Range = 1–7) = higher values indicate more acceptance of the measures; Trust Gov. = trust in government (Range = 1–7), higher values indicate more trust in the government; and Guideline Adherence = adherence to the social distancing guidelines (Range = 0–5), higher values indicate more adherence. indicates p < 0.01; indicates p < 0.001. Bold highlighted values are at least p < 0.10. Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; MK-HAI = health anxiety (Range = 1–5), higher values indicate more anxiety; Acceptance (Range = 1–7) = higher values indicate more acceptance of the measures; Trust Gov. GENERAL DISCUSSION Furthermore, future studies in the COVID-19 context should investigate the influence of information sources such as social network platforms in the context of trust (Bunker 2020; Limaye et al 2020) officials should therefore use approaches that underpin trust to promote the acceptance of measures, especially in view of long-term challenges like the COVID-19 pandemic. In particular, the pandemic should be politized at any point of time. Rather authorities as well as media and press should focus on a communication that promotes trust. Betsch et al. (2020d) recommend transparent communication and the emphasis on jointly achieved successes. Adherence to social distancing guidelines was higher among people who were older, female, less right-wing orientation, and more accepting of the measures (Study 2). Betsch et al. (2020d) also reported small positive effects of age and (marginally significant effect) of being female on safety behavior (i.e., using face covering) in the context of the COVID-19 pandemic. Further analyses showed that the association between acceptance of the measures and guideline adherence was moderated by political orientation (Study 2). It should be noted that the variable political orientation was not normally distributed but slightly right-skewed left-leaning distributed. However, low values (1 SD below mean) can be interpreted as more left- wing oriented, average values (mean) as neutral and high values (1 SD above mean) as more right-wing oriented. Thus, the results can be interpreted as follows: for politically left-wing- oriented participants acceptance of the measures had no effect on their guideline adherence, whereas data from politically neutral and right-wing-oriented participants showed a positive link between acceptance of the measures and guideline adherence. Interestingly, the antecedents of social distancing changed over the course of a year. Gender and political orientation no longer predicted adherence to guidelines in Study 3, while trust in government became more relevant. These findings are particularly important for the current COVID-19 pandemic and for future considerations in dealing with pandemics. Obviously, the importance of political orientation decreased as the Coronavirus pandemic progressed. From a practical perspective, policymakers should periodically review and challenge their assumptions about the public’s perception of the pandemic situation. In this way, communication of the necessary measures can be adjusted in the best possible way. Here, it is of particular importance to maintain the trust of the public, especially when support for anti-Coronavirus measures decline. GENERAL DISCUSSION = trust in government (Range = 1–7), higher values indicate more trust in the government; and Guideline Adherence = adherence to the social distancing guidelines (Range = 0–5), higher values indicate more adherence. indicates p < 0.01; *indicates p < 0.001. Bold highlighted values are at least p < 0.10. TABLE 11 \| Multiple linear regressions on adherence to the social distancing guidelines Study 3. TABLE 11 \| Multiple linear regressions on adherence to the social distancing guidelines Study 3. Study 3 Unstandardized coefficients Standardized coefficients B SE(B) β t p Guid. Adh. (Constant) 2.01 0.29 6.80 0.000 Gender 0.10 0.07 0.06 1.38 0.167 Age 0.03 0.00 0.19* 4.51 0.000 Trust Info. 0.08 0.02 0.15** 3.11 0.002 Left–Right −0.02 0.02 −0.04 1.03 0.303 Acceptance 0.06 0.02 0.10* 2.07 0.038 adj. R2 0.075 F 9.49*** 0.000 Guid. Adh. = adherence to the social distancing guidelines (Range = 0–5), higher values indicate more adherence, Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; Acceptance = acceptance of the measures (Range = 1–7), higher values indicate more acceptance. indicates p < 0.05; indicates p < 0.01; *indicates p < 0.001. Bold highlighted values are at least p < 0.10. Guid. Adh. = adherence to the social distancing guidelines (Range = 0–5), higher values indicate more adherence, Gender: 1 = male, 2 = female; Trust Info. = trust in official information (Range = 1–7), higher values indicate more trust; Left-Right = political orientation (Range = 1–10), higher values indicate more right orientation; Acceptance = acceptance of the measures (Range = 1–7), higher values indicate more acceptance. indicates p < 0.05; indicates p < 0.01; *indicates p < 0.001. Bold highlighted values are at least p < 0.10. November 2021 \| Volume 12 \| Article 769206 13 Reactions to COVID-19 in Germany Lermer et al. can be done by conceptual or exact replications (e.g., Stroebe and Strack, 2014). We assume that especially conceptual ones are important. That means that not that exactly same thing was done, but from the basic idea the same results are found. At the time of our data collection, it was not yet possible to foresee what the research on the COVID-19 context would be like. GENERAL DISCUSSION We very much welcome the fact that so many scientists are taking up this relevant topic. This will increase the likelihood of reducing the negative consequences of future challenges such as this pandemic. Some limitations of the study must be mentioned. All three studies were correlative cross-sectional studies. Therefore, no cause–effect relationships can be proven, and future studies should consider longitudinal studies. As in many psychological studies, our samples were convenience samples and consisted of students. However, since this institution where participants were recruited is a part-time university, the students are all employed and on average older than full-time students. Furthermore, in all studies, most of the participants were female. Women tend to perceive higher risks, show more risk-averse behavior than men (Byrnes et al., 1999; Harris and Jenkins, 2006) and are more anxious than men (Maaravi and Heller, 2020) which may have influenced the study’s results. In general, there is a high consistency between our results and those of similar studies. For example, other studies have shown that women report higher levels of social distancing than men (Pedersen and Favero, 2020; Guo et al., 2021). This is in line with our findings regarding the fact that being female is a predictor for greater adherence to social distancing guidelines and behavior change in order not to infect others. Therefore, the unequal gender distribution in our sample does not seem to have distorted the results. Nevertheless, more emphasis should be put on a balanced gender distribution in future studies. Since we asked relatively personal questions (e.g., prosocial behavior), it cannot be guaranteed that there is no social desirability bias in the data. Socially desirable responding to questionnaire items is a general problem in studies relying on self-report. Consequently, future studies should aim to replicate our research findings with more indirect measures. However, the consistency of our results with the current state of research suggests that findings can be successfully replicated. Another important limitation concerns the fact that we only measured behavioral intentions but not actual behavior. Thus, future research should focus on identifying variables that can be used to observe actual behavior. Another interesting approach for future research is to consider individualism and collectivism. The results of a recently published study analyzing data from 69 countries show that the more individualistic (vs. collectivistic) a country is, the higher the COVID-19 infection rates were (Maaravi et al., 2021). Frontiers in Psychology \| www.frontiersin.org ETHICS STATEMENT by Warren et al. (2020) in the COVID-19 vaccine context. A review paper by Bish and Michie (2010) conducted to identify key determinants of safety behavior in the context of the 2009 H1N1 influenza pandemic reports that being female and of older age is linked to adopting safety behaviors. This is also confirmed by the results of present studies for the COVID-19 context. In addition, trust, less right-wing political orientation, and acceptance of measures were shown to be relevant variables for safety behavior. These findings show how important it is to consider individual differences when it comes to prevention measures implemented on a large scale for the sake of a greater good. Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. REFERENCES (17.03.2020). PsychArchives [Preprint]. Available at: https://www.psycharchives. org/handle/20.500.12034/2478 (Accessed November 09, 2021). (17.03.2020). PsychArchives [Preprint]. Available at: https://www.psycharchives. org/handle/20.500.12034/2478 (Accessed November 09, 2021). Abramowitz, J. S., Deacon, B. J., and Valentiner, D. P. (2007). The short health anxiety inventory: psychometric properties and construct validity in a non-clinical sample. Cogn. Ther. 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AUTHOR CONTRIBUTIONS All authors developed the study concept, contributed to the study design, and interpreted the results. Material testing and data collection were performed by EL and MH. The data were analyzed by EL and MH. EL drafted the manuscript, and MH, MR, SG, and FB provided critical revisions. All authors contributed to the article and approved the submitted version. DATA AVAILABILITY STATEMENT The original contributions presented in the study are publicly available. This data can be found at: https://osf.io/y7hxe/. GENERAL DISCUSSION In addition to general trust in the government, however, trust in the government’s competencies is especially relevant. Fancourt et al. (2020, p. 464) summarize: “Public trust in the government’s ability to manage the pandemic is crucial as this trust underpins public attitudes and behaviors at a precarious time for public health.” We see further practical implications of these study findings primarily in that the results presented here may be helpful in developing and communicating interventions. The results confirm that perceptions and behavioral responses differ in Germany, both at the onset of the COVID-19 pandemic and 1 year later. As other studies (e.g., Warren et al., 2020) suggest, the government should not only ensure that trust in the government is and remains high but also consider how different groups of people are addressed in campaigns. Today, more than ever, researchers are called upon to h Overall, the present findings are helpful to target specific groups for preventive campaigns in the context of a pandemic. 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Sci. 11, 454–463. doi: 10.1177/1948550619877412 Statista (2020a). Absatzveränderung ausgewählter Produkte im Lebensmittelhandel durch Hamsterkäufe während des Coronavirus in Deutschland (in der 12.KW/2020 im Vergleich zum Vorjahr) [Graph]. Available at: https://de. statista.com/statistik/daten/studie/1102965/umfrage/umsatzveraenderung- durch-coronavirus-hamsterkaeufe/ (Accessed November 09, 2021). Zannakis, M., Wallin, A., and Johansson, L.-O. (2015). the public’s acceptance of environmental rules: political trust and environmental rule acceptance. Environ. Policy Gov. 25, 424–438. doi: 10.1002/eet.1676 Frontiers in Psychology \| www.frontiersin.org November 2021 \| Volume 12 \| Article 769206 REFERENCES Political trust and perceptions of the quality of institutional arrangements—how do they influence Frontiers in Psychology \| www.frontiersin.org November 2021 \| Volume 12 \| Article 769206 17 Lermer et al. Reactions to COVID-19 in Germany the public’s acceptance of environmental rules: political trust and environmental rule acceptance. Environ. Policy Gov. 25, 424–438. doi: 10.1002/eet.1676 be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2021 Lermer, Hudecek, Gaube, Raue and Batz. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. 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https://openalex.org/W3035259269	https://www.scielo.br/j/aabc/a/PG4Bwh4hS8QHJcTcPRnjk9h/?lang=en&format=pdf	English	null	Ecological risk assessment of glyphosate in surface water when it is used to control floating aquatic macrophytes	Anais da Academia Brasileira de Ciências	2,020	cc-by	6,222	MARINA ANDRADA MARIA, SAMUEL R. CASTRO, LISÉTE C. LANGE, CLÁUDIA L.F. SIÚVES & AYLTON CARLOS SOARES Abstract: The present work evaluated the ecological risk of glyphosate by its commercial formulation (Roundup Original®) used to control floating aquatic macrophytes. Exposure analysis and ecological effects were performed from microcosm studies. The risk characterization was performed based on the calculation of the risk quotient. The commercial formulation of glyphosate had high toxicity when it was assessed separately. On the other hand, ecotoxicological evaluation of water samples from microcosms did not present toxicity to any tested organisms, however, glyphosate application is recommended exclusively to water bodies that have the surface completely covered by macrophytes. Key words: herbicide, macrophytes, toxic effects, ecological risk assessment, aquatic toxicology. BIOLOGICAL SCIENCES Ecological risk assessment of glyphosate in surface water when it is used to control floating aquatic macrophytes An Acad Bras Cienc (2020) 92(2): e20180445 DOI 10.1590/0001-3765202020180445 Anais da Academia Brasileira de Ciências \| Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc \| www.fb.com/aabcjournal An Acad Bras Cienc (2020) 92(2): e20180445 DOI 10.1590/0001-3765202020180445 Anais da Academia Brasileira de Ciências \| Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc \| www.fb.com/aabcjournal ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE MARINA ANDRADA MARIA et al. According to Peterson & Hulting (2004) and Qu et al. (2011), ecological risk assessment is a study that should be evaluated by the risk quotient, which can be calculated for more than one substance, and being able to use the appropriate toxicity measure rather than only the acute toxicity. According to Solomon et al. (2013), the risk assessment must always be expressed as a probability. The main challenge in risk characterization is the analysis of both exposure and effects, being desirable to consider differences of sensitivity among the species (Hayashi et al. 2016). Water bodies are not usually treated as a focus area, once herbicides are commonly used in agriculture, and it is a new perspective. Therefore, the active ingredients from herbicides have their evaluated risk related to the primary contact with soil and then the surface of the water by leaching or drifting. Thus, it is necessary to evaluate the environmental risk inherent in the use of commercial formulation of herbicides for aquatic plant management. Current models for calculation of herbicide availability in surface water consider data of partition coefficient, adsorption, solubility and half-life. These parameters estimate the environmental exposure from the soil runoff. In this case, it considers the effects related to the concentration reduction of the pesticides due to the adsorption in soil or sediment, the degradation before reaching the water, as well as the immediate deposition of the spray drift in the surface of the water bodies (Peterson & Hulting 2004). In July 2015, the Brazilian Resolution nº 467 from the National Council of the Environment (CONAMA 2015) was published and it establishes criteria for the use authorization of products or agents of physical, chemical or biological processes in controlling of organisms or contaminants in surface water bodies, and makes other provisions. Such Resolution indicates the need of the risk assessment of the intended intervention considering the peculiarities of the aquatic environment. Brock (2013) reported that there are two main routes of exposure to pesticides, which may indicate risks to different communities of aquatic and terrestrial fauna, including plant species - food and water supplies. Evaluation of ecological effects estimates indirect risks from herbicide exposure and it should be analyzed for different groups of non-target, terrestrial and aquatic organisms, including birds, mammals, fish, aquatic invertebrates and plants (Peterson & Hulting 2004). ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE At this sense, the present work aims to evaluate the ecological risk of glyphosate in surface water in order to control floating aquatic macrophytes by its commercial formulation, Roundup Original®. This study allows the identification of causes, risks sources, and favors the management plan to use this herbicide. Organisms are often exposed to complex mixtures of chemical contaminants, in a synergic way, causing combined effect in the environment. Toxic effects of the joint action of different molecules and additives should be considered and evaluated (Panizzi et al. 2016). Authors such as Peterson and Hulting (2004) reported that is also important to evaluate the commercial formulation instead the active ingredient, as usual. According to such authors, this formulation may be more toxic. INTRODUCTION or soil) because it assumes that the products are directed to unspecific regions. However, relevant information of the product is required including potential uses, instructions for use, physicochemical properties and persistence, destination, transport and even information regarding effects in the environment by results of ecotoxicological tests (IBAMA 2012). Ecological risk assessment of pesticides is a legal requirement in Brazil, recommended in the Decree nº 4,074/2002 and in specific legislation of Brazilian Institute of Environment and Natural Renewable Resources - IBAMA (Ordinance nº 84/1996). In this sense, some routines and procedures must be developed, data and information must be organized, evaluated, tested and all the uncertainties related to the pesticides behavior in the environment minimized, as well as their possible ecological effects in order to simplify, facilitate decision-making and to ensure a systematic implementation of legislation (IBAMA 2012). Risk assessment is carried out through a three steps process: problem formulation, analysis and characterization of the risk. (USEPA 2004). Analysis and characterization steps of ecological risk assessment must be described in quantitative terms (Peterson & Hulting 2004). Risk quantification is performed in comparing the acute effect with the estimated environmental concentration that can be calculated by environmental models, mesocosms or field measurements (Inao et al. 2008). The problem formulation on ecological risk assessments does not include a broad characterization of the environment (climate An Acad Bras Cienc (2020) 92(2) MATERIALS AND METHODS The ecological risk assessment was performed considering the contamination of the surface water by the use of the commercial formulation An Acad Bras Cienc (2020) 92(2) e20180445 2 \| 12 ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE MARINA ANDRADA MARIA et al. of glyphosate, Roundup Original®, for the management of floating aquatic macrophytes. It was considered the risk of the commercial formulation of glyphosate, its active ingredient and metabolite aminomethylphosphonic acid (AMPA) through the methodology proposed by IBAMA (2012) in four steps: (i) problem formulation, (ii) exposure analysis, (iii) ecological effects analysis and (iv) risk characterization. of the use pattern and recognition of non-target organisms. Half-life of glyphosate was obtained in laboratory-scale experiments performed by the application of its commercial formulation in microcosms (as described below) and the quantification of the active ingredient solution in deionized water. Others parameters were also analyzed (Table I). Analysis of ecological effects Ecological effects analysis was carried out through ecotoxicological studies in order to evaluate the stressor-response relationship. Each aquarium (microcosm) had 35 L of water and 4 kg of sediment in a 20 and 2 cm- columns, respectively, totaling 22 cm high. Moreover, it contained ten individuals of fish genus Poecilia, ten individuals of fish species Danio rerio, ten snails and eight individuals of aquatic macrophytes with average weight equal to 70 g (total of 560 g of Eichhornia crassipes (Mart.) Solms (1883) or 300 g of Salvinia sp). E. crassipes was chosen because it is the macrophyte with highest occurrence in Brazil. All the aquariums were laterally covered and kept in controlled environment: temperature of 23 ± 2°C and luminosity of 3000 lux with photoperiod of 16/8 hours light and dark. Acute and chronic tests were made by using microcrustaceans and bacteria. Working solutions were prepared at concentrations of 50 mL.L-1 for the commercial formulation (360g e.a.L-1 of glyphosate) and 50 mg.L-1 for both glyphosate and AMPA metabolite. Studies involving AMPA were conducted because it is the main glyphosate metabolite. In the case of the commercial formulation, it may have higher toxicity than the active ingredient. Ecotoxicological assays were carried out with the same species and methodology in samples collected from the microcosms after 24 hours of the commercial formulation application, being evaluated the effects related to the environmental exposure to the product. Experiments were performed in triplicate for both aquatic macrophyte species (E. crassipes and Salvinia sp.) in addition to the experimental controls, totaling six experiments. The microcosms had a unique application of the commercial formulation of glyphosate (Roundup Original®), at concentration of 100 L.ha-1, and were monitored during to 20 days. This concentration was established under laboratory tests that aimed at finding the lowest concentration with satisfactory effect on macrophytes in the tested period without evidence of regrowth. The doses commonly used in agriculture had no effect for macrophytes, being necessary a concentration higher than that indicated for terrestrial control. Survival, reproduction and growth are the main effects evaluated in ecotoxicological studies, which may be performed at community, micro or mesocosms levels. (IBAMA 2012). It is important to select bioindicators species present in the exposure routes hypothetically plotted in the conceptual model diagram. Moreover, it enable the evaluation of effects of exposure to the stressor agent, considering the routes closest to the reality and non-target organisms. Exposure analysis In this stage, the risk assessment was performed for both the glyphosate (active ingredient) and its metabolite AMPA. The estimation of the environmental concentration of these substances in water was carried out by simulating the reality. Thus, the commercial formulation of glyphosate was applied in microcosms for controlling aquatic macrophytes (target organism) and it enabled to evaluate and predict values of residual concentration of the active ingredient and its metabolite in water. These values were considered in the risk assessment study. The problem formulation consists of a prior step of the product assessment aiming at predicting its adverse effects on the environment and exposure risks. In this step, the physicochemical characteristics of glyphosate were evaluated regarding its interaction with the environment. As a result, the hypothesis of environmental risk related to the use of the active ingredient and the conceptual model diagram were obtained. Hypothesis of possible risks were supported by information such as persistence, destination and transport of the herbicide in the environment, as well as the establishment Table I. Physicochemical properties of Glyphosate. Properties Values Reference Molar mass 169.1 g.mol-1 Teófilo et al. 2008 apud Santos 2012 pH (in aqueous solution 1% m.v-1) a 25°C 4.68 FISPQ 2016 Relative density 0.5 g.cm-3 Teófilo et al. 2008 apud Santos 2012 Density at 20°C 1.17 g.cm-3 FISPQ 2016 Solubility in water (Sw) 12 g.L-1 (25°C) Teófilo et al. 2008 apud Santos 2012 Acidity (pKa) <2.0, 2.32, 5.86, 10.86 Teófilo et al. 2008 apud Santos 2012 Pressure value (P) 1.94 x 10-7 mmHg FISPQ 2016 Partition coefficient n-octanal-water (Kow) Log Kow = 2.58 FISPQ 2016 Henry’s Law (KH). <2.10-7 Pam3.mol-1 Cabrera et al. 2008 Half-life (deionized water) 21.6 Author Half-life (microcosms) 3.6 Author Table I. Physicochemical properties of Glyphosate. An Acad Bras Cienc (2020) 92(2) e20180445 3 \| 12 ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE MARINA ANDRADA MARIA et al. The microcosms were constructed in a standardized way and simulating an existing pond located in Belo Horizonte/MG, Brazil, with a hot and temperate climate, at the geographical coordinate 19°53’15.702‘’S 43°54’57.906’’W. Average annual temperature is around 20.5°C and average annual rainfall about 1,430 mm (https://pt.climate-data.org/location/2889/, accessed on April 11, 2017). of glyphosate and AMPA, respectively, by ion chromatography. Water samples have been collected from the six microcosms that were used as a simplification of the reality in order to evaluate and to predict the behavior and environmental destination of the product. An Acad Bras Cienc (2020) 92(2) e20180445 4 \| 12 RQ=EC/ ECNO ou EC(I)50 Where: Where: RQ = Risk quotient EC = Environmental concentration (mg.L-1) ECNO = Effect concentration not observed in chronic toxicity test EC(I)50 = Median effective initial concentration of acute toxicity tests According to Hayashi et al. (2016), the risk quotient (RQ) is calculated by the ratio between the maximum environmental concentration and the concentration related to 50% of inhibition (IC50). A quotient value higher than 1.0 means that the maximum environmental concentration exceeds the IC50, and then the studied site has a high risk involved. The result of the chronic test was given by ECNO(I) (initial effect concentration not observed), obtained through statistical analysis using the TOXTAT 3.3 program (West & Dave 1994b). Analysis of ecological effects However, in this study, only aquatic organisms were assessed, because of the environment susceptibility, using invertebrate The environmental concentrations of the active ingredient and the metabolite in water were obtained through the daily quantification ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE MARINA ANDRADA MARIA et al. organisms as bioindicators that present a higher sensitivity. The results can be discussed on an acute or chronic basis. Acute ecotoxicity tests were carried out using standardized methodology and standard species of different trophic levels: Daphnia simillis (ABNT 2016a, NBR 12713) and Aliivibrio fischeri (ABNT 2012, NBR 15411-2). Chronic ecotoxicity was evaluated with Ceriodaphnia dubia species applying the methodology in accordance with ABNT 2016b, NBR 13373. All the related species have an ecological relevance in aquatic ecosystems and are widely used in standardized ecotoxicological tests. Risk characterization procedure was the same for the active ingredient, metabolite and commercial formulation of glyphosate. For this last one, the calculation was not done aiming at assessing the specific risk, but understanding the effect in order to find explanations in ecosystem studies related to the risk of the active ingredient. It needs to be highlighted that such risk assessments are related to the use of the active ingredient of glyphosate in surface water bodies for controlling aquatic macrophytes. The rate and the use pattern must be considered for each specific product (IBAMA 2012). The result of the acute test was presented by the median effective concentration EC(I)50 that was 48h for D. similis and 5 to 30 minutes for A. fischeri. In the first case, immobility and/or mortality were the observed effects supported by a statistical analysis using PROBIT 1.5 program (West & Dave 1994a). For A. fischeri bacteria, the decrease of the luminescence due to the exposure time was the considered criteria, evaluated by a correlation factor calculated using the Omni software and supported by equipment such as luminometer of the Microtox® M500 Analyzer System and lyophilized Biolux® biomass NRRL-B-11177. An Acad Bras Cienc (2020) 92(2) e20180445 5 \| 12 Risk characterization The RQ obtained must be compared to the risk assumption and levels of concern adopted in according to USEPA (2004). The acute risk assumption (LOC) for aquatic organisms is equal to 0.5 and the chronic risk for all animals is equal to 1.0. In this study, the RQ was compared using the LOC value of 1.0 for both the acute and chronic analyzes. The LOC value equal to The risk characterization stage comprises the effective integration of exposure to the product with ecological effects. In a deterministic approach, the most restrictive result is considered and this integration means the calculation of the risk quotient as the ratio between the estimated exposure and the considered effect by the ecotoxicological study. An Acad Bras Cienc (2020) 92(2) e20180445 5 \| 12 ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE MARINA ANDRADA MARIA et al. 1.0 means that the maximum environmental concentration is the same as that presents an ecotoxicological effect with imminent risk. In this way, aquatic organisms are subject to risks when RQ > 1.0. commercial formulation, glyphosate is classified as a kind of glycine with substituted chemical group included in the toxicological class III, being considered moderately toxic. Chemical Product Safety Information File Card (FISPQ) of the Roundup Original® classifies the mixture as extremely toxic and with Environmental Hazard Potential III, being a dangerous product to the environment. Glyphosate has routes of exposure and absorption by respiration, digestion, dermis and mucosa. The references of maximum tolerable value for the active ingredient of glyphosate that can be found in the state and federal environmental legislations from Brazil are lower than the estimated concentration of exposure. This observation is due to the fact that legislations are based on the risk of indirect contamination of the water bodies and are mainly related to the use of herbicides in agricultural activities (Ministério da Saúde 2011, Ordinance nº 2,914/2011 and CONAMA 2005, Resolution nº 357/2005, CONAMA 2011, Resolution nº 430/2011 and COPAM 2008, 01/2008). For this reason, these concentrations were not considered as the maximum concentration of glyphosate in water. The main physicochemical properties determine the herbicides behavior in the environment and are directly related to their persistence and/or degradation. In this sense, it is important to know and understand how each property and characteristic influences the herbicides behavior. The main glyphosate properties are listed in Table I. Risk characterization The solubility (Sw) of glyphosate is 12g.L-1 in water at 25°C. This is the maximum amount of glyphosate that can be solubilized in water at such temperature, above which two distinct phases will coexist. Finally, results of ecotoxicological studies, acute or chronic basis, must be assessed separately because of their different conduction times and sensitivity levels. Glyphosate has low volatility and it can be ratified by the value of vapor pressure (P) equal to 1.94 x 10-7 mmHg, which indicates a tendency of volatilization of the compound in its pure normal state. According to Beltrão (2010), the vapor pressure can be classified in: P >10-2 mmHg are considered very volatile at room temperature; 10-4 <P <10-3, moderately volatile; 10-7≤P≤10-5, low volatile; and P<10-8, non-volatile. In this scenario, it would be desirable to assess the risks to birds that can feed from the affected macrophytes and non-target plants, especially those of margin, which may be affected by drift during the application process, but these evaluations were not performed due to the absence of exposure data and effect for these groups. The partition coefficient n-octanol-water (Kow) is a parameter that is related to the herbicides behavior and destination in the environment. Values of log Kow > 4.0 indicate an accumulation of the herbicide in the organic fraction of sediments, on the other hand, log Kow values <1.0 indicate more solubility in water with low adsorption in sediments. Glyphosate Kow has An Acad Bras Cienc (2020) 92(2) e20180445 6 \| 12 RESULTS AND DISCUSSION Glyphosate is a phosphonate (C3H8NO5P), called N-(phosphonomethyl) glycine by IUPAC. It is an herbicide for agricultural use with high efficiency in weeds control acting in a non-selective and systemic manner. According to the label of its An Acad Bras Cienc (2020) 92(2) e20180445 6 \| 12 ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE MARINA ANDRADA MARIA et al. particles), emulsifiers (making compatible polar and nonpolar fractions), thickeners (increasing viscosity), solvents (turning the active ingredient soluble), wetting agents (putting the product in contact with water) and buffering (keeping the pH value within a desired range). a mean value equal to 2.58, which represents its good solubility in water; however, it often tends to be adsorbed by sediments. The ionization constant (pKa) indicates the pH values that promote different states of ionization. The ionization of glyphosate occurs in a wide pH range (2 to 10), culminating in rapid changes. Risks can be minimized by the direct application of the herbicide in the target organism avoiding excesses that can reach water bodies, where it becomes soluble and more available to the aquatic biota even if it is mainly adsorbed on suspended particles and sediments. Henry’s law constant refers to the partition coefficient of the herbicide in air-liquid or vapor- liquid. The higher is the KH value, the higher is the volatilization potential of the molecule, however, KH value decreases with increasing herbicide solubility in the liquid phase (Prata 2002). Glyphosate has low KH values, which reflects its characteristics such as low volatilization capability and the consequent high solubility in liquid. Generally, the soluble compounds have low sorption coefficients, except for those, even soluble, that have high affinity for adsorption in soil such as glyphosate (Beltrão 2010). Figure 1 has the conceptual model diagram with the environmental risk related to the use of the commercial formulation of glyphosate in controlling floating aquatic macrophytes. It is relevant to note that much of the risk is related to the application and the inherent risk of direct contact with non-target organisms. In this way, the risk is associated not only to aquatic organisms but also to terrestrial organisms, including riparian vegetation (ciliary forest), birds and insects. The possible effects are the same for all exposed organisms, including different symptoms range from death to reduction in reproductive capacity. An Acad Bras Cienc (2020) 92(2) e20180445 7 \| 12 RESULTS AND DISCUSSION The half-life is an indicative of the molecule persistence and it is the time required for the concentration of a substance to halve its original value in a particular environmental medium. Glyphosate is rapidly degraded in natural environments and can remain stable for longer periods in environments free of microorganisms and ionic species. A volume of 1.8 mL of the commercial formulation of glyphosate (648 mg equivalents) were applied by spraying in the microcosms, resulting in a final concentration about 18.5 mg e.a.L -1. However, part of this concentration remains in the macrophytes leaves during the application process that could cause lower concentrations in the water. It is important to highlight that the commercial formulation of herbicides is a mixture of “several ingredients”, not informed in respect to the trade secret, which makes it difficult to assess their behavior, physicochemical characteristics or toxicity. Concentration data of glyphosate were taken 24 hours after application, when an initial decrease in the measures was identified. Table II has the descriptive statistics for glyphosate and AMPA data obtained from the microcosm experiments. The mean value recorded for glyphosate concentration in the six microcosms According to Carvalho (2013), commercial formulations have adjuvants that are substances that aimed at maintaining stability in order to facilitate application, increase efficiency and decrease risks. These substances can influence the herbicide behavior in several ways, for instance, dispersants (avoiding agglomeration of ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE MARINA ANDRADA MARIA et al. Figure 1. Diagram of the conceptual model describing the environmental risk related to the glyphosate use to control floating aquatic macrophytes. Figure 1. Diagram of the conceptual model describing the environmental risk related to the glyphosate use to control floating aquatic macrophytes. was equal to 15.6 ± 1.5 mg.L-1 (mean ± std. dev.), ranging from 13.8 to 18.2 mg.L-1. For AMPA metabolite, the mean value recorded was 0.322 ± 0.130 mg.L-1. During all the experimental period, the highest concentration values for the AMPA metabolite were verified from 5th to 10th day with a maximum value equal to 0.553 mg.L-1. The maximum environmental concentrations obtained in the microcosms were used for the RQ calculation. Some effects on diversity of species can be tolerated. At this sense, measures of survival, reproduction and/or growth tend to be more useful than those based on physiological or biochemical responses that are not clearly related to specificities of the population or community. ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE MARINA ANDRADA MARIA et al. freshwater aquatic environments. However, this study becomes relevant because it represents another trophic level that may be affected in a similar way. the commercial formulation, which has other substances in its content. AMPA metabolite did not present any chronic or acute environmental risks, possibly because of its low concentration of formation in water. The commercial formulation of glyphosate had a higher toxicity when compared to its active ingredient. Therefore, this first had its RQ calculated in order to support the understanding of the dynamics and potential risks related to its use for the control of aquatic floating macrophytes. For the risk characterization for glyphosate, it is important to understand the risk quotient results for both the active ingredient and its commercial formulation in order to establish a use risk assessment. Analyzes of samples collected 24 hours after the application of the commercial formulation of glyphosate in microcosms were crucial and results did not indicate chronic or acute effect for any tested species, even with glyphosate concentrations higher than the EC(I)50 and ECNO values. Table IV has the results of chronic and acute RQ for the three compounds evaluated. Results obtained for the active ingredient of glyphosate, as recommended by the Environmental Protection Agency of the United States, did not indicate acute or chronic risk for microcrustaceans. However, once A. fischeri species had greater sensitivity some acute risk may exist. All microcosms had their surface completely covered by floating macrophytes at the time of application. This fact may have worked as a filter in retaining the surfactant. Finally, observations of the ecotoxicological effect were performed at community level in the microcosms after the herbicide application and no abnormalities were detected. Commercial formulation of glyphosate had acute and chronic RQ higher than the risk assumption (RQ > 1.0). Thus, this result may be an alert due to the difference between the risk presented by the active ingredient and An Acad Bras Cienc (2020) 92(2) e20180445 9 \| 12 Table II. Glyphosate environmental concentration and AMPA measured in the microcosms. Parameter Mean mg.L-1 Minimum mg.L-1 Maximum mg.L-1 Standard deviation mg.L-1 Glyphosate 15.6 13.8 18.2 1.5 AMPA 0.322 0.186 0.553 0.130 Table III. Ecotoxicological results. Sample C. dubia CENO D. similis CE(I)50 A. RESULTS AND DISCUSSION For instance, the decrease of the luminescence observed for A. fischeri. (IBAMA 2012). The relevance of making ecotoxicological assessments with more than one species is because some bioindicators are less sensitive to certain substances, as occurred to the A. fischeri species in the assay with the commercial formulation of glyphosate. It may be due to the lower toxic effect of the inert substance present in the commercial formulation of glyphosate for this species. Table III has the results of the ecotoxicological assays for the active ingredient of glyphosate, AMPA metabolite and the commercial formulation Roundup Original®. The acute risk quotient was calculated for the two evaluated species, taking into account the most sensitive species for each product or the most relevant toxic response, when the focus falls on the survival levels about the biochemical responses. A. fischeri species, saltwater bacteria, is less representative than D. similis in evaluating An Acad Bras Cienc (2020) 92(2) e20180445 8 \| 12 ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE Acknowledgments The authors are grateful for the valuable contributions of the Botanical Garden of the Zoo-Botanic Foundation from Belo Horizonte/MG and Foundation for Research Support of Minas Gerais/Brazil - FAPEMIG. By the risk quotient, the study indicated that glyphosate does not have risk to microcrustaceans survival or reproduction, but it may cause adverse effects to non-target and more sensitive organisms. Thus, further studies specifically designed to examine the considered effects as well as testing non-target organisms are needed. CONCLUSIONS Physicochemical characteristics of glyphosate corroborate its low volatility and high solubility in water with potential to be adsorbed by sediments. Glyphosate presents an acidic characteristic with four dissociation constants, which indicates possible ionic changes in environments with pH ranging from 2 to 10. This latter condition may reflects the glyphosate susceptibility to pH and its short half-life in natural environments, about 3.6 days. The maximum environmental concentrations reached in the aquatic microcosm study by using commercial formulation of glyphosate, applied at 100 L.ha-1, for controlling aquatic macrophytes do not demonstrate any risk for aquatic invertebrates. Finally, it must be highlighted that the study considered a condition of unique application of glyphosate in environments with the surface of the water completely covered by the floating aquatic plants. The conceptual model diagram has indicated indirect ecological risk related to the application by drift or direct contact with non- target organisms, not only to aquatic organisms but also to terrestrial organisms. ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE fischeri CE(I)50 Glyphosate 27.5 mg.L-1 35.13 mg.L-1 11.46 mg.L-1 AMPA 12.5 mg.L-1 52.80 mg.L-1 20.25 mg.L-1 Roundup 3.6 mg e.a.L-1 9.00 mg e.a.L-1 20.16 mg e.a.L-1 Table II. Glyphosate environmental concentration and AMPA measured in the microcosms. Parameter Mean mg.L-1 Minimum mg.L-1 Maximum mg.L-1 Standard deviation mg.L-1 Glyphosate 15.6 13.8 18.2 1.5 AMPA 0.322 0.186 0.553 0.130 Table II. Glyphosate environmental concentration and AMPA measured in the microcosms. environmental concentration and AMPA measured in the microcosms. Table III. Ecotoxicological results. Sample C. dubia CENO D. similis CE(I)50 A. fischeri CE(I)50 Glyphosate 27.5 mg.L-1 35.13 mg.L-1 11.46 mg.L-1 AMPA 12.5 mg.L-1 52.80 mg.L-1 20.25 mg.L-1 Roundup 3.6 mg e.a.L-1 9.00 mg e.a.L-1 20.16 mg e.a.L-1 ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE MARINA ANDRADA MARIA et al. Table IV. Risk quotient results (RQ); Table IV. Risk quotient results (RQ); Compound RQ - chronic CA / ECNO RQ - acute - D.similis CA / EC(I)50 RQ – acute - A.fischeri CA / EC(I)50 Glyphosate standard 0.66 0.52 1.59 AMPA standard 0.04 0.01 0.03 Commercial formulation 5.06 2.02 0.90 an area completely covered with floating aquatic macrophytes. An Acad Bras Cienc (2020) 92(2) e20180445 10 \| 12 ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE MARINA ANDRADA MARIA et al. Universidade Federal de Goiás. Escola de Agronomia e Engenharia de Alimentos. Goiás: 111 p. (Unpublished). Universidade Federal de Goiás. Escola de Agronomia e Engenharia de Alimentos. Goiás: 111 p. 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It was also observed that the high toxicity can be removed when the commercial formulation is applied correctly in ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. 2016b. NBR 13373. Ecotoxicologia Aquática - Toxicidade crônica - Método de ensaio com Ceriodaphnia spp (Crustacea. Cladocera). BELTRÃO DS. 2010. Efeitos da aplicação de glifosato na química de solos alagados. Dissertação (Mestrado). An Acad Bras Cienc (2020) 92(2) e20180445 10 \| 12 ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE MARINA ANDRADA MARIA et al. american surface waters. Enrivon Toxicol Chem 32(1): 10-11. Author contributions This work was part of the doctoral project of researcher Marina Andrada Maria, headed by professor Liséte Celina Lange. Ayton Carlos Soares and Cláudia Lauria Fróes Siúves supported in conducting and carrying out the experiments and Professor Samuel Rodrigues Castro contributed to the data analysis and writing of this document. USEPA - U. S. ENVIRONMENTAL PROTECTION AGENCY. 2004. Overview of the Ecological Risk Assessment Process in the Office of Pesticide Programs. U. S. Environmental Protection Agency: Endangered and Threatened Species Effects Determinations. Office of prevention. pesticides and toxic substances. Office of pesticide programs. Washington. D.C., 92 p. WEST I & DAVE G. 1994a. EPA PROBIT Version 1.5. Wyoming: University of Wyoming. WEST I & DAVE G. 1994a. EPA PROBIT Version 1.5. Wyoming: University of Wyoming. WEST I & DAVE G. 1994b. TOXTAT Version 3.3. Wyoming: University of Wyoming. WEST I & DAVE G. 1994b. TOXTAT Version 3.3. Wyoming: University of Wyoming. ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE Ecological risk assessment of atrazine in north An Acad Bras Cienc (2020) 92(2) e20180445 11 \| 12 ECOLOGICAL RISK ASSESSMENT OF GLYPHOSATE Manuscript received on May 5, 2018; accepted for publication on September 30, 2018 Manuscript received on May 5, 2018; accepted for publication on September 30, 2018 Manuscript received on May 5, 2018; accepted for publication on September 30, 2018 MARINA ANDRADA MARIA1,2 https://orcid.org/0000-0002-9373-1819 LISÉTE C. LANGE2 https://orcid.org/0000-0002-5768-8729 CLÁUDIA L.F. SIÚVES1 https://orcid.org/0000-0001-5074-062X CLÁUDIA L.F. SIÚVES1 https://orcid.org/0000-0001-5074-062X AYLTON CARLOS SOARES1 https://orcid.org/0000-0002-3477-3277 1Institute SENAI of Technology in Environment, Innovation and Technology Center, SENAI-FIEMG, 2000 José Cândido da Silveira Avenue, Horto, 31035-536 Belo Horizonte, MG, Brazil 1Institute SENAI of Technology in Environment, Innovation and Technology Center, SENAI-FIEMG, 2000 José Cândido da Silveira Avenue, Horto, 31035-536 Belo Horizonte, MG, Brazil 2Federal University of Minas Gerais, Department of Sanitary and Environmental Engineering, 6627 Presidente Antônio Carlos Avenue, Pampulha, 31270-901 Belo Horizonte. MG, Brazil 3Federal University of Juiz de Fora, Department of Sanitary and Environmental Engineering, José Lourenço Kelmer Street, São Pedro, 36036-900 Juiz de Fora, MG, Brazil 2Federal University of Minas Gerais, Department of Sanitary and Environmental Engineering, 6627 Presidente Antônio Carlos Avenue, Pampulha, 31270-901 Belo Horizonte. MG, Brazil 3Federal University of Juiz de Fora, Department of Sanitary and Environmental Engineering, José Lourenço Kelmer Street, São Pedro, 36036-900 Juiz de Fora, MG, Brazil How to cite MARIA MA, CASTRO SR, LANGE LC SIÚVES CLF & SOARES AC. 2020. Ecological risk assessment of glyphosate in surface water when it is used to control floating aquatic macrophytes. An Acad Bras Cienc 92: e20180445. DOI 10.1590/0001-3765202020180445. Correspondence to: Maria Andrada Maria E-mail: mamaria@fiemg.com.br Correspondence to: Maria Andrada Maria E-mail: mamaria@fiemg.com.br Correspondence to: Maria Andrada Maria E-mail: mamaria@fiemg.com.br An Acad Bras Cienc (2020) 92(2) e20180445 12 \| 12
https://openalex.org/W4308886926	http://uu.diva-portal.org/smash/get/diva2:1721247/FULLTEXT01	English	null	To calibrate or not to calibrate, that is the question	Water research	2,023	cc-by	11,647	A R T I C L E I N F O Keywords: Sensor maintenance Process control Wastewater treatment plant Measurement error Condition-based maintenance Interlaboratory test Sensors used for control have become widespread in water resources recovery facilities during the strive for resource efficient operations. However, their accuracy is reliant on uncertain laboratory measurements, which are used for calibration and, in turn, to correct for sensor drift. At the same time, current sensor calibration practices are lacking clear theoretical understanding of how measurement uncertainties impact the final control action. The effects of a customarily, and ad hoc, applied calibration threshold are unknown, leading to the current situation where many wastewater treatment processes are controlled by measurements with unknown accuracy. To study how sensor accuracy is affected by calibration, including varying calibration thresholds, we developed a simple theoretical model with closed-form expressions based on the variance and bias in sensor and laboratory measurements. The model was then simulated to yield the results, which showed no practical gain of using a calibration threshold, apart from the situation when calibration is more time-consuming than validation. By contrast, the best accuracy was obtained when consistently executing calibration, which opposes common practice. Further, the sensor calibration error was shown to be transferred to the process, causing a similar deviation from the setpoint when the same sensor was used for control. This emphasizes the importance of minimizing laboratory measurement uncertainties during calibration, which otherwise directly impact opera tions. Due to these findings we strongly advice shifting mindset from considering calibration as a sequential detection and correction approach, towards an estimation approach, aiming to estimate bias magnitude and drift speed. accuracy, and whether these actions can be tailored to achieve a desired accuracy. Oscar Samuelsson a,b,, Erik U. Lindblom b,c, Anders Bj¨ork b, Bengt Carlsson a Oscar Samuelsson a, a Uppsala University, Sweden b IVL Swedish Environmental Research Institute, Sweden c Lund University, Sweden a Uppsala University, Sweden b IVL Swedish Environmental Research Institute, Sweden c Lund University, Sweden Corresponding author at: Uppsala University, Sweden. E-mail addresses: oscar.samuelsson@it.uu.se, oscar.samuelsson@ivl.se (O. Samuelsson). Water Research 229 (2023) 119338 Water Research 229 (2023) 119338 * Corresponding author at: Uppsala University, Sweden. E-mail addresses: oscar.samuelsson@it.uu.se, oscar.sam Available online 7 November 2022 0043-1354/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.watres.2022.119338 Received 13 July 2022; Received in revised form 1 November 2022; Accepted 6 November 2022 2.2.1. True concentration update The true concentration xt was assumed to be constant between two calibration occasions k and k + 1, apart from an adjustment, δc, xt(k + 1) = xt(k) + δc(k) (1) (1) xt(k + 1) = xt(k) + δc(k) However, these errors are not quantified or considered during the validation and calibration procedure. Therefore, the final sensor accu racy after calibration is unclear. This leads to the situation where the treatment process is controlled based on measurements with unknown accuracy. This further lowers the incentive to improve existing cali bration routines since any improvement would go unnoticed. The adjustment δc, represents the effect of the control signal on the true concentration. The control signal can either be manual or auto matically executed via a feedback controller and corresponds here to a change in amount of wasted activated sludge. For simplicity, other disturbances acting on the true sludge concentrations, such as variability in influent flow rate, concentrations, temperature, and operational changes in the return activated sludge flow were excluded. It is, how ever, straightforward to extend the model with such additional factors. Ultimately, current calibration practices are lacking a clear theo retical understanding of how the lab uncertainties impact the final sensor accuracy, and whether a threshold can be applied to improve or control the accuracy. Therefore, we analysed calibration from both a practical and theoretical perspective to clarify what an optimal threshold value may be. Then, we analysed the impact from the un certainties during calibration and how these are transferred to the sensor, as well as the process, when the sensor is used in a feedback control loop or for monitoring. More specifically, we developed a model describing the instant steps during calibration, which was then simu lated and analysed theoretically. The calibration model was illustrated with a fictive total suspended solids sensor used to control a constant total suspended solids concentration in an activated sludge tank. Table 1 List of variables used in the calibration model. Variable Description k Discrete time index enumerating calibration occasions xt True concentration x* r Reference measurement (incl. bias and noise) xr Reference measurement (excl. 2.1. Example system and limitations 2.1. Example system and limitations comparing the sensor value with a reference measurement. In practice, the more common reference is a grab sample, e.g., for TSS, ammonium, and nitrate. It is possible to also use a standard solution with a known concentration if this is available (common for pH-sensors). This study focuses on evaluating current practices, and we here assume that the reference measurement is a grab sample. The validation step is intended to decide whether the sensor provided a ‘good enough’ measurement or needs calibration. What good enough means is subjective and applica tion specific and is encoded in a so-called calibration threshold γ (henceforth referred to as the threshold). It is common to only calibrate the sensor when the difference during validation exceeds the threshold. The adjustment is then executed via the sensor signal processing unit, which we here assume to consist of a simple offset adjustment. The calibration model is illustrated with a TSS sensor used for monitoring and control of the TSS in the bioreactor of an activated sludge process. The results are also applicable to a general sensor- control system where sensor validation and calibration is conducted with uncertain reference measurements. A deliberate limitation is that we here only considered off-set calibration, which is sufficient to correct a constant error, i.e., an error that is independent of the measured value. However, for a sensitivity error (slope error) where the error magnitude depends on the measured value, two-, or multipoint calibration is needed (e.g. an ultraviolet light sensor). The implications of this limi tation, with relation to the results, are discussed in Section 4. It should be noted that available calibration methods differ among sensor makes. It is also common to have the choice of either conducting an off-set or slope calibration. This study is exemplified by a TSS-sensor calibrated with an offset, which resembles, e.g., a Cerlic ITX sensor. Despite the prevalent usage of thresholds, we have, to the best of our knowledge, not seen a theoretical explanation for how the threshold γ should be selected to produce a desired accuracy. On the contrary, we have seen a wide range of thresholds in practice, instead motivated by practical experience, ad hoc procedures, or by sensor supplier infor mation (unpublished results by Andersson, 2019). 2.1. Example system and limitations A common suggestion is that a too tight threshold (or even omitting the threshold) would lead to excessive calibrations with increased variations in the measured signal, because of the more frequent calibrations. Thus, the threshold is not only intended to control the desired accuracy. A list of the used model variables and related abbreviations is given in Table 1. 2.2. Sensor validation and calibration model A model for the sequential steps of sensor validation and calibration is successively introduced in this section together with an overview of how the model was simulated (Section 2.2.10). A second challenge with the current validation and calibration practice is that uncertainties in the reference measurement are not considered in a systematic manner. One or two grab laboratory (lab) samples are commonly used for validation and calibration. Lab samples contain both systematic and random errors through the sampling and analytical procedures (Eurachem, 2019). For example, a systematic error is present if the concentration always differs between two mea surement points such as the surface of an activated sludge tank and 0.5 m below the surface. Similarly, influent variations causing dynamic variations in the tank concentration would induce a random sampling error if the sensor and lab sample are not taken simultaneously at the same location. i An important model feature is that one time step k in the model corresponds to one validation-calibration sequence (henceforth referred to as a calibration occasion). This is commonly executed at a fixed in terval (weekly or monthly) although this is not a requirement for the method. 1. Introduction Biased sensors operating in a feedback control loop have been shown to have negative impact on the treatment and resource efficiency in water resource recovery facilities (WRRFs) (Samuelsson et al., 2021). At the same time, on-line sensors and advanced control are technologies that enable optimized resource efficient operations (Stentoft et al., 2021). The challenge is to manage the increasing abundance of sensor data, and in particular, their accuracy. We here refer to accuracy as the combination of trueness (lack of systematic errors, bias) and precision (lack of random errors) in line with standardized nomenclature (Inter national Organization for Standardization, 1994). Sensor calibration in WRRFs can be conducted by linear regression (Rieger et al., 2005). This is also a widely accepted method in general when the measurement used for validation (also denoted reference measurement) has a low precision, as compared to the sensor (BIPM, 2008). Alternative methods exist that consider precision in both sensor and reference (Orear, 1982), although they are scarce in practice. A drawback with the linear regression approach is that at least two reference samples are needed. Preferably, three or more samples are desired that should further be spread out in the full measurement range. Possibly due to these time-consuming requirements, a simpler approach has become standard in the WRRF domain, which is described next. The evaluation of sensor measurement quality and accuracy is known as validation, which is commonly followed by calibration. These are the most important actions for reaching a high accuracy in practice, together with the mandatory sensor cleaning routines. The aim of this paper is to show how validation and calibration impact the sensor The current practice for sensor calibration is a two-step sequence, commonly executed on a fixed time interval. This approach applies to most sensors such as total suspended solids (TSS), ammonium, nitrate, and dissolved oxygen (DO). The first step – validation - is conducted by O. Samuelsson et al. Water Research 229 (2023) 119338 2.2.1. True concentration update bias and noise) br Bias in reference measurement vr(k) Reference noise value at time k Rr Variance of reference measurement Rs Total variance of sensor measurement Rs a Variance of apparent sensor noise Rs min Variance of sensor repeatability Rs max Variance of sensor reproducibility x* s Sensor measurement (incl. bias and noise) xs Sensor measurement (excl. bias and noise) xs,calib. Calibrated sensor value vs(k) Sensor noise value at time k γ Calibration threshold δa Offset adjustment during calibration δc Change induced by the controller δd Validation difference sp Controller setpoint Table 1 List of variables used in the calibration model. 2.2.6. Sensor calibration in a feedback control loop The repeatability is the minimum variance for repeated measurements at constant conditions. The reproducibility is the maximum variance of the precision when different sensor and operator individuals reproduce equivalent measurements in the same media. Therefore, the repeat ability, Rs min, sets the lower bound and the reproducibility, Rs max, the upper bound for the added variance. Thus, the total variance, Rs, lies between Rs a + Rs min ≤Rs ≤Rs a + Rs max, depending on the sensor validation routines. For example, well-trained staff executing docu mented routines with a high reproducibility (Rs max→Rs min) would yield lower random errors, as compared to ad hoc actions conducted without caring for the sensor’s condition. The main consequence of (8) is that δc(k) will depend on the sensor drift and the adjustment during calibration δc(k) = sp −(xs(k) + δa(k) + bs) (9) (9) This can be understood by recalling the sequence of made changes in the sensor during one calibration occasion. First, the sensor xs(k) was adjusted during calibration with δa(k) as described by (6a–7a). Next, the sensor receives a bias (7b) and the controller compensates for the changes to fulfil (8), that is xs(k + 1) = sp. Finally, the impact on the true concentration with a feedback controller is again described by (1), with δc from (9). For validation (omitting calibration), (9) was revised by simply setting δa(k) to zero according to (6b). The sensor was further assumed to get a bias bs between each cali bration occasion k and k + 1, 2.2.7. Validation and calibration errors during monitoring and feedback control i 2.2.6. Sensor calibration in a feedback control loop 2.2.6. Sensor calibration in a feedback control loop As described in (1), the true concentration was changed via the effect of a manual or automatic control signal, δc. For the situation with feedback control (e.g. using a PI-controller), the sensor’s accuracy will impact the true concentration via the controller, as described below. (3) x* s(k) = xs(k) + vs(k), (3) x* s(k) = xs(k) + vs(k), was also modelled with a noise-free component xs and white Gaussian noise vs with variance Rs. Note that the noise variance is measured with respect to k, and not conventional time t in (3). Therefore, Rs represents the sensor variance between validation occasions. First, the controller setpoint, sp, was assumed to be met prior each calibration occasion. That is xs(k) = sp, (8) (8) The apparent measurement noise is the conventional measurement noise and is the variance when reading the sensor value used for the validation. Commonly, the sensor value during a validation is obtained by manually reading the sensor display during a short time interval. Mathematically, the apparent sensor measurement noise, Rs a = ( σr̅̅̅ N √ )2 , is a function of the N sensor measurements executed to assess the sensor mean value during one validation occasion, where σr is the standard deviation of Rs a. Hence, Rs a approaches zero when N→∞. xs(k) = sp, i.e., a perfect control without constraints on the control signal. Note that in (8) we used the noise-free measurement in (3) and assume that the sensor measurement noise does not influence the controller’s ability to reach the setpoint. This assumption is valid for PI-controllers when the sensor’s apparent measurement noise frequency is much larger than the controller’s speed (i.e., its integration time), which is the case for most controlled and slow wastewater treatment processes. i.e., a perfect control without constraints on the control signal. Note that in (8) we used the noise-free measurement in (3) and assume that the sensor measurement noise does not influence the controller’s ability to reach the setpoint. This assumption is valid for PI-controllers when the sensor’s apparent measurement noise frequency is much larger than the controller’s speed (i.e., its integration time), which is the case for most controlled and slow wastewater treatment processes. In addition to the apparent sensor noise, random errors may add due to the so-called repeatability and reproducibility of the sensor validation. 2.2.5. Calibration In general, the so-called accepted reference value is the most accu rate reference value that can be used for comparison, i.e., sensor vali dation and calibration (International Organization for Standardization, 1994). Here, the concentration was assumed to be measured with one or several lab measurements in the media close to the sensor, i.e., the sensor’s ambient water. We use the ISO nomenclature and the term reference measurement for such lab measurements. The reference measurement Having identified δd, the sensor was adjusted with a constant, δa, if the difference was larger than a user defined calibration threshold γ, \|δd(k)\| > γ, δa(k) = δd(k) (6a) \|δd(k)\| ≤γ, δa(k) = 0 (6b) \|δd(k)\| > γ, δa(k) = δd(k) \|δd(k)\| ≤γ, δa(k) = 0 \|δd(k)\| > γ, δa(k) = δd(k) (6a) 1994). Here, the concentration was assumed to be measured with one or several lab measurements in the media close to the sensor, i.e., the sensor’s ambient water. We use the ISO nomenclature and the term reference measurement for such lab measurements. The reference measurement (6b) The adjustment δa in (6a) is known as an off-set calibration and provides a calibrated sensor xs,calib.(k) as x* r(k) = xt(k) + br + vr(k) = xr(k) + vr(k), (2) (2) xs,calib.(k) = xs(k) + δa(k) (7a) xs,calib.(k) = xs(k) + δa(k) (7a) Recall from (4) that between every calibration occasion, the sensor gets a bias in addition to the change in true concentration was modelled with a bias br and measurement noise vr which is assumed to be Gaussian with zero mean and variance Rr. Furthermore, xr is the noise-free (but biased) reference measurement. The measurement noise is the sum of all random errors including sampling and analytical errors. Similarly, we assumed systematic errors due to sampling and analysis to be summed in the bias term br. xs(k + 1) = xs,calib.(k) + bs + δc(k) = xs(k) + δa(k) + bs + δc(k) (7b) (7b) Eq. (7b) describes how the sensor signal changes when calibration is applied by including δa(k), as compared to (4), in which the sensor is not calibrated but only validated (i.e. δa(k) = 0). 2. Materials and methods A general discrete-time model for calibration is introduced in Section 2.2, which is illustrated by an example containing a TSS sensor measuring in an activated sludge tank (Section 2.1). The calibration model was then simulated and evaluated as described in Section 2.3 to assess the impact of threshold values on the sensor accuracy, and indi rectly, the controlled TSS concentration. 2 O. Samuelsson et al. Water Research 229 (2023) 119338 2.2.7. Validation and calibration errors during monitoring and feedback control i xs(k + 1) = xs(k) + bs + (xt(k + 1) −xt(k)), (4) (4) The final expressions for how calibration (7b) or only validation (4) impact sensor accuracy is given by (10) and (11), based on (1)–(9) with a complete derivation in Appendix A. where xt(k +1) −xt(k) from (1) shows the change in true concentration due to the impact of the control signal. Note that when the true con centration is constant, i.e., xt(k + 1) = xt(k), the sensor measurement is only changed by bs between the calibration occasions. Sensor used for monitoring. When the sensor is only used for monitoring and not control, the validation and calibration errors were here defined as the difference between the true and measured concentration. Vali dation error (no calibration) To simplify, the bias bs was assumed to reach the same value between each calibration occasion, although a time varying bias, bs(k), is likely and could straightforwardly be included in the model. xt(k + 1) −x* s(k + 1) = xt(k) −xs(k) −bs −vs(k + 1) (10a) (10a) 2.2.2. Reference measurement 2.2.2. Reference measurement 2.2.3. Sensor measurement The sensor measurement 2.2.6. Sensor calibration in a feedback control loop 2.3. Performance evaluation Note that the only difference between (10) and (11) is the sensor noise vs(k + 1), which is part of the sensor monitoring example but not the feedback control example. This is a direct consequence of the assumption made in (8). How well δd measures the true difference xt −xs, i.e., whether a bias can be detected, was analysed with the receiver operating characteristics (ROC) and a two-sample Z-test. The improvement in accuracy by con ducting several repeated lab measurements for the reference measure ment (replicates) as well as for the sensor was analysed for one, three and five repeated samples. The ROC visualizes the detection and false alarm rates as a function of calibration threshold value (Kay, 1998). 2.2.8. Calibration rule The decision to calibrate or not to calibrate can be answered by assessing the error magnitudes in (10) and (11). When the error is larger during validation than for calibration, the sensor should be calibrated and vice versa. Interestingly, the same simple decision rule was obtained for both (10) and (11), namely The impact from calibration thresholds, on the true concentration, was analysed by simulating the calibration model for K=1 000 time steps and M=10 000 Monte Carlo repetitions with the settings in Table 2. The root mean squared error (RMSE) \|xt(k) −xs(k)\| > \| −br −vr(k) + vs(k)\|, Calibrate \|xt(k) −xs(k)\| < \| −br −vr(k) + vs(k)\|, Validate (12) (12) RMSE = ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ 1 M ∑ M m=1 ( 1 K ∑ K k=1 (xt(k) −sp)2 ) ) √ √ √ √ , (14a) (14a) That is, when the absolute sensor bias, \|xt(k) −xs(k)\|, is larger than the bias in the reference measurement plus the noise realizations; cali bration should be executed. for a sensor used for control was then used to assess the accuracy and impact from calibration thresholds of the calibration and validation error in (11). Taking expectation of the right side of (12) yields E[\| −br −vr(k) + vs(k)\|] = E[br], (13) E[\| −br −vr(k) + vs(k)\|] = E[br], (13) The RMSE was also analysed by its bias and variance proportions. In general, the mean squared error (MSE) is the sum of the squared bias and variance, see e.g. (Gustafsson, 2000) which demonstrates the logic decision to only calibrate when the reference measurement is more accurate than the sensor. However, the expected value of the left side of (12) is not obvious and depend on the calibration threshold in (6). For this reason, the model was simulated as described in the next sections. MSE(X) = E[X]2 + E[X −E[X]]2 (14b) (14b) Further, the impact from calibration thresholds on the variation in true concentration (xt) and controller correction (δc) was evaluated by their respective standard deviations during the simulations. Finally, the expected value of (11) when γ = 0 was derived as reference to the nu merical estimate in (13) obtained from the simulations. 2.2.9. Motivation of model parameters To the best of our knowledge, we have not found any studies or practical validation results that provide error estimates of the needed model parameters, that is, the bias and variance of TSS sensor and reference measurements. Therefore, these parameter values were assumed according to our reasoning in the next paragraphs. Firstly, the reference measurement variance was assumed to be the sum of the analytical and sampling error variance for a lab measure ment. A typical standardized analytical protocol specifies 10–15 % measurement uncertainty for TSS, which includes both systematic and random errors. The sampling errors are less studied, and the only example we found was Rossi et al. (2011), which indicates a 20–100 % sampling error, depending on flow conditions. Here, we selected a variance error for the reference measurement of 150 mg/L. This corre sponds to a 10 % measurement uncertainty completely based on random errors, which is a deliberate underestimation of the true value. Based on the limited data about systematic proportion of sampling and analytical errors we used the same error value for both the reference bias, as for the variance. 2.2.4. Validation During sensor validation, the noisy sensor measurement is compared with the reference measurement. The difference, δd, was identified as Calibration error (calibration) xt(k + 1) −x* s(k + 1) = −br −vr(k) + vs(k) −bs −vs(k + 1) (10b) (10b) δd(k) = x* r(k) −x* s(k) = xt(k) + br + vr(k) −(xs(k) + vs(k)) (5) (5) δd(k) = x* r(k) −x* s(k) = xt(k) + br + vr(k) −(xs(k) + vs(k)) 3 O. Samuelsson et al. Water Research 229 (2023) 119338 Sensor used for feedback control. For a sensor operating in a feedback control loop, the validation and calibration errors are instead defined as the difference between the true and desired setpoint concentration, i.e., how the sensor error is transferred to the process. Although this study uses the TSS sensor as example, the challenge to assess random and systematic errors is general for most water quality sensors. 2.2.10. Model simulations and software 2.2.10. Model simulations and software Validation error (no calibration) Validation error (no calibration) xt(k + 1) −sp = xt(k) −xs(k) −bs (11a) The simulated actions during one calibration occasion, i.e., the simulated calibration model, are outlined in Table 3 with related parameter values in Table 2. The model was implemented in MATLAB version R2020 and is available as Supplementary materials. xt(k + 1) −sp = xt(k) −xs(k) −bs (11a) (11a) xt(k + 1) −sp = xt(k) −xs(k) −bs Calibration error (calibration) Calibration error (calibration) xt(k + 1) −sp = −br −vr(k) + vs(k) −bs (11b) (11b) 3. Results alarm rate equals the significance level (Kay, 1998). An appropriate test was to assess the absolute difference during validation like a two-sample Z-test. The null hypothesis (H0) was that: there is no difference between the sensor and reference measurement (no bias, or a too small bias). The alternative hypothesis (H1) was that a certain difference exists, e.g., that the bias equals 350 mg/L or above. Fig. 1(b) illustrates the probability distributions for such test with the same settings as used in Fig. 1(a), where a bias of 350 mg/L was assumed to be present when the alter native hypothesis is true. The results are presented in the same order as calibration is executed in practice. That is, first, the bias detection performance during sensor validation is analysed (Section 3.1). Next, the selection of an optimal calibration threshold is studied (Section 3.2). Last, the impact from calibration thresholds on the process variations is analysed (Section 3.3). Fig. 1(b) explains the difference in detection performance for the three black lines in Fig. 1(a). First, the power approaches one as the threshold is lowered to cover H1, see the arrow in Fig. 1(b). Next, if H0 and H1 are perfectly separated, e.g., due to an assumed large bias in H1 (>>350 mg/L), optimal detection performance can be obtained with power one and zero significance level. Last, the overlap between H0 and H1 is also decreased if the variance of either hypothesis is decreased. This was the reason for the improved detection performance with three repeated measurements in Fig. 1(a). In general, the standard deviation of a Gaussian decreases with σ/ ̅̅̅n √ when replicate samples are taken, where σ is the original standard deviation and n is the number of replicate samples. Table 4 shows the dramatic improvement in power when three or more samples were used. i 3.1. Sensor bias detection during validation The calibration rule in (12) requires that the difference xt(k) −xs(k) can be quantified, i.e., that the sensor bias can be detected. This is in practice assessed via the validation and the difference δd(k) = x* r(k) − x* s (k) in (5). How well bias of 100 and 350 mg/L were detected during sensor validation with simulations and the settings in Table 2 is shown in Fig. 1(a) as the receiver operating characteristics. In Fig. 1(a), a false alarm is when the difference during validation calls for a calibration, although there is no true difference, i.e., no bias. The opposite situation is denoted detection, i.e., when a bias is correctly detected during validation. The optimal detection performance is when the detection ratio is one and the false alarm rate is zero for all threshold values. The lowest acceptable performance is indicated by the diagonal grey line in Fig. 1(a), which is obtained when the decision to calibrate is decided randomly. It is possible to get an even lower performance if deliberately violating the goal of validation. However, as a benchmark, we should expect any validation method to be better than just flipping a coin. Ultimately, Fig. 1 and Table 4 indicates that a sufficiently large bias (>350 mg/L) or repeated measurements during validation are needed for a reliable detection (a power above 0.8 is commonly required in statistical tests) for the settings in Table 2. 3.2. Identifying the optimal calibration threshold The detection performance (high detection rate and low false alarm rate) increased with an increasing bias (Fig. 1(a)). Thus, it was easier to correctly detect a large bias, which is expected. Likewise, a lowered threshold value improved the detection rate, but at the cost of an increased false alarm rate. This is indicated by the arrow in Fig. 1(a). When the threshold was at its minimum, i.e., zero, the detection and false alarm rate matched the random detection performance (upper right corner in Fig. 1(a)). That is, both the maximum detection and false alarm rates were obtained, with equal proportions of detections as false alarms. Close to optimal detection performance was obtained when three replicate measurements were taken during validation for a 350 mg/L bias (thick black solid line in Fig. 1(a)). How the calibration threshold comes into play and impact the sensor accuracy and true concentration is now analysed by evaluating simu lations with the settings in Table 2. Also, the impact from different threshold values with respect to the root mean squared error (RMSE) is analysed. Table 2 Assumed model parameter values for the case study simulations. The parameter values were subjectively chosen due to the lack of experimental results and explained in Section 2.2.10. Assumed model parameter values for the case study simulations. The parameter values were subjectively chosen due to the lack of experimental results and explained in Section 2.2.10. Variable/ parameter Value Comment sp 3 000 mg/L A common target TSS concentration in an activated sludge plant Rr (150 mg/L) 2 Corresponds to a standard 10 % analytical uncertainty comprising only random errors. Rs (50 mg/L) 2 A small sensor noise variance reflecting the variation for taking a measurement in-situ, in line with common practice. The 50 mg/L corresponds to one third of the variance for the reference measurement. br 150 mg/L An assumed systematic error magnitude due to sampling and analysis with the same magnitude as the random error. bs 100 mg/(L, k) 350 mg/(L, k) An assumed drift between each calibration occasion. Two bias values were studied illustrating a small and large bias. γ 0–1000 mg/L A common calibration threshold in practice is between 10 and 20 % deviation of the reference measurement (300–600 mg/L) Secondly, the sensor noise variance was assumed to be smaller than the reference measurement variance. As indicated in Section 2.2.3, the sensor variance can be reduced when performing a careful measure ment, e.g., during constant conditions in a bucket with wastewater, and during an extended time. We selected the sensor noise variance to one third of the assumed of reference measurement variance. Lastly, we considered two bias levels, 100 mg/L and 350 mg/L. These correspond to values that would be considered ‘small’ (requiring no action) and ‘large’ (needing a calibration) in practice. 4 O. Samuelsson et al. Water Research 229 (2023) 119338 3.2.1. Simulating the calibration model Top and bottom figure share y-axis label. O. Samuelsson et al. Fig. 2. Simulation of the calibration model with settings in Table 2 and a calibration threshold of 310 mg/L when th Measurements and the controller setpoint. (b) Sensor and controller adjustments and the sensor bias, i.e., the drift. Top Fig. 2. Simulation of the calibration model with settings in Table 2 and a calibration threshold of 310 mg/L when the sensor is used for feedback control. (a) Measurements and the controller setpoint. (b) Sensor and controller adjustments and the sensor bias, i.e., the drift. Top and bottom figure share y-axis label. • The reference measurement varies around the true concentration, but with an offset equal to its bias. more easily analysed in terms of their relative proportions of the mean squared error (MSE) since the MSE is the sum of these two components, recall (14b). The bias proportion of the error can thus be obtained by computing the ratio between the bias and the MSE. • The true concentration drops in line with the accumulated sensor drift. The bias proportion was constantly about 75 % of the MSE, regard less of the threshold value. This result indicates that the squared bias and variance errors increased equally much when the threshold was increased. i.e., that the variance proportion did not increase for a zero threshold value (when the sensor was biased). Further, Fig. 2(b) shows that Further, Fig. 2(b) shows that Further, Fig. 2(b) shows that • The periods with no sensor adjustments or controller corrections (e. g., k=3–6) coincides with the consistent sensor drift in Fig. 2(a). The periods with no sensor adjustments or controller corrections (e. g., k=3–6) coincides with the consistent sensor drift in Fig. 2(a). However, the MSE bias proportion dramatically dropped for γ > 400 mg/L in Fig. 3(b), which illustrates the same situation as in Fig. 3(a) but with an unbiased sensor. This observation simply reflects that the un biased sensor never will be calibrated if a sufficiently large threshold is applied (recall Table 3) and that the true concentration and the sensor was initialized to xs = xt= sp −br −bs. This was also the only situation when a lower RMSE was obtained with a calibration threshold larger than zero, i.e., to keep the sensor ‘as is’. 3.2.1. Simulating the calibration model The output from simulating the calibration model with the sensor operating in a feedback control loop is shown in Fig. 2, which is intended to demonstrate that the calibration model produced logic results. The top part, Fig. 2(a), shows the that An alternative to the ROC is to adopt a statistical test framework and evaluate sensor validation in the view of a statistical hypothesis test. In this regard, the detection rate is known as the test’s power and the false • The sensor measurement varies around the setpoint regardless of the true concentration. • The sensor measurement varies around the setpoint regardless of the true concentration. Fig. 1. (a) Receiver operating characteristics (ROC) for detecting a bias during sensor validation with the settings in Table 2. The ROC is shown for two bias values (100 mg/L dashed line and 350 mg/L solid black lines) and one set of measurements during valida tion (n = 1) and triplicate measurements (n = 3). The random detector (grey solid line) sets the lower performance bound. Threshold values (γ) that produce five percent false alarms and 95 % detection rate follow the grey dashed lines and are indicated with white and black circles, respectively. The grey arrow indicates the improved detection rate when the threshold value is lowered. (b) Probability distributions corresponding to the black solid line in (a) with n = 1. The grey arrow indicates how a lowered threshold increases the power of when the alternative hypothesis is true with a bias of 350 mg/L (H1:bias of 350 mg/L, grey solid line), and the significance level of the null hypothesis (H0:no bias, black line). The dashed grey line shows the original Gaussian distribution for δd, which is folded above zero due to the absolute difference in (6). 350 mg/L (H1:bias of 350 mg/L, grey solid line), and the significance level of the null hypothesis (H0:no bias, black line). The dashed grey line shows the original Gaussian distribution for δd, which is folded above zero due to the absolute difference in (6). 5 Water Research 229 (2023) 119338 O. Samuelsson et al. Water Research 229 (2023) 119338 Fig. 2. Simulation of the calibration model with settings in Table 2 and a calibration threshold of 310 mg/L when the sensor is used for feedback control. (a) Measurements and the controller setpoint. (b) Sensor and controller adjustments and the sensor bias, i.e., the drift. 3.2.1. Simulating the calibration model When the sensor is perfect there is no gain in calibrating it with uncertain reference measurement (recall the calibration decision rule in (12)). Ultimately, the results in Fig. 3(a, b) indicate that the lowest RMSE, and thus the highest accuracy, was obtained when calibration was consistently executed. In this respect, the optimal threshold value was zero when the impact from a biased sensor operating in feedback control should be minimized during calibration. The one exception (to the best of our knowledge) for when a calibration • When the true concentration has dropped low enough so that the difference between the sensor and the reference is larger than the calibration threshold, the sensor is adjusted during calibration (e.g., k=7 and k=12). A complete set of figures describing the scenarios where the sensor was used for monitoring, and with/without bias and with/without noise in the sensor and reference measurements are provided as complement in the Supplementary Materials. 3.2.3. Using a threshold to control the calibration error 3.2.3. Using a threshold to control the calibration error 3.2.3. Using a threshold to control the calibration error The deviation in true concentration and RMSE in Fig. 3(a,b) was induced by the sensor and reference measurement errors as described by (11b) and denoted calibration error. Similarly, the calibration error for the sensor, i.e., its accuracy, was described in (10b). How the sensor’s accuracy was affected by different thresholds and replicate samples is shown in Table 5. l RMSE ( xt(k + 1) −x* s(k + 1) ) = ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ (−br −bs)2 + Rr + 2Rs √ (15a) RMSE(xt(k + 1) −sp) = ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ (−br −bs)2 + Rr + Rs √ (15b) (15a) The sensor’s accuracy reflected the accuracy of the controlled true concentration (Table 5). This was expected since the model is based on similar expressions (compare (10) and (11)), where only the sensor noise differs. Further, Table 5 shows that an increasing threshold corresponds to an increased RMSE, which was also shown in Fig. 3. However, the threshold’s absolute value does not necessarily reflect the achieved ac curacy. More specifically, the RMSE decreased with decreasing variance error (increasing number of samples), see Table 5. Further, the threshold was shown to only impact the false alarm rate (Fig. 1). Altogether, these results indicate that a threshold cannot control the sensor accuracy, although it will have a direct (negative) impact on the final sensor accuracy. (15b) where the bias proportion of the MSE is (−br −bs)2 and the variance part is Rr + 2Rs for the sensor accuracy and Rr + Rs for the controlled con centration due to the assumption in (8). Note, that (15) provides a closed-form expression that quantifies the accuracy as a function of the bias and variance of sensor and reference samples, when no threshold is used. 3.3. Process variations due to sensor calibration One hypothesized complication of using γ = 0 mg/L (mentioned in Section 1) was the potential variations in the true concentration or in the change induced by the control signal δc. Fig. 3(c,d) shows how the variance in δc and the true concentration were affected by different calibration thresholds in the range 0–1 000 mg/L. Similarly, as for the RMSE, the variance in both the true concentration and δc increased with increasing threshold values in Fig. 3(c). In the end, neither a decreased RMSE, nor an increased variance, was indicated as an issue when omitting the calibration threshold. Table 4 Table 4 The only exception where a threshold can decrease RMSE can be seen in Fig. 3(b), which applies for the case with an unbiased sensor. Applying γ > 800 mg/L indicates a RMSE equal to zero (Fig. 3(b)). This corresponds to a sufficiently large threshold where no calibrations are executed due to random variations in the sensor and reference mea surements. Complementary computations (data not shown) showed a linear correlation between the total sensor and reference standard de viation and the threshold producing zero RMSE as γ = 4.8 ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ Rr + Rs √ (R2=99.74 %). Thus, as a rule-of-thumb, a threshold needs to be at least five time times larger than the standard deviation of the random errors if calibration-on-noise should be avoided. Note, for such large threshold, the detection rate is below the random detector performance (Fig. 1(a, b)) and incapable of detecting a bias of 350 mg/L. Threshold (γ) and power values (1 −β) for a statistical hypothesis test, akin to a two-sample Z-test, reflecting Fig. 1(b) at different significance levels (α) and number of repeated measurements (n) and bs = \|350\|. Both the mean values and standard deviations were assumed to be known and different. Bold numbers indicate the settings in Fig. 1 (thin black solid line in (a)). α (Significance level) γ (Calibration threshold) 1 −β (Power) 1 3 5 1 3 5 0.1 260 150 120 0.71 0.99 1.0 0.05 310 180 140 0.59 0.97 1.0 0.01 410 240 180 0.36 0.89 0.99 threshold may be favourable was when the sensor trueness was better than what can be obtained from a calibration, and when bias drift is not expected. The actual sensor accuracy is studied in the next section. Table 3 Table 3 Description of the actions during one validation-calibration sequence and time step k executed during simulation. Variables were defined in Section 2.2.1–2.2.6. Time step Action Related equation Values to be set prior simulation Setpoint (sp), bias in reference (br) and sensor (bs), noise variance in reference (Rr) and sensor (Rs). Initiate Set true concentration to xt= sp −br −bs Set sensor measurement xs = xt k 1 Identify the true concentration xt(k) and the noise-free sensor measurement xs(k) (1) 2 Compute the biased, but noise free, reference measurement xr (2) 3 Sample and add Gaussian noise to the sensor and reference measurements vs(k) ∼N(0,Rs), vr(k) ∼N(0,Rr) (2), (3) 4 Perform validation by computing the difference δd between sensor and reference measurement (5) 5 Assess whether the difference is larger than the calibration threshold γ and decide whether to calibrate or not to calibrate. (6) 6 Calibrate or skip calibration (7a) (4) In practice - use sensor for monitoring and control 7 Add bias to sensor (4) 8 Adjust true concentration with δc (1), (9) k + 1 9 Identify the true concentration xt(k +1) and the noise-free sensor measurement xs(k + 1) (1) 3.2.4. Expected values for zero threshold value 3.2.4. Expected values for zero threshold value The RMSE for a zero threshold obtained from simulations can be verified by comparing the expected value for the calibration error in (10b) and (11b) with the RMSE value for γ = 0 mg/L in Fig. 3(a,b). As Hastie et al. (2009) describe, the standard expression for the mean squared error is MSE(X) = E[X]2 + E[X −E[X]]2, which can then be used to quantify the accuracy in (10b) and (11b) as Table 5 Comparison of the impact of a threshold on the sensor’s accuracy (middle col umn) and the inherited accuracy in the controlled process (right column) when measured as RMSE. The difference for varying number of replicate samples (n), which reduce the variance, is also shown. γ RMSE Process (xt −sp) Sensor (xt −x* s ) n = 1 n=3 n = 5 n = 1 n = 3 n = 5 0 296 266 260 300 268 261 200 311 311 318 315 312 319 400 383 417 428 386 418 428 600 487 529 540 489 530 541 800 598 643 654 600 643 654 1 000 711 756 767 713 757 767 4. Discussion 4.1. Targeting sensor bias detection or estimation? Not surprisingly, the results showed that it is challenging to detect a 3.2.2. Root mean squared error for different threshold values 3.2.2. Root mean squared error for different threshold values The impact of RMSE with different threshold values when the sensor was used for feedback control is shown in Fig. 3(a,b).The RMSE increased with increasing threshold (Fig. 3(a)). The RMSE consists of a bias and a variance part. These are however Fig. 3. (a) Root mean squared error (RMSE) (black solid line) and the percentage of mean squared error (MSE) due to bias (grey dashed line) for calibration thresholds in the range 0–1 000 mg/L and the settings in Table 2. (b) Same as in (a) but without sensor bias. (c,d) Standard deviation (SD) of the true concentration (xt, black solid line) and controller induced correction (δc, grey solid line) with (c) and without bias (d). Fig. 3. (a) Root mean squared error (RMSE) (black solid line) and the percentage of mean squared error (MSE) due to bias (grey dashed line) for calibration thresholds in the range 0–1 000 mg/L and the settings in Table 2. (b) Same as in (a) but without sensor bias. (c,d) Standard deviation (SD) of the true concentration (xt, black solid line) and controller induced correction (δc, grey solid line) with (c) and without bias (d). 6 Water Research 229 (2023) 119338 O. Samuelsson et al. 4.2. Optimizing the practical trade-off in accuracy When regarding calibration as an estimation problem, a natural follow-up question is what the optimal trade-off is with regards to achieved accuracy for spent resources. i The (15) gave a closed-form expression for the final accuracy, depending on the noise and bias errors in the sensor and the reference measurement. First, the equation is a good tool to assure that the desired accuracy is reached. For example, a previous simulation study showed that the bias above 500 mg/L in a total suspended solids sensor was problematic for the nitrogen removal efficiency (Samuelsson et al., 2021). The equation can be used to decide the maximum acceptable bias and variance in the sensor and reference measurement to maintain the desired treatment efficiency. Cost can be traded for accuracy during calibration, and thereby identify the minimum needed effort. For example, the improved accuracy from triplicate reference samples can be compared to the increased analysis cost, or an alternative action. The improvements for revised calibration strategies can be analysed through the bias and variance components in (15). A good example is the frequently debated ‘bucket approach’, and whether it is worth the efforts during calibration. Disregarding the unlikely situation in the previous paragraph, the results showed that both the highest accuracy as well as the smallest variation, in true concentration were obtained when no threshold was applied, i.e., when calibration always was executed. This contrasts the common perception that a calibration threshold can be used to somehow improve the accuracy in the sensor, or reduce the variations induced during calibration. It is unclear why the usage of calibration thresholds has reached wide acceptance in the wastewater domain. One reason could be saving time if validation is quicker than calibration. Another reason could be that it is a consequence of considering validation as a bias detection test and assess whether calibration is needed, i.e., a detection problem. In this regard, statistical hypothesis testing is a widely taught method that produces a threshold via the decided significance level. However, a statistical test is commonly executed for more than one observation when the mean and variances are estimated from data. This can cause confusion for how such a test should be executed for one sample (which is common practice during validation) and may be one reason for the lack of documented procedures for choosing the optimal threshold. 4. Discussion 4. Discussion 4.1. Targeting sensor bias detection or estimation? Not surprisingly, the results showed that it is challenging to detect a 7 Water Research 229 (2023) 119338 O. Samuelsson et al. dataset used during estimation by applying a threshold would violate the best estimation practice and bias the training dataset. It is only customary to remove outliers. This is the opposite effect to what a threshold induces. For example, when applying a 310 mg/L threshold, only values and outliers outside this limit are used for calibration. The reason for these outliers is more likely caused by inaccurate reference measurement and not a large sensor bias. Also, reference measurements close to the sensor’s (within the threshold range) are not used for cali bration and effectively wastes part of the available data for estimation. small bias when the reference measurement has a large uncertainty. The detection rate could be increased by lowering the calibration threshold, and optimally reached one when set to zero - at the expense of an increased false alarm rate. Then, the detection performance approached the random detector’s performance with 50 % false alarms. Half of the executed calibrations would then be carried out, despite that the sensor is unbiased, and hence, in no need for calibration. In practice, this would be a poor strategy if calibration is time-consuming or costly. However, for most water quality sensors, the preceding validation and reference measurement sample is the more costly action. The calibration effort only amounts to manually entering the validation data in the sensor’s digital processing unit (and even this step has been automated in some Swedish WRRFs). In the end, negligible negative effects can be assumed from excessive calibrations in practice. In the end, we could ask what the main goal with sensor validation and calibration is. Do we want to know whether the sensor has a bias (yes/no), or do we want to produce a sensor measurement as accurately as possible? We recommend the latter and to shift our mindset towards regarding sensor validation and calibration as an estimation problem rather than a detection problem. The logic calibration rule in (12)–(13) stated that calibration should only be executed when the reference measurement is more accurate than in the sensor. 4.2. Optimizing the practical trade-off in accuracy The results showed that the significance level and the false alarm rate could be controlled by the threshold, but not the detection rate or the sensor accuracy. The bucket approach refers to when the sensor validation and cali bration measurements are taken in a bucket filled with wastewater, instead of directly from the process. When analysing the bucket approach from (15), we can see that it improves the accuracy in several ways. First, the sensor’s apparent noise can be reduced to zero if the wastewater concentration is constant and the measurement is conducted sufficiently long (recall the sensor measurement Eq. (3)). Second, the lab sampling error decreases, as compared to when a grab sample is taken in the process. As a reference, the sampling error in sewers is indicated to be between 20 % (good turbulent sampling conditions) and up to 100% (laminar flow) in (Rossi et al., 2011). Last, note that a reduced sampling error impacts both the bias and variance in the reference measurement, when the sampling error contributes to both br and Rr. However, a remaining key challenge is to quantify the sampling er rors, as well as the bias and variance proportions in the analytical errors. Without good error estimates, also the estimated accuracy in (15) be comes unreliable. Therefore, we recommend both on-site and academic research efforts to bridge the knowledge gap in lab sample error quan tification. The following actions are recommended An alternative view is to instead consider validation and calibration as an estimation problem. This is logical since most water quality sensors are indirect measurements and rely on calibration (estimation) via reference measurements. In such estimation viewpoint, all efforts are focused around estimating the bias and maximizing the sensor accuracy for the given data, rather than worrying about whether the sensor is sufficiently biased or not. By continuously using all available data for calibrating the sensor, the lowest RMSE is obtained on average (Fig. 3), while accepting that variations in the adjustments in Fig. 2(b) are a natural consequence from the uncertain reference measurement and calibration procedure. (1) Routinely execute interlaboratory assessments (‘round-robin’) to identify systematic and random errors in the analytical chain. (2) Quantify the sensor validation variance Rs in practice via the reproducibility and repeatability for the used validation approach. (3) Assess the variation in different sampling methods, using, e.g., the variogram approach as described in (Petersen and Esbensen, 2005). 4. Discussion One such situation in practice when bias detection, as a pre-step for calibration, could be motivated is when a sensor has been calibrated to a very high accuracy, e.g., when directly supplied from the supplier. One example is airflow rate sensors, which can be adjusted to a high accuracy in factory (where a good reference sensor is available). Then, calibrating the same sensor with on-site routines (lower accuracy) would deteriorate the supplier calibration in line with Eq. (15) and the results for the unbiased sensor (Fig. 3(b)). If instead a calibration threshold value is used, the sensor should only be calibrated when it has lost its supplier provided accuracy and obtained a sufficiently large bias. Then, the rule-of-thumb in Section 3.2.3 could be used to select a suf ficiently large threshold. Note, however, that a large bias is required for a reliable detection when the reference measurement has a low accuracy (Fig. 1). Thus, it would be a gamble (and a guess) to know when the sensor has lost enough accuracy to gain from on-site calibration. 4.3. Aiming towards a best sensor calibration practice An assumption in the calibration model was that the same bias was obtained at each calibration occasion k. This reflects the ideal situation when calibration is executed just-in-time to maintain a desired accuracy. A challenge, which was not considered here, amounts to estimate the drift speed. In practice, the sensor drift varies for different reasons, although only few studies have made detailed assessments about the exact relationships (Cecconi and Rosso, 2021; Ohmura et al., 2019; Samuelsson et al., 2018). Further, the calibration interval k is commonly fixed, leading to different bias values for a fixed time, depending on the drift speed. Thus, assessing the time to reach a certain bias is critical in addition to the actual bias quantification. A good thing is that the drift speed can be estimated from historic calibration data when including validation measurements information about how the sensor signal was adjusted during calibration. Tracking these so-called metadata are essential for assessing the sensor’s condition. Today, routines are un fortunately lacking in practice on how to fully exploit the values in metadata, which could, when used, be a game-changer for best sensor calibration practices. The importance of this topic is emphasized by the on-going task group on Metadata collection and organization (MetaCO, supported by International Water Association [IWA]). i Note that (15) is generally applicable regardless of calibration strategy. Here, an offset calibration was assumed, but also a slope cali bration for a slope error would give the same results - as long as the bias is corrected during the calibration. This is because we here evaluated the RMSE at the calibration instants. However, as a complement, it would be interesting to also study how the RMSE differs when the time between calibration occasions is considered. Then, the effects of using an appropriate calibration strategy could be studied (in a dynamic model) where the time to attain the ideal setpoint in (8) would impact the results. Assuming an ideal situation with quantified lab sample uncertainties, metadata governance systems in place, and extending the results and discussions in this study, we can sketch a simple six-step-recipe best calibration practice for mitigating sensor drift with off-set calibrations An additional, and timely, study would analyse how the errors in reference measurements and the inherited sensor calibration errors, are transferred during model calibration. 4.3. Aiming towards a best sensor calibration practice Assessing model accuracy is becoming a burning question when real-time process models gaining wide-spread usage, popularly denoted digital-twins. There are similar ities between model and sensor calibration, but more reference mea surements and constraints during model calibration complicates assessment of the final model accuracy. It would therefore be interesting to see whether the findings here could be extended and revised to also benefit model calibration. (1) Decide the needed sensor accuracy using process expertise or simulations. (2) Assess and quantify the errors in (15). (2) Assess and quantify the errors in (15). (3) Decide the most cost-effective way to achieve the needed accu racy in step 1, e.g., by replicate samples during calibration or tailored sampling approaches. i (4) Execute calibrations at an initially fixed interval, e.g., monthly, and record the estimated sensor drift speed during different seasons. 5. Conclusion (5) Extrapolate the drift speed and identify the calibration interval corresponding to the maximum accepted bias and desired accuracy. Uncertainties in the lab measurements used for sensor calibration induced errors in the sensor as well as the process controlled by a controller with the very same sensor. This calibration error could not be mitigated by using a calibration threshold, which on the contrary, decreased the sensor accuracy for large threshold values. Thus, the best choice to reach maximum sensor accuracy was to violate current prac tices and skip the calibration threshold, and instead consistently execute calibration. The only realistic situation that motivated a threshold was when the needed time for calibration was large, as compared to vali dation. Setting the calibration threshold to zero did neither increase variations in the process nor in the control signals, which has been a common concern. (6) Iterate step 4–5 and continuously improve the calibration routine while evaluating the metadata produced during calibration. The calibration practice could also be adopted for a slope calibration if one expects the bias to vary with varying concentrations. This would require adjustments of step 1–2 and step 5 in how the bias and variance are computed. The bias may change depending on the considered concentration. Regardless of calibration method, the suggested calibration strategy aims at correcting slow and minor sensor drift, which will likely result in extended calibration intervals. Further, the sensor’s calibration error was transferred to the process as a similar deviation between the setpoint and true concentration (when controlled by a feedback control loop). The transfer of calibration errors was seen in simulations as well as demonstrated analytically. The expected sensor accuracy and the errors transferred to the process were quantified via a closed-form expression when the bias and variance are known for the sensor and reference measurement. This provides a theoretical tool to assess and improve calibration strategies in practice. As a complement to the low frequency calibration, we suggest using data-driven methods such as the software sensor-based approaches reviewed in (Haimi et al., 2013), for high frequency (real-time) moni toring and to detect sudden large bias. 4.2. Optimizing the practical trade-off in accuracy It also becomes clear in the estimation viewpoint why thresholds can be problematic. For example, excluding part of the measurements in a 8 Water Research 229 (2023) 119338 O. Samuelsson et al. (4) Differentiate between systematic and random errors in the ana lyses when assessing the lab coverage factor (confidence inter val). This may require new uncertainty quantification methods. (4) Differentiate between systematic and random errors in the ana lyses when assessing the lab coverage factor (confidence inter val). This may require new uncertainty quantification methods. is executed. This is true when the controller is perfect and maintains the concentration at the setpoint, which applies for the studied model. However, in practice, the controller will not be perfect, and an offset calibration will be inappropriate for a slope error. Further, the bias di rection may shift between positive and negative direction, as well as with different magnitudes, e.g., depending on season. Altogether, the effects of these realistic issues need to be further analysed, especially during the time between the calibration occasions that were not considered here. For such analysis, dynamic simulations are desired. It is likely that these issues will induce deviations in the desired true con centration, and potentially also create larger variations in the control signal. Still, we hypothesize that such negative effects won’t be reduced by applying a calibration threshold in the light of the presented results in this study. Rather, executing the correct mitigating action (such as a slope calibration for a slope error) should be strived for. Such informed decision is reliant upon accurate validation data and metadata as described in the previous sections. 4.3. Aiming towards a best sensor calibration practice References Ohmura, K., Thürlimann, C.M., Kipf, M., Carbajal, J.P., Villez, K., 2019. Characterizing long-term wear and tear of ion-selective pH sensors. Water Sci. Technol. 80 (3), 541–550. Bureau International des Poids et Mesures, International Electrotechnical Commission, International Federation of Clinical Chemistry, International Laboratory Accreditation Cooperation, International Organization for Standardization, International Union of Pure and Applied Chemistry, International Union of Pure and Applied Physics, International Organization of Legal Metrology. (2008) Evaluation of measurement data — guide to the expression of uncertainty in measurement. 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Eurachem/EUROLAB/CITAC/Nordtest/AMC Guide: Measurement Uncertainty Arising from Sampling: a Guide to Methods and Approaches, 2nd ed. Eurachem. S. Andersson, L.Å., Samuelsson, O., Nilsson, S., 2019. Instrumentera R¨att på Avloppsreningsverk. IVL Swedish environmental research institute, Svenskt Vatten. Gustafsson, F., 2000. Adaptive Filtering and Change Detection. John Wiley & Sons Ltd, West Sussex, England. S. Andersson, L.Å., Samuelsson, O., Nilsson, S., 2019. Instrumentera Ratt på Avloppsreningsverk. IVL Swedish environmental research institute, Svenskt Vatten. Samuelsson, O., Bj¨ork, A., Zambrano, J., Carlsson, B., 2018. Fault signatures and bias progression in dissolved oxygen sensors. Water Sci. Technol. 78 (5-6), 1034–1044. Samuelsson, O., Olsson, G., Lindblom, E., Bj¨ork, A., Carlsson, B., 2021. Sensor bias impact on efficient aeration control during diurnal load variations. Water Sci. Technol. 83 (6), 1335–1346. Samuelsson, O., Bj¨ork, A., Zambrano, J., Carlsson, B., 2018. Fault signatures and bias progression in dissolved oxygen sensors. Water Sci. Technol. 78 (5-6), 1034–1044. 4.4. Limitations and future work on the calibration model Finally, the bias detection rate during validation was low due to the uncertainties in the lab measurement. More knowledge about the pro portions of random and systematic sampling and measurement errors are needed to optimize both validation and calibration procedures. i The calibration model assumes an offset calibration, which is also used for the assumed TSS-sensor make. Such calibration is appropriate for an offset error, i.e., an offset bias. When the bias is instead caused by a slope error or a nonlinearity in the sensor, an offset correction will only fully correct the bias at the specific concentration where the calibration Because of the findings, we argue that the current calibration view point needs to shift focus from the existing sequential detection and 9 Water Research 229 (2023) 119338 Water Research 229 (2023) 119338 O. Samuelsson et al. available as supplementary materials. available as supplementary materials. correction approach, towards an estimation approach with the goal to instead estimate bias magnitude and drift speed. In this respect, methods for automatically quantifying the apparent measurement errors based on metadata will be central for adopting best calibration practices, which, in effect, are needed to optimize resource efficient operations. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. i Oscar Samuelsson reports financial support from Stiftelsen Åforsk, grant 21-366, for finalising this research initiated during his PhD- studies. The PhD studies were funded by the Foundation for IVL (SIVL), Stockholm Vatten och Avfall, K¨appala association, Syvab, and Svenskt Vatten. Data availability The model used for producing the simulation and related data is Acknowledgments We gratefully acknowledge the time and extensive review by the three anonymous reviewers, as well as the review and feedback from John Copp and Gustaf Olsson. The questions and comments were valu able and significantly improved the manuscript. i Appendix A: derivation of validation and calibration errors A) Validation of sensor used for monitoring by combining (1)–(4) xt(k + 1) −x* s(k + 1) = xt(k) + δc(k) −(xs(k + 1) + vs(k + 1)) = = xt(k) + δc(k) −(xs(k) + bs + δc(k) + vs(k + 1)) = = xt(k A) Validation of sensor used for monitoring by combining (1)–(4) xt(k + 1) −x* s(k + 1) = xt(k) + δc(k) −(xs(k + 1) + vs(k + 1)) = = xt(k) + δc(k) −(xs(k) + bs + δc(k) + vs(k + 1)) = = xt(k) −xs(k) −bs −vs(k + 1) (10a) B) Calibration of sensor used for monitoring by combining (1)–(7) xt(k + 1) −x* s(k + 1) = xt(k) + δc(k) −(xs(k) + δa(k) + bs + δc(k) + vs(k + 1)) = A) Validation of sensor used for monitoring by combining (1)–(4) xt(k + 1) −x* s(k + 1) = xt(k) + δc(k) −(xs(k + 1) + vs(k + 1)) = = xt(k) + δc(k) −(xs(k) + bs + δc(k) + vs(k + 1)) = = xt(k) −xs(k) −bs −vs(k + 1) (10a) B) C lib ti f d f it i b bi i (1) (7) B) Calibration of sensor used for monitoring by combining (1)–(7) + 1) = xt(k) + δc(k) −(xs(k) + δa(k) + bs + δc(k) + vs(k + 1)) = = xt(k) + δc(k) −(xs(k)+(xt(k) + br + vr(k) −xs(k) −vs(k)) + bs + δc(k) + vs(k + 1)) == −br −vr(k) + vs(k) −bs − xt(k + 1) −x* s(k + 1) = xt(k) + δc(k) −(xs(k) + δa(k) + bs + δc(k) + vs(k + 1)) = = xt(k) + δc(k) −(xs(k)+(xt(k) + br + vr(k) −xs(k) −vs(k)) + bs + δc(k) + vs(k + xt(k + 1) −xs(k + 1) = xt(k) + δc(k) −(xs(k) + δa(k) + bs + δc(k) + vs(k + 1)) = = xt(k) + δc(k) −(xs(k)+(xt(k) + br + vr(k) −xs(k) −vs(k)) + bs + δc(k) + vs(k + 1)) == −br −vr(k) + vs(k) −bs −vs(k + 1) (10b) ( ) s( ) ( ) ( ) ( ( ) ( ) ( ) ( )) = xt(k) + δc(k) −(xs(k)+(xt(k) + br + vr(k) −xs(k) −vs(k)) + bs + δc(k) + vs(k + 1)) == −br −vr(k) + vs(k) −bs −vs(k + 1) (10b) = xt(k) + δc(k) −(xs(k)+(xt(k) + br + vr(k) −xs(k) −vs(k)) + bs + δc(k) + vs(k + 1)) == −br −vr(k) + vs(k) −bs −vs(k + 1) (10b) When the same sensor is used for control, the error in the true concentration as a consequence of the sensor accuracy is When the same sensor is used for control, the error in the true concentration as a consequence of the sensor accuracy is C) Validation of sensor used for control sensor is used for control, the error in the true concentration as a consequence of the sensor accuracy is sensor used for control When the same sensor is used for control, the error in the true concentration as a consequence of the sens C) Validation of sensor used for control xt(k + 1) −sp = xt(k) + δc(k) −sp = = xt(k) + δc(k) −sp = xt(k) + (sp −xs(k) −bs) −sp = = xt(k) + sp −xs(k) −bs − xt(k + 1) −sp = xt(k) + δc(k) −sp = = xt(k) + δc(k) −sp = xt(k) + (sp −xs(k) −bs) −sp = = xt(k) + sp −xs(k) −bs −sp == xt(k) −xs(k) −bs (11a) D) Calibration of sensor used for control xt(k + 1) −sp = xt(k) + δc(k) −sp == xt(k) + δc(k) −sp = xt(k) + (sp −xs(k) −δa(k) −bs) + δc(k) −sp = D) Calibration of sensor used for control D) Calibration of sensor used for control xt(k + 1) −sp = xt(k) + δc(k) −sp == xt(k) + δc(k) −sp = xt(k) + (sp −xs(k) −δa(k) −bs) + δc(k) −sp = = xt(k) + sp −xs(k) −xt(k) −br −vr(k) + xs(k) + vs(k) −bs −sp == −br −vr(k) −bs + vs(k) (1 xt(k + 1) −sp = xt(k) + δc(k) −sp == xt(k) + δc(k) −sp = xt(k) + (sp −xs(k) −δa(k) −bs) + δc(k) −sp = = xt(k) + sp −xs(k) −xt(k) −br −vr(k) + xs(k) + vs(k) −bs −sp == −br −vr(k) −bs + vs(k) (11b) Kay, S.M., 1998. Appendix A: derivation of validation and calibration errors Fundamentals of Statistical Signal Processing: Detection Theory. Prentice-Hall PTR, Upper Saddle River, NJ, USA. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.watres.2022.119338. Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.watr Appendix A: derivation of validation and calibration errors Derivations of the final four expressions Eqs. (10a) and (10b) and Eqs. (11a) and (11b) are given here using the same equation numbers as in Section 2. xpressions Eqs. (10a) and (10b) and Eqs. (11a) and (11b) are given here using the same equation numbers as in Derivations of the final four expressions Eqs. (10a) and (10b) and Eqs. (11a) and (11b) are given here using Section 2. References S l O Ol G Li dbl E Bj¨ k A C l B 2021 S bi Haimi, H., Mulas, M., Corona, F., Vahala, R., 2013. Data-derived soft-sensors for biological wastewater treatment plants: an overview. Environ. Model. Softw. 47, 88–107. Samuelsson, O., Olsson, G., Lindblom, E., Bj¨ork, A., Carlsson, B., 2021. Sensor bias impact on efficient aeration control during diurnal load variations. Water Sci. Technol. 83 (6), 1335–1346. Hastie, T., Tibshirani, R., Friedman, J.H., 2009. The Elements of Statistical Learning: Data Mining, Inference, and Prediction. Springer. Stentoft, P.A., Munk-Nielsen, T., Møller, J.K., Madsen, H., Valverde-P´erez, B., Mikkelsen, P.S., Vezzaro, L., 2021. Prioritize effluent quality, operational costs or global warming? ... Using predictive control of wastewater aeration for flexible management of objectives in WRRFs. Water Res. 196, 116960. International Organization for Standardization, 1994. Accuracy (Trueness and Precision) of Measurement Methods and Results — Part 1: General Principles and definiTions (ISO 5725-1:1994). International Organization for Standardization. 10
https://openalex.org/W1582944693	https://hal.inria.fr/hal-01055410/file/0209--Zhiyong_He.pdf	English	null	The Service Architecture of Agricultural Informatization	IFIP advances in information and communication technology	2,010	cc-by	3,291	To cite this version: Zhiyong He, Lecai Cai, Hongchan Li, Jujia Xu. The Service Architecture of Agricultural Informa- tization. Third IFIP TC 12 International Conference on Computer and Computing Technologies in Agriculture III (CCTA), Oct 2009, Beijing, China. pp.32-39, 10.1007/978-3-642-12220-0_6. hal- 01055410 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01055410 https://inria.hal.science/hal-01055410v1 Submitted on 12 Aug 2014 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Abstract˖ Keywords: agricultural informatization, service architecture, framework THE SERVICE ARCHITECTURE OF AGRICULTURAL INFORMATIZATION Zhiyong He ,* 1 ,Lecai Cai1, Hongchan Li 1, Jujia Xu 1 1 Institute of Computer Application, Sichuan University of Science and Engineering, Zigong Sichuan, P. R. China 643000 * Corresponding author, Address: Institute of Computer Application, Sichuan University of Science and Engineering, Zigong 643000, Sichuan Province, P. R. China, Tel: +86- 13778520180, Fax: +86-813-5505966, Email: hzy@suse.edu.cn Zhiyong He ,* 1 ,Lecai Cai1, Hongchan Li 1, Jujia Xu 1 1 Institute of Computer Application, Sichuan University of Science and Engineering, Zigong Sichuan, P. R. China 643000 * Corresponding author, Address: Institute of Computer Application, Sichuan University of Science and Engineering, Zigong 643000, Sichuan Province, P. R. China, Tel: +86- 13778520180, Fax: +86-813-5505966, Email: hzy@suse.edu.cn Agricultural informatization is the basis for the modernization of agriculture, at present there are many different ways of construction and models in agricultural information systems. Agricultural informatization for the Theory and Application of the status quo, a comparative analysis of a typical agricultural information system, agricultural information service system architecture is advanced, discussed in detail the operating mechanism of service, standards and application of norms, information platform architecture and other major issues, for the construction of agricultural informatization technology standards and norms of the project has a reference value. Keywords: agricultural informatization, service architecture, framework 1. INTRODUCTION Agricultural informatization is the strategy of rejuvenating the implementation and prospering rural market economy, promote solve problems of "agriculture, countryside and farmers", promoting the construction of rural well-off, agricultural science and technology service system is the important part of the construction. It is the vital significance to construct the agriculture information service system of agricultural informationization, and to improve the level of agricultural modernization and agricultural comprehensive competitiveness. 2 Zhiyong He ,Lecai Cai , Hongchan Li , Jujia Xu At present, the agriculture information service system has no unifying definition. Some articles which are related to agricultural information service system aims at agricultural informationization, of development; provides agricultural information service all kinds of main agricultural information service as the core, consist the organic system according to certain rules and system(Yingbo Li et al., 2005). Rural information service system is an integral component which it is engages in the organization of the rural information service, personnel, information infrastructure, information resources and the necessary information technology (Liang Xue et al., 1998). An organic whole is integrated by the agricultural information service system, it has agricultural information resource, agriculture information service participants, policies and regulations, operation mechanism, information infrastructure, and information technology, etc, agricultural information collection and processing, the spread and application are realized, its purpose is to provide technology and organization of agricultural informatization and security. 2.1 Overseas status The agriculture information service system as an important support system in the market economy developed countries. The market information service system construction is strengthened, the agriculture information legislation is noticed, International cooperation and agricultural market information service are strengthened, and the forceful measures construction of economic restructuring is promoted (Jing Zhao et al., 2007). g p g A huge market information network and information network of perfect function have been established by The United States, the policy of the development of agricultural information system is stringier (Xiangyu Zhang et al., 2005). The Japanese government attaches great importance to build a perfect agricultural market information service system, plays a folk in provide market information, and the role of computer in the popularization and application of rural (Yi Yang et al., 2005), the information service system of agricultural market as one of the key service system construction, Japanese agricultural informatization development in these successful experience is worth our using for reference. French formed government, folk, university diversified and comprehensive information service pattern and diversity of information service main body，which include the national agricultural subject, agricultural chamber, agricultural scientific research and the Service Architecture of Agricultural Informatization 3 teaching units, various agricultural industry organizations and professional associations, folk information media and various agricultural production cooperatives and mutual (Zuoyu Guo et al., 2000), etc. Europe officials are the subject of agricultural information service (Li Bai. et al., 2006), in addition to the official, agriculture information service industry association (including organization, agricultural chamber) and futures market and insurance institutions to provide information service also occupied very big one part, play an important role in the agricultural risk prevention. In addition, South Korea established level from agricultural service system, the collection scientific research, promotion and training trinity, unified in agricultural service system, process is simplified and concentrated, promote the new leadership of rural sports. the common features are formed when all the agriculture information service system are constructed, namely government organizations from macroscopically management and coordination in the executive departments, and clear responsibilities of collaboration; Second, the information service, information service, diversification of diversification, such as France; Third, pay attention to strengthen service environment from scientific research, education to ensure service system. 2.2 China’s status Compared with the developed countries, the Chinese rural information service system construction starts relatively late, construction of agricultural information system has made significant achievements. Since 1995, China agriculture proposed involving agricultural informatization construction "JinNong project", based on Countries with large agricultural products market, emphasis, main agricultural education scientific research units and the agricultural professional societies, associations, agricultural and rural informatization are accelerated and promoted, agriculture comprehensive management and service information system" is established. The agricultural basic database, the national agricultural monitoring, forecast and warning, the macroeconomic regulation and control and decision-making service application system, agricultural production situation and crop production forecast system for agricultural development are established and perfection, and play an important role in recent 5 years. At present, the provincial department of agriculture, 97% of the prefecture (city) and 80% of the county agriculture department has information management and service, 64 percent of the information service, the township set up more than 20 million people in the development of rural feedback team, preliminary build from central to local agricultural information system. Information technology development and application achieve positive progress. the information application system of the administrative Zhiyong He ,Lecai Cai , Hongchan Li , Jujia Xu 4 examination and approval and the government office network office automation, scheduling NongQing scheduling, satellite communication, remote sensing, agriculture and animal epidemic information use the information technology are developed by the agriculture department. Department of agriculture, according to the management and service requirement, information system is developed which is related soil testing formula, pest control, basic farmland management. It effectively promotes the agricultural department of e-government, for accelerating the development of modern agriculture, the narrowing of "digital divide" plays a positive role in promoting (Ministry of Agriculture PRC., 2008). p p g ( y g ) At present, information service work of agriculture, countryside and Peasant is developing, it covers market monitoring warning system of the main agricultural products, and provides strong basis for the leadership, macro decision˗Information release which provides timely information industry of agricultural economy for farmers by the ministry of economic information standard calendar; It plays more and more major role in the promoting agricultural, the rural economy development, and the construction of new countryside by coordinating the agriculture information service work. 2.2 China’s status There are 4 million the industrialization of agriculture leading enterprise,17 million rural cooperative intermediary organizations, 61 million administrative villages, 95 million agricultural production and operation, and 240 million rural agent in our country, they can accept the department of agriculture information service by information networks and other forms. 3. THE CURRENT PROBLEMS Agricultural informatization construction of our country is still in the initial, but compared service ability of the information service system with government, enterprises and the demand of agriculture information service, compared modern agriculture and the construction of socialist new countryside with the overall requirements of information work, compared with the developed countries in market economy, there is still a relatively large gap. Current outstanding problems mainly include: 1) The agricultural informatization regulations system is not perfect, the agriculture information standardization is lag; 2) Weak infrastructure and farmers in developing the information service of the "last mile" problems have not been solved; 3) Information collection and collection channels are not standardization, information resource is shortage, information content of practicality is weak, quality need to be promoted, and information resource integration is difficult; 4) The share degree of Existing hardware resources and information resources is low, and does not form the effective mechanism of sharing; 5 the Service Architecture of Agricultural Informatization 5) The function of agricultural information, for example Radio, television, newspapers, telecommunications, media communication, has not been fully developed and integration, support ability of information platform technology is not adequately; 5) The function of agricultural information, for example Radio, television, newspapers, telecommunications, media communication, has not been fully developed and integration, support ability of information platform technology is not adequately; gy q y 6) Peasants culture quality is low, awareness of information is weak, and information ability is not strong; 7) The percentage of The agricultural population of Internet users is low, agricultural sites are less, practical contents are also less, unwrought depth, using low degree, application service ability is bad; 8) The construction of agricultural team is lag, the demand of work can not satisfy by the personnel quantity, and service ability; 9) The degree of marketing is not high, the agricultural product market of electronic business affairs still not completely, commodities and services network level is low; 10) Agricultural information service system is not perfect, the agricultural informatization development level of regional of the middle, east and west is serious imbalance, and differences of regional development are very serious. 3. THE CURRENT PROBLEMS In view of the above problems, regional agricultural comprehensive information public service platform model and application research is put forward in this paper, and the agriculture information service of the "last mile" problem of agricultural information platform, and integration of information resources sharing, and agricultural informatization service standard lag issues are solved, it is an important realistic significance for perfecting the agriculture information service system. 4. DEVELOPMENT OF DYNAMIC ANALYSIS The tendency of our agricultural, rural informatization construction is following: 1) The government's leading tendency. Public information service is provided as the important functions of the government, the government must attach great importance to, overall planning, and investment, speed up the construction on which the government must play a leading role g p y g 2) The complicated trend of demand. Different departments, various industries, of all kinds’ enterprise, cooperative organizations and farmers have different subjects such information needs. On the demand of information quality, effective customized information becomes main stream. On the demand of information on the subject, the strong demand of farmers, various enterprises and intermediary organizations are also increasingly urgent needs. 6 Zhiyong He ,Lecai Cai , Hongchan Li , Jujia Xu 3) The trend of the diverse channels. In the information services, multi- channel, more comprehensive application, modes means are emerge. "SanDian unity" information service in the background is the one of the effective way. y 4) The trend of work. Agricultural information system construction includes agricultural production, processing, distribution and scientific education, promoting technology, consumption and other aspects, enrich the connotation of work, the extension of agricultural industries, and combining the closer, interweave propulsion trend. 5) The development trend of socialization. In recent years, the development of government leading practice showed that, the social forces in agricultural information system construction is the inevitable choice. 6) Precision trend of Production. Precision agriculture based on the global positioning system (GPS) and geographic information system (GIS), information collection and management system of farmland, intelligent decision support system of agricultural machinery system and system integration technology, becomes the core of agricultural informatization development. 5.1 The nature of agricultural information construction framework Agricultural information construction framework is a whole of many elements, which integrates information collection, possessing, communication, application and so on; the purpose is to provide technology and organization of agricultural informatization and security. Its main function is to collect information on the rural economy, processing, storage and transmission by feedback. Firstly, the governments and the relevant departments for the agricultural development plan and guide to provide the scientific basis as farmers, merchants and agricultural market. Secondly, it provides timely and accurate information service for farmers, merchants and agricultural market, accelerates the process of marketing of rural economy. Rural information service system provides information environment of the order transparent, information network of perfect accessibility and information service of high efficient, it is significant and profound influence on rural economic development. 7 the Service Architecture of Agricultural Informatization y 2) System interface layer y 2) System interface layer System interface layer is a system which the agricultural informatization construction to meet different levels of real-time information collection, transmission and exchange and sharing, service. 5.2 The mode of agricultural information construction framework Notice of The national agricultural and rural informatization construction framework (2007-2015) by the issuance of agriculture of March 2008(Ministry of Agriculture PRC., 2008),pointed out: basic framework of our agricultural and rural informatization construction consists mainly in information infrastructure, information resources, talents, service and application system, and the development of the system, operation mechanism, whose rural economic, political, cultural and social fields, etc. study and formulate relevant software and hardware technology standards, data acquisition and processing standards, information, formulate standards, pay attention on information acquisition, storage, processing, processing standards and norms, accelerate information services, agricultural information classification and coding standard can be formulated quickly. Now, there is not standard of agricultural information construction framework in China, through the research and analysis standard of e- government standard (rules) and the ministry of agricultural and rural informatization construction, the general framework of agricultural information service system frame model is put forward, as shown in Fig1. Agricultural comprehensive reform and operation mechanism The agricultural informatization standards and norms System interface (middleware) Information infrastructure Information security Information resource database Services and applications People team Fi 1 Th d f i lt l i f ti t ti f k Fig.1: The mode of agricultural information construction framework Agricultural informatization basic framework consists mainly in rural economic, political, cultural and social fields of information infrastructure, information resource database, system interface, service and application system, talent team, agricultural comprehensive reform and the operation mechanism of agricultural informatization, information security and standard and standard etc. 1) The information infrastructure It mainly includes network infrastructure, information technology equipment. Network infrastructure mainly has the computer network, the communication network and broadcast television, newspapers, magazines 8 Zhiyong He ,Lecai Cai , Hongchan Li , Jujia Xu and billboards. Information technology basic equipment mainly refers to the development of information technology and popularization and application of the necessary facilities. 3) Information resource database 3) Information resource database It is mainly includes science, technology, markets, policies, regulations, culture, education, health, and other information resources database. It serves the participants of production and living, and other social activities in the agriculture and rural. 4) People team 4) People team A practical and efficient service team is built, the information service mode innovation is strengthened, the applicability and effectiveness and scientific of the rural information service is improved. 5) Services and applications system It mainly includes service and information technology application. Information service combines with rural economy, social development of the actual, the development of modern agriculture, basic conditions of the existing information, the industrialization of agriculture and rural economic, the need of cooperation organization. p g 6) Information security 6) Information security It mainly includes information security facilities, technical solutions, systems and equipment in order to ensure safe operation. 7) The standards and norms of agricultural informatization It consists of the general standard, application standards, safety standards and infrastructure construction, management and service integration of standard; including agriculture information resources development and sharing, network (station) construction management, information service, information technology development and application, safety and security, etc. 8) Agricultural comprehensive reform and operation mechanism It mainly includes the mechanism which is the agricultural informatization development, deepening reform of administrative system and promoting each other and common development; and forms the government-leading, market and other social forces joint participation of multiple input mechanism, and forms sustainable development mechanism of agricultural and rural informatization. 8) Agricultural comprehensive reform and operation mechanism It mainly includes the mechanism which is the agricultural informatization development, deepening reform of administrative system and promoting each other and common development; and forms the government-leading, market and other social forces joint participation of multiple input mechanism, and forms sustainable development mechanism of agricultural and rural informatization. 9 the Service Architecture of Agricultural Informatization 6. CONCLUSION Information infrastructure is the basic conditions of agricultural informatization construction. System interface layer is the data exchanging platform of agricultural informatizition. It implements the data exchange function between the different applications. Information resource databases are the important content of agricultural informatization construction. Service and application system is the starting point and the foothold of agricultural informatization construction. Information security is the running guarantee for agricultural informatization construction. Agricultural informatization standardization is the key link of agricultural informatization construction and development. Agricultural comprehensive reform and the operation mechanism of agricultural informatization is the fundamental guarantee. Agricultural information service system is the basic work of agricultural informatization construction, from the theory and application situation of agricultural informatization, compares and analyses the typical agricultural information system, the agricultural informatization service system structure is put forward, relevant laws and regulations of agricultural informationization construction is accelerate formulate, the perfect work system is established, agricultural and rural informatization construction standardization and institutionalized are promoted, it is an important meaning to construct the agricultural informatization engineering standards and norms. Ministry of Agriculture PRC. The notices about National Agriculture and Rural Informatization Construction Framework (2007-2015) by the ministry of agriculture, http://www.agri.gov.cn/jhgb/t20080321_1029943.htm, 2008,(in Chinese) Zhiyong He ,Lecai Cai , Hongchan Li , Jujia Xu REFERENCES Yingbo Li, Study of agricultural information service system, China Agricultural University, PhD thesis, 2005,:34-35(in Chinese) Liang Xue, Yu Fang, Agricultural Informatization, Beijing˖Jinghua Press，1998,2:218- 230(in Chinese) Jing Zhao, Yuping Wang, Summary Research on Agricultural Informatization in and Abroad, Document, Information & Knowledge, 2007,6:80-85(in Chinese) Xiang-yu Zhang, Study on strengthening the construction of agricultural information system. Agricultural economic problems, 2003,1:22-24(in Chinese) Yi Yang, Concerning the development and enlightenment of Japanese agricultural informatization. Modern Japan's economy,2005,6:62-64(in Chinese) Zuoyu Guo, see the agriculture information service in the agriculture information service network construction from France—training report of the agriculture information service network in France, Feed wide-angle, 2000,4:28-29(in Chinese) Li Bai, et al. Mode and enlightenment of Euro agriculture information service. World Agriculture, 2006,1:33-35(in Chinese) Ministry of Agriculture PRC. Construction Planning of The "11th five-year plan" period of National Agricultural Information System by the ministry of agriculture. http://www.agri.gov.cn/jhgb/t20080321_1029961.htm, 2008,(in Chinese) Zhiyong He ,Lecai Cai , Hongchan Li , Jujia Xu 10 Ministry of Agriculture PRC. The notices about National Agriculture and Rural Informatization Construction Framework (2007-2015) by the ministry of agriculture, http://www.agri.gov.cn/jhgb/t20080321_1029943.htm, 2008,(in Chinese)
https://openalex.org/W4362364690	https://aacr.figshare.com/ndownloader/files/39870732	English	null	Supplementary Figures 1-5 from Netrin-1 and Its Receptor DCC Are Causally Implicated in Melanoma Progression	null	2,023	cc-by	1,996	Supplementary Figure 1 WM239A melanoma cell line A C * E ctl DCC rTetR 2A peptide HygR DCC TRE prom hPGK prom DCC GAPDH 0 µg/ml 0.5 µg/ml 2 µg/ml Doxycycline D * ** Doxycycline B Supplementary Figure 1: DCC behaves as a tumor suppressor in melanoma. A. Schematic diagram of the DCC inducible construct. Expression of DCC is controlled by the reverse tetracycline-controlled transactivator (rTetR) which activates the TRE promoter in presence of doxycycline. Infected cells are selected by hygromycine (HygR). rTetR and HygR are under the control of a ubiquitous hPGK promoter and the cleavage between the two proteins is Supplementary Figure 1 WM239A melanoma cell line A C * E ctl DCC rTetR 2A peptide HygR DCC TRE prom hPGK prom DCC GAPDH 0 µg/ml 0.5 µg/ml 2 µg/ml Doxycycline D * Doxycycline B Supplementary Figure 1: DCC behaves as a tumor suppressor in melanoma. A. Schematic diagram of the DCC inducible construct. Expression of DCC is controlled by the reverse tetracycline-controlled transactivator (rTetR) which activates the TRE promoter in presence of doxycycline. Infected cells are selected by hygromycine (HygR). rTetR and HygR are under the control of a ubiquitous hPGK promoter and the cleavage between the two proteins is Supplementary Figure 1 WM239A melanoma cell line A rTetR 2A peptide HygR DCC TRE prom hPGK prom DCC GAPDH 0 µg/ml 0.5 µg/ml 2 µg/ml Doxycycline Doxycycline B Supplementary Figure 1 WM239A melanoma cell line A rTetR 2A peptide HygR DCC TRE prom hPGK prom DCC GAPDH 0 µg/ml 0.5 µg/ml 2 µg/ml Doxycycline ** Doxycycline B B C * E C 2A peptide RE prom hPGK prom DCC GAPDH 0 µg/ml 0.5 µg/ml 2 µg/ml Doxycycline D * Doxycycline Supplementary Figure 1: DCC behaves as a tumor suppressor in melanoma. A. Schematic diagram of the DCC inducible construct. Expression of DCC is controlled by the reverse tetracycline-controlled transactivator (rTetR) which activates the TRE promoter in presence of doxycycline. Infected cells are selected by hygromycine (HygR). rTetR and HygR are under the control of a ubiquitous hPGK promoter and the cleavage between the two proteins is ensured by a 2A peptide. Below, stable melanoma cell lines for DCC (amelanotic WM239A) were treated with 0.5 and 2 µg/ml of doxycycline for 24 h and DCC protein levels were evaluated by Western blot analysis. DCC protein, normalized against GAPDH, increased strongly following doxycycline treatment. B. Scid mice were engrafted with stable melanoma cell lines containing the DCC construct (amelanotic WM239A) by subcutaneous injection. When tumor size reached around 100 mm3, expression of DCC was induced by daily intraperitoneal injection of doxycycline (n = 9), the control (clt) group was treated with PPI water (n = 9). Results are presented as mean tumor volumes. Two-way ANOVA: Time p < 10-4, Cell type p = 0.0087, Interaction *** p = 10-4. C. Photograph of tumor-bearing mice. D. Supplementary Figure 1 WM239A melanoma cell line A C * E ctl DCC rTetR 2A peptide HygR DCC TRE prom hPGK prom DCC GAPDH 0 µg/ml 0.5 µg/ml 2 µg/ml Doxycycline D * ** Doxycycline B Supplementary Figure 1: DCC behaves as a tumor suppressor in melanoma. A. Schematic diagram of the DCC inducible construct. Expression of DCC is controlled by the reverse tetracycline-controlled transactivator (rTetR) which activates the TRE promoter in presence of doxycycline. Infected cells are selected by hygromycine (HygR). rTetR and HygR are under the control of a ubiquitous hPGK promoter and the cleavage between the two proteins is Tumor volumes were measured at day 18 (end of the experiment) after sacrifice of mice and taking only the size of tumors without skin. * p = 0.0226 Two-tailed Unpaired t test. E. Expression of DCC is confirmed by q-RT-PCR, relatively to the GUSB housekeeping gene, in tumors from a group of mice treated with doxycycline whereas no expression was detected in the control group. * p = 0.0167 Two-tailed Unpaired t test. D * * E D * E D * C ctl DCC C ctl D E C ctl mentary Figure 1: DCC behaves as a tumor suppressor in melanoma. Supplementary Figure 1: DCC behaves as a tumor suppressor in melanoma. A. Schematic diagram of the DCC inducible construct. Expression of DCC is controlled by the reverse tetracycline-controlled transactivator (rTetR) which activates the TRE promoter in presence of doxycycline. Infected cells are selected by hygromycine (HygR). rTetR and HygR are under the control of a ubiquitous hPGK promoter and the cleavage between the two proteins is ensured by a 2A peptide. Below, stable melanoma cell lines for DCC (amelanotic WM239A) were treated with 0.5 and 2 µg/ml of doxycycline for 24 h and DCC protein levels were evaluated by Western blot analysis. DCC protein, normalized against GAPDH, increased strongly following doxycycline treatment. B. Scid mice were engrafted with stable melanoma cell lines containing the DCC construct (amelanotic WM239A) by subcutaneous injection. When tumor size reached around 100 mm3, expression of DCC was induced by daily intraperitoneal injection of doxycycline (n = 9), the control (clt) group was treated with PPI water (n = 9). Results are presented as mean tumor volumes. Two-way ANOVA: Time ** p < 10-4, Cell type p = 0.0087, Interaction *** p = 10-4. C. Photograph of tumor-bearing mice. D. Tumor volumes were measured at day 18 (end of the experiment) after sacrifice of mice and taking only the size of tumors without skin. * p = 0.0226 Two-tailed Unpaired t test. E. Expression of DCC is confirmed by q-RT-PCR, relatively to the GUSB housekeeping gene, in tumors from a group of mice treated with doxycycline whereas no expression was detected in the control group. * p = 0.0167 Two-tailed Unpaired t test. Supplementary Figure 2 Supplementary Figure 2: Photograph of tumor-bearing mice. Phenotype of BRAFV600E expressing mice, BRAFV600E;DCCmut mice and BRAFV600E;tgNETRIN-1 mice. Supplementary Figure 2: Photograph of tumor-bearing mice. Supplementary Figure 1 WM239A melanoma cell line A C * E ctl DCC rTetR 2A peptide HygR DCC TRE prom hPGK prom DCC GAPDH 0 µg/ml 0.5 µg/ml 2 µg/ml Doxycycline D * ** Doxycycline B Supplementary Figure 1: DCC behaves as a tumor suppressor in melanoma. A. Schematic diagram of the DCC inducible construct. Expression of DCC is controlled by the reverse tetracycline-controlled transactivator (rTetR) which activates the TRE promoter in presence of doxycycline. Infected cells are selected by hygromycine (HygR). rTetR and HygR are under the control of a ubiquitous hPGK promoter and the cleavage between the two proteins is Phenotype of BRAFV600E expressing mice, BRAFV600E;DCCmut mice and BRAFV600E;tgNETRIN-1 mice. Supplementary Figure 3 Supplementary Figure 3 A B 0% 10% 20% 30% 40% 50% 60% 70% Weak moderate strong 44% 44% 11% 44% 44% 13% 0,8% 34% 65% Netrin-1 tumor cell intensity Benign melanocytic lesions Melanoma without metasta Melanoma with metastasis 0% 20% 40% 60% 80% 00% 0-25% 25-50% 50-75% 75-100% 6% 36% 42% 17% 1% 11% 64% 24% 0,8% 0% 9% 90% Netrin-1 tumor cell score Benign melanocytic lesions melanoma without metastasis melanoma with metastasis Supplementary Figure 3: Netrin-1 is overexpressed in most advanced stages of melanoma. A and B. Histograms (A, B) describe the scores and tumor intensity of netrin-1 in benign melanocytic lesions vs melanoma with or without metastasis at time of study. The analyses show a correlation between the levels of netrin-1 (intensity or score) and the level of malignancy of the nevi. A B 0% 10% 20% 30% 40% 50% 60% 70% Weak moderate strong 44% 44% 11% 44% 44% 13% 0,8% 34% 65% Netrin-1 tumor cell intensity Benign melanocytic lesion Melanoma without metas Melanoma with metastasi 0% 20% 40% 60% 80% 100% 0-25% 25-50% 50-75% 75-100% 6% 36% 42% 17% 1% 11% 64% 24% 0,8% 0% 9% 90% Netrin-1 tumor cell score Benign melanocytic lesions melanoma without metastasis melanoma with metastasis B A Supplementary Figure 3: Netrin-1 is overexpressed in most advanced stages of melanoma. A and B. Histograms (A, B) describe the scores and tumor intensity of netrin-1 in benign melanocytic lesions vs melanoma with or without metastasis at time of study. The analyses show a correlation between the levels of netrin-1 (intensity or score) and the level of malignancy of the nevi. Supplementary Figure 4 Supplementary Figure 4 Supplementary Figure 4 cCaspase-3 BRAFV600E BRAFV600E;tgNETRIN-1 * A B Supplementary Figure 4: Netrin-1 up-regulation reduces caspase-3 activity in Braf mice tumors. A. Representative images of cCaspase-3 positive staining in sections performed on the respective tumors described in Figure 4 are presented. Scale bar represents 50 μm. B. cCaspase- 3 positive cells described in A are quantified. (* p = 0.0179 Unpaired t test). B cCaspase-3 BRAFV600E BRAFV600E;tgNETRIN-1 A A * B cCaspase-3 BRAFV600E;tgNETRIN-1 Supplementary Figure 4: Netrin-1 up-regulation reduces caspase-3 activity in Braf mice tumors. A. Representative images of cCaspase-3 positive staining in sections performed on the respective tumors described in Figure 4 are presented. Scale bar represents 50 μm. B. cCaspase- 3 positive cells described in A are quantified. (* p = 0.0179 Unpaired t test). * Skmel3 melanoma cell line WM793 melanoma cell line C A Supplementary Figure 5 Skmel5 melanoma cell line B Supplementary Figure 5: Netrin-1 interference (alone or in combination with chemotherapy) inhibits tumor growth in melanoma xenografted models. A. Effect of netrin-1-interfering antibody NET1-H-mAb (n = 10) on Skmel5 xenograft tumor growth compared to Iso-mAb control group (n = 10). Results are presented as mean tumor volumes. Two- way ANOVA: Time **** p < 10-4, Antibody * p = 0.0409, Interaction **** p < 10-4. B. Survival curve comparison between the group of mice treated with DTIC + NTN1-H-mAb (n = 13) and the one treated with DTIC + Iso-mAb (n = 13), in mice xenografted with amelanotic WM793 melanoma cell Supplementary Figure 5 * A Supplementary Figure 5 Skmel5 melanoma cell line * A Skmel5 melanoma cell line A Skmel3 melanoma cell line WM793 melanoma cell line C B Supplementary Figure 5: Netrin-1 interference (alone or in combination with chemotherapy) inhibits tumor growth in melanoma xenografted models. A. Effect of netrin-1-interfering antibody NET1-H-mAb (n = 10) on Skmel5 xenograft tumor growth compared to Iso-mAb control group (n = 10). Results are presented as mean tumor volumes. Two- way ANOVA: Time **** p < 10-4, Antibody * p = 0.0409, Interaction **** p < 10-4. B. Survival curve comparison between the group of mice treated with DTIC + NTN1-H-mAb (n = 13) and the one treated with DTIC + Iso-mAb (n = 13), in mice xenografted with amelanotic WM793 melanoma cell line. * p = 0.0329. C. Survival curve comparison between the group of mice treated with DTIC + NTN1-H-mAb (n = 12) and the one treated with DTIC + Iso-mAb (n = 12), in mice xenografted with amelanotic Skmel3 melanoma cell line. P = 0.0515 (ns). Statistical significance of differences between both groups was determined by Mantel-Cox test in B and C. Skmel3 melanoma cell line WM793 melanoma cell line C B WM793 melanoma cell line B Skmel3 melanoma cell line C C B Skmel3 melanoma cell line Supplementary Figure 5: Netrin-1 interference (alone or in combination with chemotherapy) inhibits tumor growth in melanoma xenografted models. A. Effect of netrin-1-interfering antibody NET1-H-mAb (n = 10) on Skmel5 xenograft tumor growth compared to Iso-mAb control group (n = 10). Results are presented as mean tumor volumes. Two- way ANOVA: Time **** p < 10-4, Antibody * p = 0.0409, Interaction **** p < 10-4. B. Survival curve comparison between the group of mice treated with DTIC + NTN1-H-mAb (n = 13) and the one treated with DTIC + Iso-mAb (n = 13), in mice xenografted with amelanotic WM793 melanoma cell line. * p = 0.0329. C. Survival curve comparison between the group of mice treated with DTIC + NTN1-H-mAb (n = 12) and the one treated with DTIC + Iso-mAb (n = 12), in mice xenografted with amelanotic Skmel3 melanoma cell line. P = 0.0515 (ns). Statistical significance of differences between both groups was determined by Mantel-Cox test in B and C.
https://openalex.org/W2810922572	https://europepmc.org/articles/pmc6097723?pdf=render	English	null	Revisiting the Children-of-Twins Design: Improving Existing Models for the Exploration of Intergenerational Associations	Behavior genetics	2,018	cc-by	14,417	1 For a detailed introduction to the use of children-of-twins data, the analytical methods frequently applied to such data, and a review of the empirical literature we refer readers to McAdams et al. (2014). Revisiting the Children-of-Twins Design: Improving Existing Models for the Exploration of Intergenerational Associations Received: 14 September 2017 / Accepted: 13 June 2018 / Published online: 30 June 2018 © The Author(s) 2018 * Tom A. McAdams tom.mcadams@kcl.ac.uk Abstract Datasets comprising twins and their children can be a useful tool for understanding the nature of intergenerational associa- tions between parent and offspring phenotypes. In the present article we explore structural equation models previously used to analyse Children-of-Twins data, highlighting some limitations and considerations. We then present new variants of these models, showing that extending the models to include multiple offspring per parent addresses several of the limitations dis- cussed. Accompanying the updated models, we provide power calculations and demonstrate with application to simulated data. We then apply to intergenerational analyses of height and weight, using a sub-study of the Norwegian Mother and Child Cohort (MoBa); the Intergenerational Transmission of Risk (IToR) project, wherein all kinships in the MoBa data have been identified (a children-of-twins-and-siblings study). Finally, we consider how to interpret the findings of these models and discuss future directions. Keywords Children-of-twins · Extended family design · Intergenerational transmission · Parent · Offspring · The Norwegian Mother and Child Cohort Study (MoBa) · The Intergenerational Transmission of Risk (IToR) project Keywords Children-of-twins · Extended family design · Intergenerational transmission · Parent · Offspring · The Norwegian Mother and Child Cohort Study (MoBa) · The Intergenerational Transmission of Risk (IToR) project transmission and ask whether associations between parent and child remain after accounting for their genetic relatedness. Studying samples of twin pairs with children can provide valuable insight into the nature of intergenerational associa- tions. Using samples of monozygotic (MZ) and dizygotic (DZ) twins with children (or indeed any samples of differen- tially related siblings/cousins with offspring), it is possible to evaluate hypotheses regarding the nature of intergenerational The use of samples of twins with children to examine intergenerational associations has been discussed in depth elsewhere (D’Onofrio et al. 2003; Fischer 1971; Gottes- man and Bertelsen 1989; McAdams et al. 2014; Silberg and Eaves 2004).1 However, briefly, the offspring of MZ twins are as related to their parent’s co-twin as they are to their own parent (they share 50% of their DNA). In contrast, the offspring of DZ twins share 25% of their genetic variance with their parent’s co-twin. By comparing MZ avuncular correlations (correlations between uncle/aunt and niece/ nephew) with DZ avuncular correlations, it is possible to estimate the role of genetic factors in explaining intergenera- tional associations. It is also possible to estimate the extent to which parent–child associations remain after accounting for genetic transmission. Behavior Genetics (2018) 48:397–412 https://doi.org/10.1007/s10519-018-9912-4 Behavior Genetics (2018) 48:397–412 https://doi.org/10.1007/s10519-018-9912-4 ORIGINAL RESEARCH Abstract Where a1′ is significant, this indicates that genetic factors influencing the parental phenotype also explain variance in the offspring phenotype, and the association between parent and child phenotype is (at least partially) attributable to shared genes. Where p is significant, this indicates that the parent–child association (at least partially) persists after accounting for shared genes. possible to use siblings or even cousins with children, wher- ever samples are large enough. That is, rather than (or as well as) comparing avuncular relationships in MZ and DZ families, siblings, half-sibling and cousins can be used. Chil- dren share 25, 12.5 and 6.25% of their genetic variance with their parent’s sibling, half-sibling and cousin respectively. These differences in relatedness can be used to estimate the role of genetic factors in explaining intergenerational asso- ciations in the same way that twin data is used (e.g. Kuja- Halkola et al. 2014). A variety of techniques can be used to analyse children- of-twin (CoT) and/or children-of-sibling (CoS) data and investigate the nature of intergenerational associations. While various multilevel regression models have been applied to such data, in keeping with behavioural genetic tradition, structural equation models (SEMs) have been most widely used, and it is these models that we focus on in this article. Biometric SEMs are advantageous in that they allow researchers to quantify the relative importance of genetic and environmental influences in explaining variance on a trait and covariance between traits. Our intention in this article is to describe the SEMs thus far applied to CoT data, discuss their limitations, and suggest extensions to the mod- els that help to overcome these limitations. We then apply these extended models, first demonstrating their power using simulated data, and then using real data originating from the Norwegian Intergenerational Transmission of Risk (IToR) project, a sub study of the Norwegian Mother and Child Cohort Study (MoBa) (Magnus et al. 2016). Throughout, we highlight and discuss remaining limitations and issues to consider for those interested in making use of CoT models and those interested in collecting CoT and/or CoS data. We provide OpenMx scripts for all models via the Supplemen- tary Materials section of this article. The model in Fig. 1a and other CoT/CoS models like it decompose the intergenerational association into a genetic component and a phenotypic component (Eley et al. 2015; Hannigan et al. 2016; McAdams et al. 2015, 2017; Silberg et al. 2010, 2012). In Fig. Abstract 1b we adapt this model to allow for the possibility that shared environmental influences on the parent generation may influence the offspring genera- tion as well, via a new path, c1′, that captures the effect of the extended family environment. Parent–child covariance is thus explained by a combination of p, a1′, a1, c1′ and c1. The effects of the extended family environment have been included in suggested CoT models previously (D’Onofrio et al. 2003). Indeed, it is possible to model multiple sources of shared environment using CoT/CoS data. Intergenera- tional models including a variety of shared environmental factors have previously been applied to CoS data derived from the Swedish national population registries (Chang et al. 2014; Kuja-Halkola et al. 2014; Latvala et al. 2015). In the present study we include one intergenerational shared- environment path and explore the impact that significant extended family environmental effects may have on CoT models. We believe that now is a good time to revisit and explore extensions to these models for two reasons. First, inter- generational datasets now exist that are appropriate for the application of CoT models, but that are also larger and more complex than previous datasets (e.g. registry data, ITOR). Second, many on-going twin studies have participants who are in, or are entering, adulthood. When these participants begin having children, such datasets will have the potential to teach us a great deal about the nature of intergenerational transmission. It is therefore worthwhile exploring how best to model these associations, and how best to collect data for this purpose. Abstract Edited by Brian D’Onofrio. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10519-018-9912-4) contains supplementary material, which is available to authorized users. * Tom A. McAdams tom.mcadams@kcl.ac.uk 1 Social, Genetic & Developmental Psychiatry Centre, Institute of Psychiatry, Psychology & Neuroscience, King’s College, De Crespigny Park, Box PO80, SE5 8AF London, UK 2 Department of Mental Disorders, Norwegian Institute of Public Health, Oslo, Norway 3 Department of Psychology, University of Oslo, Oslo, Norway 4 School of Pharmacy, University of Oslo, Oslo, Norway Edited by Brian D’Onofrio. Edited by Brian D’Onofrio. Edited by Brian D’Onofrio. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10519-018-9912-4) contains supplementary material, which is available to authorized users. * Tom A. McAdams tom.mcadams@kcl.ac.uk 1 Social, Genetic & Developmental Psychiatry Centre, Institute of Psychiatry, Psychology & Neuroscience, King’s College, De Crespigny Park, Box PO80, SE5 8AF London, UK While twins with children have most often been the focus of genetically informed intergenerational studies, it is also 2 Department of Mental Disorders, Norwegian Institute of Public Health, Oslo, Norway 3 Department of Psychology, University of Oslo, Oslo, Norway 4 School of Pharmacy, University of Oslo, Oslo, Norway (0121 3456789) 3 3456789) 3 Behavior Genetics (2018) 48:397–412 398 Study in Sweden (TOSS) (Eley et al. 2015; McAdams et al. 2015, 2017). TOSS is a study of adult twins and their ado- lescent children (Neiderhiser and Lichtenstein 2008). The TOSS sample comprises data collected on one child per twin, these children being cousins to one another. The model in Fig. 1a decomposes variance on the parental (twin) pheno- type into additive genetic influences (A1; MZ twins share all genetic influences, DZ twins share half), common or shared environmental influences (C1; environmental influences shared between MZ and DZ twins alike), and non-shared or unique environmental influences (E1; environmental influ- ences that make twins different to one another). Variance in the offspring phenotype is decomposed into genetic influ- ences shared in common with the parental phenotype (A1′), genetic influences specific to the offspring phenotype (A2), and non-shared environmental influences (E2). A path also runs from parent phenotype to offspring phenotype (p). Par- ent–child covariance is therefore explained by a combination of p, a1′ and a1. Genetic transmission is modelled via the a1 and a1′ path, and a path fixed to 0.50. This is because par- ents pass on half of their DNA to their children. Multiple‑children‑of‑twins structural equation models with one another so share, on average, 25% of their genetic variance (where their parents are MZ twins), or 12.5% of their genetic variance (where their parents are DZ twins). These relatedness coefficients (0.25 and 0.125), and the dif- ference between them, are lower than those of twins (1.00 and 0.50) and result in considerably less power to detect genetic effects unique to the offspring generation than there is for the parent generation. Similarly, the estimation of A1′ (genetic effects shared between parent and child) relies upon comparisons between MZ and DZ avuncular (aunt/uncle-to- niece/nephew) relationships, comprising relatedness coeffi- cients of 0.50 and 0.25, so again, there is less power to detect these paths than there is to detect e.g. a genetic correlation between two phenotypes in the parent generation. Because cousins do not share a nuclear family environment it is also not possible to estimate the role of environmental effects shared by siblings in the offspring generation (C2). with one another so share, on average, 25% of their genetic variance (where their parents are MZ twins), or 12.5% of their genetic variance (where their parents are DZ twins). These relatedness coefficients (0.25 and 0.125), and the dif- ference between them, are lower than those of twins (1.00 and 0.50) and result in considerably less power to detect genetic effects unique to the offspring generation than there is for the parent generation. Similarly, the estimation of A1′ (genetic effects shared between parent and child) relies upon comparisons between MZ and DZ avuncular (aunt/uncle-to- niece/nephew) relationships, comprising relatedness coeffi- cients of 0.50 and 0.25, so again, there is less power to detect these paths than there is to detect e.g. a genetic correlation between two phenotypes in the parent generation. Because cousins do not share a nuclear family environment it is also not possible to estimate the role of environmental effects shared by siblings in the offspring generation (C2). In Fig. 2 we present an extension of the CoT model in Fig. 1b that allows for the inclusion of 2 or more children per parent—the multiple-children-of-twins (MCoT) model.2 In this model, the inclusion of siblings in the offspring gen- eration provides sibling covariances with which to estimate the influence of the shared environment on the offspring phenotype (C2). Siblings also share more genetic variance than cousins, increasing power to detect genetic effects in the offspring generation. 2 In the figures and examples we provide, our MCoT models include two children per parent. However, it is perfectly possible to include three or more offspring per parent by simply extending the accompa- nying matrices (see OpenMx scripts for more details). Children of twins structural equation models Note: A1 = additive genetic effects on parental phenotype; C1 = shared- environmental effects on parental phenotype; E1 = nonshared envi- ronmental effects on parental phenotype; A1′ = genetic effects com- mon to parental phenotype and offspring phenotype; C1′ = extended family effects whereby the shared environment of the parents influ- ences offspring phjenotype; A2 = familial effects specific to offspring phenotype; C2 = shared-environmental effects on offspring pheno- type (not estimable using cousin data); E2 = nonshared environmen- Fig. 1 Children-of-twins structural equation models—for use with samples comprising twin pairs with a single child per twin. Note: A1 = additive genetic effects on parental phenotype; C1 = shared- environmental effects on parental phenotype; E1 = nonshared envi- ronmental effects on parental phenotype; A1′ = genetic effects com- mon to parental phenotype and offspring phenotype; C1′ = extended family effects whereby the shared environment of the parents influ- ences offspring phjenotype; A2 = familial effects specific to offspring phenotype; C2 = shared-environmental effects on offspring pheno- type (not estimable using cousin data); E2 = nonshared environmen- tal effects on offspring phenotype; p = phenotypic effect of parent on offspring; rE = within-parent correlation between E1 for parenting of child 1 and 2. Allows parenting of each child to differ (when neces- sary this should be allowed to vary according to offspring zygosity). NB the pathway between A1 and A1′ is fixed to 0.50 because parents and children share 50% of their genome. To avoid over complicating path diagrams, variance paths have been omitted, but for all latent factors variance = 1. For A1′ this means that residual variance (after accounting for the path between A1 and A1′) is 0.75 Children of twins structural equation models Several CoT SEMs have previously been published. In Fig. 1a, we present a path diagram of the model applied in several published articles to data from the Twin Offspring A key limitation of the models presented in Fig. 1a, b is that they have low power to accurately estimate offspring aetiology. This is because the offspring of twins are cousins 3 399 Behavior Genetics (2018) 48:397–412 Fig. 1 Children-of-twins structural equation models—for use with samples comprising twin pairs with a single child per twin. Note: A1 = additive genetic effects on parental phenotype; C1 = shared- environmental effects on parental phenotype; E1 = nonshared envi- ronmental effects on parental phenotype; A1′ = genetic effects com- mon to parental phenotype and offspring phenotype; C1′ = extended family effects whereby the shared environment of the parents influ- ences offspring phjenotype; A2 = familial effects specific to offspring phenotype; C2 = shared-environmental effects on offspring pheno- type (not estimable using cousin data); E2 = nonshared environmen- tal effects on offspring phenotype; p = phenotypic effect of parent on offspring; rE = within-parent correlation between E1 for parenting of child 1 and 2. Allows parenting of each child to differ (when neces- sary this should be allowed to vary according to offspring zygosity). NB the pathway between A1 and A1′ is fixed to 0.50 because parents and children share 50% of their genome. To avoid over complicating path diagrams, variance paths have been omitted, but for all latent factors variance = 1. For A1′ this means that residual variance (after accounting for the path between A1 and A1′) is 0.75 Fig. 1 Children-of-twins structural equation models—for use with samples comprising twin pairs with a single child per twin. Note: A1 = additive genetic effects on parental phenotype; C1 = shared- environmental effects on parental phenotype; E1 = nonshared envi- ronmental effects on parental phenotype; A1′ = genetic effects com- mon to parental phenotype and offspring phenotype; C1′ = extended family effects whereby the shared environment of the parents influ- ences offspring phjenotype; A2 = familial effects specific to offspring phenotype; C2 = shared-environmental effects on offspring pheno- type (not estimable using cousin data); E2 = nonshared environmen- Fig. 1 Children-of-twins structural equation models—for use with samples comprising twin pairs with a single child per twin. Multiple‑children‑of‑twins structural equation models Including two or more children per parent also provides much more information with which to estimate offspring aetiology: In the MCoT model with two offspring there are two sibling covariances and four cousin covariances per family, compared to one cousin covariance in the standard CoT model. Similarly, the MCoT model 1 3 400 Behavior Genetics (2018) 48:397–412 Fig. 2 Multiple-children-of-twins structural equation model. Parent phenotype is invariant across offspring (MCoT inv) Note: A1=addi offspring phenotype; p = phenotypic effect of parent on offspring rE=within parent correlation between E1 for parenting of child Fig. 2 Multiple-children-of-twins structural equation model. Parent phenotype is invariant across offspring (MCoT-inv). Note: A1 = addi- tive genetic effects on parental phenotype; C1 = shared-environmental effects on parental phenotype; E1 = nonshared environmental effects on parental phenotype; A1′ = genetic effects common to parental phenotype and offspring phenotype; C1′ = extended family effects whereby the shared environment of the parents influences offspring phjenotype; A2 = familial effects specific to offspring phenotype; C2 = shared-environmental effects on offspring phenotype (not esti- mable using cousin data); E2 = nonshared environmental effects on offspring phenotype; p = phenotypic effect of parent on offspring; rE = within-parent correlation between E1 for parenting of child 1 and 2. Allows parenting of each child to differ (when necessary this should be allowed to vary according to offspring zygosity). NB the pathway between A1 and A1′ is fixed to 0.50 because parents and children share 50% of their genome. To avoid over complicating path diagrams, variance paths have been omitted, but for all latent factors variance = 1. For A1′ this means that residual variance (after account- ing for the path between A1 and A1′) is 0.75 Fig. 2 Multiple-children-of-twins structural equation model. Parent phenotype is invariant across offspring (MCoT-inv). Note: A1 = addi- tive genetic effects on parental phenotype; C1 = shared-environmental effects on parental phenotype; E1 = nonshared environmental effects on parental phenotype; A1′ = genetic effects common to parental phenotype and offspring phenotype; C1′ = extended family effects whereby the shared environment of the parents influences offspring phjenotype; A2 = familial effects specific to offspring phenotype; C2 = shared-environmental effects on offspring phenotype (not esti- mable using cousin data); E2 = nonshared environmental effects on delinquency). However, many parental phenotypes have the potential to vary between children within the same family (e.g. 3 Application to simulated data: the power to distinguish routes of intergenerational transmission The CoT models we present are typically used to assess the extent to which an intergenerational correlation of inter- est may be attributable to genetic relatedness, and whether an association remains after accounting for relatedness. As discussed, they can also be used to assess the role of the extended family environment, but first we focus on the distinction between genetic vs. phenotypic transmission in the absence of extended family effects. The ability of the model to accurately distinguish phenotypic transmission (i.e. transmission of a social or other nature not involving the passing of genes from parent to child) from genetic trans- mission is dependent upon the power to detect each of these pathways. For phenotypic transmission, this means the p path. For genetic transmission, this involves genetic effects in the parent generation (a1) and the transmission of these effects to the next generation (a1′). Of all these paths (p, a1, a1′), there is least power to detect a1′. Power to detect the a1′ pathway is dependent upon many factors, includ- ing the magnitude of the parent-offspring correlation; the proportion of the correlation that is attributable to genetic overlap; the size of the avuncular relatedness coefficients (0.50 and 0.25 for families in which parents are MZ and DZ twins respectively); the size of the sample; the size of the families within the sample, the MZ:DZ ratio; and the herit- ability of the phenotypes. In the following power analyses we use simulated data to first demonstrate how the power to detect a1′ is increased by increasing the number of offspring included per parent. We then introduce shared environmental effects into parent and child phenotypes, and explore models including significant extended family effects (c1′) as these are often omitted from CoT models. While previous studies have not included multiple off- spring per twin parent in CoT models, some researchers (Hannigan et al. 2016; Silberg et al. 2010, 2012) have taken another approach to increase their ability to accurately esti- mate the aetiology of the offspring phenotype, by combin- ing a CoT dataset with data from a children-as-twins (CaT) dataset. In these studies, the CaT dataset comprised data on twins who were of the same age group as the offspring in the CoT dataset, collected using the same instrument. Multiple‑children‑of‑twins structural equation models This does not affect the estimation of A, C or E and does not involve as many assumptions, or require the estimation of as many additional paths, as the Cholesky decomposition. Where the offspring generation comprises multiple zygosities (MZ, DZ, sibs etc.), and where the within-person correlation on the parent phenotype is known to differ by child zygosity (e.g. parenting and any other phenotypes where child-driven evocative rGE is present), a separate rE correlation should be estimated for each zygosity to ensure that estimates are unbiased. Yet another alternative specification would be to include an additional latent factor (N for nuclear family)3 to capture the within-person correlation. This would have the benefit of distinguishing between the unique environmental component that makes parents’ behaviour consistent for each of their children (N) vs. the unique environmental compo- nent that makes their behaviour different for each child (E1). Such an approach could be useful if the parent phenotype varies between offspring because it was assessed at different time points. However, it would not be appropriate for data in which child zygosity predicts within-person differences in parent phenotype. child phenotype, they provide no direct information on the nature of parent–child covariance. As such, wherever possi- ble we would advise researchers who are considering setting up a CoT/CoS study to collect information on multiple chil- dren per parent, as doing so will provide additional sources of covariance with which to decompose intergenerational associations. That said, the use of a CaT dataset does offer obvious advantages in terms of increased power to estimate genetic and environmental effects in the offspring genera- tion, and avoids complications of age difference between sib- lings/cousins attenuating the estimates if they change with age. It is worth noting here that DZ twinning is heritable, so MCoT studies will generally have the advantage of including twins in both generations. 3 Credit goes to an anonymous reviewer for this suggestion. Multiple‑children‑of‑twins structural equation models aetio- logical differences in the parent phenotype for the first vs. second child where data is sorted by birth-order. However, for most situations we advocate a third option, presented in Fig. 3 (MCoT-var). Here, the within-person association is estimated as a combination of A, C, and E, where the correlation between E for e.g. the parenting of child 1 and child 2, is freely estimated (see rE in Fig. 3), thus allow- ing for differences between the two phenotypes. This does not affect the estimation of A, C or E and does not involve as many assumptions, or require the estimation of as many additional paths, as the Cholesky decomposition. Where the offspring generation comprises multiple zygosities (MZ, DZ, sibs etc.), and where the within-person correlation on the parent phenotype is known to differ by child zygosity (e.g. parenting and any other phenotypes where child-driven evocative rGE is present), a separate rE correlation should be estimated for each zygosity to ensure that estimates are unbiased. Yet another alternative specification would be to include an additional latent factor (N for nuclear family)3 to capture the within-person correlation. This would have the benefit of distinguishing between the unique environmental component that makes parents’ behaviour consistent for each of their children (N) vs. the unique environmental compo- nent that makes their behaviour different for each child (E1). Such an approach could be useful if the parent phenotype varies between offspring because it was assessed at different time points. However, it would not be appropriate for data in which child zygosity predicts within-person differences in parent phenotype. thus brings within-person correlations into the estimation of A, C and E. Another alternative would be to specify the within-person correlation as a Cholesky decomposition, thus freely estimating the association between A, C and E on the first parent phenotype and the second. This may be of use to researchers interested in the effects of parity, e.g. aetio- logical differences in the parent phenotype for the first vs. second child where data is sorted by birth-order. However, for most situations we advocate a third option, presented in Fig. 3 (MCoT-var). Here, the within-person association is estimated as a combination of A, C, and E, where the correlation between E for e.g. the parenting of child 1 and child 2, is freely estimated (see rE in Fig. 3), thus allow- ing for differences between the two phenotypes. Multiple‑children‑of‑twins structural equation models many parenting phenotypes, psychopathology meas- ured at different time points). As such, the MCoT model in Fig. 2 (hereon referred to as MCoT-inv) must be further extended to deal with parent phenotypes that are variant across offspring. with two offspring includes twice as many intergenerational covariances per family (parent-offspring and avuncular) as the CoT model. As such, the MCoT model should have more power to decompose intergenerational associations into a1′, c1′ and p, and to estimate offspring aetiology (see Table 1 for actual power calculations, introduced below). It is worth noting here that in the MCoT model, offspring correlate at 0.25 on the A1′ factor. This is because although both chil- dren are equally related to their parent, each child shares a random 50% of their genes with their parent. Importantly, where siblings in the offspring generation are MZ twins they will correlate at 1 on the A1′ factor because they share all of their genes, including the 50% shared with their parent. Such data will need to be modelled accordingly. f In extending the MCoT model for use with parent pheno- types that vary between offspring, it is important to consider how to specify the ‘within-person’ correlation that occurs between, for example, Twin 1’s parenting of their first and second child. In some CoT models (Narusyte et al. 2008) such correlations have been specified as A1 + C1, meaning that differences in e.g. the parenting of child 1 and parenting of child 2 are explained by unique environmental influences (E1). This specification is straightforward and makes a cer- tain amount of sense (i.e. it is the same parent). However, it also has the undesirable consequence of constraining within- person correlations to be the same as MZ correlations, and The MCoT model presented in Fig. 2 will be useful to researchers interested in associations between parent and offspring phenotypes where the parental phenotype in ques- tion is invariant across children (e.g., certain diagnoses, or ‘historic’ parental phenotypes such as history of juvenile 1 3 Behavior Genetics (2018) 48:397–412 401 thus brings within-person correlations into the estimation of A, C and E. Another alternative would be to specify the within-person correlation as a Cholesky decomposition, thus freely estimating the association between A, C and E on the first parent phenotype and the second. This may be of use to researchers interested in the effects of parity, e.g. Application to simulated data: the power to distinguish routes of intergenerational transmission Note: A1 = addi- tive genetic effects on parental phenotype; C1 = shared-environmental effects on parental phenotype; E1 = nonshared environmental effects on parental phenotype; A1′ = genetic effects common to parental phenotype and offspring phenotype; C1′ = extended family effects whereby the shared environment of the parents influences offspring phjenotype; A2 = familial effects specific to offspring phenotype; C2 = shared-environmental effects on offspring phenotype (not esti- mable using cousin data); E2 = nonshared environmental effects on first few power analyses (1-1a, 2-2b, and 3-3b) in Table 1 we specify this association to be 40% attributable to genetic transmission, and 60% attributable to phenotypic transmis- sion. That is, although this association is primarily pheno- typic, it would be useful for researchers to be able to identify that over one-third of the association is genetic in origin. intergenerational associations are attributable to genetic and phenotypic effects (models 1-1a, 2-2b, and 3-3b in Table 1); genetic, extended family and phenotypic effects (models 1c, 2c, and 3c); and to extended family and phenotypic effects (models 1d, 2d, and 3d). We present these power analyses with the caveat that in complex SEMs, varying the magni- tude of any path can have an impact on the power to detect any other path, so interpretation is not straightforward. We therefore present these analyses simply as an aid to under- stand how the power to detect the paths of intergenerational transmission (a1′, c1′, p) varies for each of our models. Scripts for running all of the models and power analyses presented in this paper are provided via the supplementary materials. Scripts are designed to simulate data according to given model parameters, so as well as exploring issues of power, readers can explore the covariance structure that might be expected in particular scenarios. In Table 1 it can be seen that power to detect a1′ was lowest in the 4 variable CoT model (model 1a: one-child- per-twin), was greater in the six variable MCoT-inv model (model 2a: two-children-per-twin with invariant parent phenotype), and greater still in the 8 variable MCoT-var model (model 3a: two-children-per-twin with variant par- ent phenotype). To achieve 80% power to detect a1′ in our example required 947 twin families (twin pairs with chil- dren) if using the CoT model, but only 551 when using the MCoT-inv model, and 469 families when using the MCoT- var model. Application to simulated data: the power to distinguish routes of intergenerational transmission The introduction of the CaT dataset increases power to detect genetic effects in the offspring generation (A2), and allows shared environmental effects to be estimated (C2). A CaT dataset also has the advantage that twins are always the same age as one another, whereas cousins are often not. However, the children-as-twins data inform only on the variance of the In Table 1 we present the results of power analyses of the models presented in Figs. 1, 2 and 3 in scenarios in which 1 402 Behavior Genetics (2018) 48:397–412 Fig. 3 Multiple-children-of-twins structural equation model. Parent phenotype is variant across offspring (MCoT-var). Note: A1 = addi- tive genetic effects on parental phenotype; C1 = shared-environmental effects on parental phenotype; E1 = nonshared environmental effects on parental phenotype; A1′ = genetic effects common to parental phenotype and offspring phenotype; C1′ = extended family effects whereby the shared environment of the parents influences offspring phjenotype; A2 = familial effects specific to offspring phenotype; C2 = shared-environmental effects on offspring phenotype (not esti- mable using cousin data); E2 = nonshared environmental effects on offspring phenotype; p = phenotypic effect of parent on offspring; rE = within-parent correlation between E1 for parenting of child 1 and 2. Allows parenting of each child to differ (when necessary this should be allowed to vary according to offspring zygosity). NB the pathway between A1 and A1′ is fixed to 0.50 because parents and children share 50% of their genome. To avoid over complicating path diagrams, variance paths have been omitted, but for all latent factors variance = 1. For A1′ this means that residual variance (after account- ing for the path between A1 and A1′) is 0.75 offspring phenotype; p = phenotypic effect of parent on offspring; rE = within-parent correlation between E1 for parenting of child 1 and 2. Allows parenting of each child to differ (when necessary this should be allowed to vary according to offspring zygosity). NB the pathway between A1 and A1′ is fixed to 0.50 because parents and children share 50% of their genome. To avoid over complicating path diagrams, variance paths have been omitted, but for all latent factors variance = 1. For A1′ this means that residual variance (after account- ing for the path between A1 and A1′) is 0.75 Fig. 3 Multiple-children-of-twins structural equation model. Parent phenotype is variant across offspring (MCoT-var). 3 Application to simulated data: the power to distinguish routes of intergenerational transmission Of course, there are more individuals per family in the MCoT-inv model than the CoT model, but still fewer were required in total (3306 vs. 3788). It is also worth con- sidering that asking twin parents enrolled in a study to pro- vide data on 2+ of their children is likely to be less challeng- ing for researchers than is the recruitment of entirely new In all power analyses we simulated data comprising a sample of 1000 twin pairs (40% MZ) with children. We have tried to simulate a scenario that we believe to be representa- tive of a real-world situation—two moderately heritable traits with an intergenerational correlation of 0.35. In the 1 3 Behavior Genetics (2018) 48:397–412 403 1 Table 1 Results from power analyses exploring power to detect phenotypic (P) genetic (A1′), and family environmental (C1′) intergenerational pathways in CoT, MCoT-inv and MCoT-var mod- els Above we test for the power to detect A1′, C1′ and p pathways using data simulated to fit particular data structures. In each scenario the correlation between parent and child phenotype is 0.35. In models 1-1a, 2-2b, and 3-3b, models are specified such that the parent–child correlation is 40% attributable to genetic relatedness and 60% attributable to phenotypic exposure. In models 1c, 2c, and 3c models are specified such that the parent–child correlation is 40% attributable to the extended family environment and 60% attributable to phenotypic exposure. In models 1d, 2d, and 3d the parent–child correlation is 40% attributable to genetic relatedness, 40% attributable to the extended family environment and 20% attributable to phenotypic exposure. Datasets simulated comprised 1000 complete twin pairs in which each twin had one child in models 1–1d, and two children in all other models. Data is simulated such that 40% of twin pairs are monozygotic. Par- ent and child phenotypes were normally distributed. All models converged on an accurate solution and all expected variance–covariance matrices mirrored those of the simulated data. As well as giving power in our simulated sample we also give the required N in terms of families (and individuals) necessary to achieve 80% power to detect A1′, p and C1′. NB values given for latent factors (A1, C1, etc.) are variance components, whereas the value given for p is a standardised path coefficient (beta). Application to simulated data: the power to distinguish routes of intergenerational transmission The effects of A1′, C1′ and p on child phenotype are given as direct effects only and do not include indirect effects e.g. via the path c1′c1p. See OpenMx scripts for more details Model A1 C1 E1 A2 C2 E2 A1′ C1′ p Power A1′ Req. N A1′ Power C1′ Req. N C1′ Power p Req. N p 1 CoT with single child per twin 0.50 0.00 0.50 0.33 – 0.41 0.16 – 0.21 0.82 947 (3788) – – 0.98 487 (1948) 1a CoT with single child per twin. Includes parental C. No C1′ 0.50 0.20 0.30 0.33 – 0.41 0.16 – 0.21 0.47 2188 (8752) – – 0.79 1029 (4116) 1c CoT with single child per twin. Includes parental C and C1′ 0.50 0.20 0.30 0.33 – 0.39 0.16 0.10 0.07 0.40 2697 (10,788) 0.79 1028 (6168) 0.16 8653 (34,612) 1d CoT with single child per twin. Includes parental C and C1′. No A1′ 0.50 0.20 0.30 0.33 – 0.47 – 0.10 0.21 – – 0.98 512 (3072) > 0.99 237 (948) 2 MCoT-inv. Two children per twin. Invariant parent phenotype 0.50 0.00 0.50 0.33 0.00 0.41 0.16 – 0.21 0.97 551 (3306) – – > 0.99 276 (1656) 2a MCoT-inv. Includes parental C. No C1′ 0.50 0.20 0.30 0.33 0.00 0.41 0.16 – 0.21 0.71 1232 (7392) – – 0.96 572 (3432) 2b MCoT-inv with two children per twin. Invariant parent phenotype. Includes child C 0.50 0.00 0.50 0.33 0.20 0.21 0.16 – 0.21 0.93 665 (3990) – – 0.98 492 (2952) 2c MCoT-inv. Includes parental C and C1′ 0.50 0.20 0.30 0.33 0.00 0.39 0.16 0.10 0.07 0.64 1452 (8712) 0.94 650 (3900) 0.25 4657 (27,942) 2d MCoT-inv. Includes parental C and C1′. No A1′ 0.50 0.20 0.30 0.33 0.00 0.47 – 0.10 0.21 – – > 0.99 253 (1518) > 0.99 144 (864) 3 MCoT-var with two children per twin. Variant par- ent phenotype 0.50 0.00 0.50 0.33 0.00 0.41 0.16 – 0.21 0.98 469 (2814) – – > 0.99 237 (1422) 3a MCoT-var. Includes parental C. No C1′ 0.50 0.20 0.30 0.33 0.00 0.41 0.16 – 0.21 0.80 1001 (6006) – – > 0.99 337 (2022) 3b MCoT-var with two children per twin. Variant par- ent phenotype. Includes child C 0.50 0.00 0.50 0.33 0.20 0.21 0.16 – 0.21 0.97 512 (3072) – – > 0.99 222 (1332) 3c MCoT-var with two children per twin. Application to simulated data: the power to distinguish routes of intergenerational transmission Variant par- ent phenotype. Includes parental C and C1′ 0.50 0.20 0.30 0.33 0.00 0.39 0.16 0.10 0.07 0.72 1218 (7308) 0.94 629 (3774) 0.30 3866 (23,196) 3d MCoT-var with two children per twin. Variant parent phenotype. Includes parental C and C1′. No A1′ 0.50 0.20 0.30 0.33 0.00 0.47 – 0.10 0.21 – – > 0.99 242 (1452) > 0.99 123 (738) Results from power analyses exploring power to detect phenotypic (P) genetic (A1′), and family environmental (C1′) intergenerational pathways in CoT, MCoT-inv and MCoT-var mod- A1 C1 E1 A2 C2 E2 A1′ C1′ p Power A1′ Req. N A1′ Power C1′ Req. N C1′ Power p Req. N p with single child per twin 0.50 0.00 0.50 0.33 – 0.41 0.16 – 0.21 0.82 947 (3788) – – 0.98 487 (1948) T with single child per twin. Includes parental No C1′ 0.50 0.20 0.30 0.33 – 0.41 0.16 – 0.21 0.47 2188 (8752) – – 0.79 1029 (4116) T with single child per twin. Includes parental and C1′ 0.50 0.20 0.30 0.33 – 0.39 0.16 0.10 0.07 0.40 2697 (10,788) 0.79 1028 (6168) 0.16 8653 (34,612) T with single child per twin. Includes parental C d C1′. No A1′ 0.50 0.20 0.30 0.33 – 0.47 – 0.10 0.21 – – 0.98 512 (3072) > 0.99 237 (948) oT-inv. Two children per twin. Invariant parent henotype 0.50 0.00 0.50 0.33 0.00 0.41 0.16 – 0.21 0.97 551 (3306) – – > 0.99 276 (1656) oT-inv. Includes parental C. No C1′ 0.50 0.20 0.30 0.33 0.00 0.41 0.16 – 0.21 0.71 1232 (7392) – – 0.96 572 (3432) oT-inv with two children per twin. Invariant rent phenotype. Includes child C 0.50 0.00 0.50 0.33 0.20 0.21 0.16 – 0.21 0.93 665 (3990) – – 0.98 492 (2952) oT-inv. Includes parental C and C1′ 0.50 0.20 0.30 0.33 0.00 0.39 0.16 0.10 0.07 0.64 1452 (8712) 0.94 650 (3900) 0.25 4657 (27,942) oT-inv. Includes parental C and C1′. No A1′ 0.50 0.20 0.30 0.33 0.00 0.47 – 0.10 0.21 – – > 0.99 253 (1518) > 0.99 144 (864) oT-var with two children per twin. Variant par- t phenotype 0.50 0.00 0.50 0.33 0.00 0.41 0.16 – 0.21 0.98 469 (2814) – – > 0.99 237 (1422) oT-var. Includes parental C. Application to simulated data: the power to distinguish routes of intergenerational transmission No A1 0.50 0.20 0.30 0.33 0.00 0.47 0.10 0.21 > 0.99 253 (1518) > 0.99 144 (864) 3 MCoT-var with two children per twin. Variant par- ent phenotype 0.50 0.00 0.50 0.33 0.00 0.41 0.16 – 0.21 0.98 469 (2814) – – > 0.99 237 (1422) 3a MCoT-var. Includes parental C. No C1′ 0.50 0.20 0.30 0.33 0.00 0.41 0.16 – 0.21 0.80 1001 (6006) – – > 0.99 337 (2022) 3b MCoT-var with two children per twin. Variant par- ent phenotype. Includes child C 0.50 0.00 0.50 0.33 0.20 0.21 0.16 – 0.21 0.97 512 (3072) – – > 0.99 222 (1332) 3c MCoT-var with two children per twin. Variant par- ent phenotype. Includes parental C and C1′ 0.50 0.20 0.30 0.33 0.00 0.39 0.16 0.10 0.07 0.72 1218 (7308) 0.94 629 (3774) 0.30 3866 (23,196) 3d MCoT-var with two children per twin. Variant parent phenotype. Includes parental C and C1′. No A1′ 0.50 0.20 0.30 0.33 0.00 0.47 – 0.10 0.21 – – > 0.99 242 (1452) > 0.99 123 (738) Above we test for the power to detect A1′, C1′ and p pathways using data simulated to fit particular data structures. In each scenario the correlation between parent and child phenotype is 0.35. In models 1-1a, 2-2b, and 3-3b, models are specified such that the parent–child correlation is 40% attributable to genetic relatedness and 60% attributable to phenotypic exposure. In models 1c, 2c, and 3c models are specified such that the parent–child correlation is 40% attributable to the extended family environment and 60% attributable to phenotypic exposure. In models 1d, 2d, and 3d the parent–child correlation is 40% attributable to genetic relatedness, 40% attributable to the extended family environment and 20% attributable to phenotypic exposure. Datasets simulated comprised 1000 complete twin pairs in which each twin had one child in models 1–1d, and two children in all other models. Data is simulated such that 40% of twin pairs are monozygotic. Par- ent and child phenotypes were normally distributed. All models converged on an accurate solution and all expected variance–covariance matrices mirrored those of the simulated data. As well as giving power in our simulated sample we also give the required N in terms of families (and individuals) necessary to achieve 80% power to detect A1′, p and C1′. Application to simulated data: the power to distinguish routes of intergenerational transmission No C1′ 0.50 0.20 0.30 0.33 0.00 0.41 0.16 – 0.21 0.80 1001 (6006) – – > 0.99 337 (2022) oT-var with two children per twin. Variant par- t phenotype. Includes child C 0.50 0.00 0.50 0.33 0.20 0.21 0.16 – 0.21 0.97 512 (3072) – – > 0.99 222 (1332) oT-var with two children per twin. Variant par- t phenotype. Includes parental C and C1′ 0.50 0.20 0.30 0.33 0.00 0.39 0.16 0.10 0.07 0.72 1218 (7308) 0.94 629 (3774) 0.30 3866 (23,196) oT-var with two children per twin. Variant arent phenotype. Includes parental C and C1′. 0.50 0.20 0.30 0.33 0.00 0.47 – 0.10 0.21 – – > 0.99 242 (1452) > 0.99 123 (738) Above we test for the power to detect A1′, C1′ and p pathways using data simulated to fit particular data structures. In each scenario the correlation between parent and child phenotype is 0.35. In models 1-1a, 2-2b, and 3-3b, models are specified such that the parent–child correlation is 40% attributable to genetic relatedness and 60% attributable to phenotypic exposure. In models 1c, 2c, and 3c models are specified such that the parent–child correlation is 40% attributable to the extended family environment and 60% attributable to phenotypic exposure. In models 1d, 2d, and 3d the parent–child correlation is 40% attributable to genetic relatedness, 40% attributable to the extended family environment and 20% attributable to phenotypic exposure. Datasets simulated comprised 1000 complete twin pairs in which each twin had one child in models 1–1d, and two children in all other models. Data is simulated such that 40% of twin pairs are monozygotic. Par- ent and child phenotypes were normally distributed. All models converged on an accurate solution and all expected variance–covariance matrices mirrored those of the simulated data. As well as giving power in our simulated sample we also give the required N in terms of families (and individuals) necessary to achieve 80% power to detect A1′, p and C1′. NB values given for latent factors (A1, C1, etc.) are variance components, whereas the value given for p is a standardised path coefficient (beta). The effects of A1′, C1′ and p on child phenotype are given as direct effects only and do not include indirect effects e.g. via the path c1′c1p. See OpenMx scripts for more details 2d MCoT inv. Includes parental C and C1 . Application to simulated data: the power to distinguish routes of intergenerational transmission NB values given for latent factors (A1, C1, etc.) are variance components, whereas the value given for p is a standardised path coefficient (beta). The effects of A1′, C1′ and p on child phenotype are given as direct effects only and do not include indirect effects e.g. via the path c1′c1p. See OpenMx scripts for more details No A1 1 3 404 Behavior Genetics (2018) 48:397–412 Fig. 4 Power to detect genetic transmission using children-of-twin data: Applying three different models to simulated data. Note: Data was simulated such that the parent–child phenotypic correlation was always 0.35. Datasets comprised 1000 complete twin pairs where 40% were monozygotic. Only the path a1′ was directly manipulated. Only paths a1′, p and e2 varied. Other specifications were as follows: A1 = 0.50, C1 = 0.00, E1 = 0.50, A2 = 0.33, C2 = 0.00, E2 = residual child variance a far smaller impact upon the ability to detect a1′, dem- onstrating that this effect is specific to the parental shared environment.f twin families. The MCoT-var model comprises just as many individuals as the MCoT-inv model, but includes more data (e.g., additional measurement occasions, or child-specific parenting questionnaire responses). However, the increase in power in the MCoT-var model was such that it required considerably fewer individuals than the MCoT-inv model. In Fig. 4 we illustrate the number of twin pairs required to reach 80% power to detect a1′ of varying magnitude using each of these 3 models. The numbers required decrease as genetic transmission explains an increasing proportion of the correlation between parent and child. In models 1c, 2c, and 3c we further explored the effect of the shared environment on these models by also including c1′ as a significant contributor to the parent–child associa- tion. In these simulations we specified the intergenerational association (rPh = 0.35) to be 40% attributable to genetic transmission, 40% attributable to extended family effects, and 20% attributable to phenotypic transmission. As with all of our analyses, results showed that MCoT-var had more power than MCoT-inv, which had more power than CoT. The inclusion of c1′ further reduced power to detect a1′ in all models. In order to reach 80% power to detect a1′, the CoT, MCoT-inv and MCoT-var models required 2697, 1452, and 1218 families respectively. Application to real data To further demonstrate the power gained by including mul- tiple children per family in CoT/CoS SEMs, we applied two of the above models to data from the Intergenerational Transmission of Risk (ITOR) project. The ITOR project is based on the Norwegian Mother and Child Cohort Study (MoBa) (Magnus et al. 2016). The MoBa is a prospec- tive population-based pregnancy cohort study conducted by the Norwegian Institute of Public Health. Participants were recruited from all over Norway from 1999 to 2008. The women consented to participation in 41% of the preg- nancies. The cohort now includes 114,500 children, 95,200 mothers and 75,200 fathers. The IToR project has linked data from portions of the MoBa to registry pedigrees (i.e. the Norwegian Population Registry and the Medical Birth Registry of Norway), and zygosity information (i.e. based on genotyping, questionnaires, and linkage to the Norwegian Twin Registry). These pedigrees include unique identifica- tion numbers for all grandparents, parents, and children that have been listed in the Norwegian Population Registry or Medical Birth Registry.f i For height, the results of model fitting (see Table S4 and accompanying text in supplementary materials) revealed that it was possible to drop A1′, C1′ or the p path from the CoT model, indicating that it was not possible to distinguish between the different routes of intergenerational transmis- sion in this analysis. Conversely, in the MCoT model, it was not possible to drop the A1′ path or the C1′ path, indicating that both of these routes were significant. Examination of the MCoT model confirms the importance of genetic fac- tors, with 87% of the mother–child covariance being attrib- utable to genetic transmission. These results would strongly suggest that genetic transmission is the primary reason for correlations between maternal height and the height of their 18-month-old children. For weight, model fitting results led to the same con- clusion for the CoT and MCoT analysis, whereby genetic transmission explained the covariance between mater- nal pre-pregnancy weight and that of their child at age 18 months. The results of model fitting and confidence intervals highlights the greater power of the MCoT model to detect a1′. While the participation rate of 41% in the MoBa study gives some cause for caution in interpreting these findings, we are not aware of any reason to expect the mechanisms underlying the correlation between mother and child height and weight to vary by participation. Application to simulated data: the power to distinguish routes of intergenerational transmission Power to detect c1′ was far greater than was power to detect a1′, with ~ half or fewer families required to achieve 80% power. Models 1d, 2d, and 3d (in which the intergenerational association was 40% attributable to extended family effects, and 60% attributable to phenotypic transmission) confirm that in the absence of a1′, power to detect c1′ remained very high and considerably higher than a1′ of a similar magnitude. Previously it has been noted that some CoT models have difficulty converging on a correct solution when parental shared environment is non-zero (Narusyte et al. 2008). For this reason we also ran models in which parental shared environment was non-zero (C1 = 0.20: models 1a, 2a, 3a in Table 1). In our simulations these models did converge on a correct solution. However, power to detect a1′ was reduced, with double the number of families required to achieve the same power as simulations where C1 = 0. In simulations where C1 = 0.20 the power was reduced more in the CoT model (1a) than MCoT-inv (2a), than MCoT- var (3a). A non-zero influence of shared environmental effects in the offspring generation (models 2b and 3b) had 1 3 3 Behavior Genetics (2018) 48:397–412 405 In the supplementary materials we explore the conse- quences of not modelling c1′ where it does in fact play a role in intergenerational associations (Table S1). We also explore the consequences of unmodelled dominance effects (Table S2). These analyses are discussed towards the end of this article. offspring generation. The sibling pairs in the offspring gen- eration also included MZ twin pairs (35), and half-sibling pairs (9). Parameter estimates from all analyses are displayed in Table 2. For both height and weight, the estimates of paren- tal aetiology were very similar when comparing across the CoT and MCoT models, as should be expected. However, the same was not true for the offspring aetiology, with notice- able differences in parameter estimates and their significance (i.e. confidence intervals).i Application to real data We examined the association between maternal and off- spring height and weight in ITOR. Maternal height and weight prior to pregnancy was self-reported in the 15th week of gestation, and offspring height and weight was obtained via maternal report at age 18 months. The cor- relation between maternal and child height was 0.28, for weight it was 0.20. First, we fitted a four variable (one child per mother) CoT model to the data. We then fitted MCoT models to the data, allowing for two children per mother. We chose the MCoT-inv model for the analysis of height because maternal height did not differ by offspring. To the weight data we fitted an MCoT-var model, because maternal weight did differ by offspring. Discussion In this article we have demonstrated how to expand CoT models to include multiple children per twin, and how doing so can increase power and solve some of the shortcomings of previous models. Using real data, we have shown that the inclusion of more than one offspring per parent can have a substantive impact on results, even when only a small proportion of the overall sample has more than one child in the study. For the association between mother and child height, the (one child per parent) CoT model could not distinguish genetic from phenotypic transmission. How- ever, once we included complete sib-ships in the offspring generation, our MCoT model clearly demonstrated that this association could best be understood as primarily genetic in nature. ff The sample for analysis comprised 10,610 mothers on whom we had data. These included MZ and DZ twin pairs, siblings, and half-siblings (see Supplementary Materials Table S3 for a complete sample breakdown). These indi- vidual mothers constituted 4875 complete sibling pairs, and 860 incomplete pairs (i.e. singletons). We had data on 8281 children. In the first analysis (one child per mother), 7090 of these children were included. In the second analysis (up to two children per mother), all were included. In the sec- ond analysis there were 1191 complete sibling pairs in the 1 3 3 406 Behavior Genetics (2018) 48:397–412 Table 2 Results of of CoT and MCoT analyses of the intergenerational association between maternal height and weight and the height and weight of offspring at age 18 months in the ITOR dataset A1, C1, E1, A2, C2, E2, A1′, C1′ are given as variance components, p is a path estimate. p2 will give the proportion of variance accounted for by p. Discussion The final three columns provide the propor- tion of parent–child covariance accounted for by genetic, extended family environmental, and phenotypic pathways Model A1 C1 E1 A2 C2 E2 A1′ C1′ p Prop rPh genetic Prop rPh fam env Prop rPh phenotypic Height CoT model: one-child per parent 0.86 (0.79, 0.93) 0.04 (0.00, 0.09) 0.10 (0.07, 0.13) 0.87 (0.00, 0.93) – 0.00 (0.00, 0.80) 0.04 (0.00, 1.00) 0.02 (0.00, 0.17) 0.15 (− 0.24, 0.29) 0.35 0.10 0.56 MCoT-inv model: two-chil- dren per parent 0.90 (0.81, 0.93) 0.01 (0.00, 0.07) 0.09 (0.01, 0.12) 0.55 (0.00, 0.72) 0.00 (0.00, 0.26) 0.12 (0.08, 0.20) 0.26 (0.08, 0.72) 0.06 (0.00, 0.18) 0.01 (− 0.16, 0.12) 0.87 0.11 0.02 Weight CoT model: one-child per parent 0.68 (0.62, 0.73) 0.00 (0.00, 0.01) 0.32 (0.27, 0.37) 0.34 (0.00, 0.89) – 0.39 (0.00, 0.85) 0.21 (0.00, 0.28) 0.07 (0.00, 0.11) 0.00 (0.00, 0.13) 0.98 0.02 0.00 MCoT-var model: two-chil- dren per parent 0.69 (0.64, 0.74) 0.00 (0.00, 0.02) 0.31 (0.26, 0.36) 0.49 (0.00, 0.81) 0.08 (0.00, 0.31) 0.17 (0.00, 0.57) 0.21 (0.05, 0.27) 0.04 (0.00, 0.08) 0.00 (0.00, 0.09) 0.97 0.03 0.00 1 3 3 407 Behavior Genetics (2018) 48:397–412 We believe that our extensions to existing CoT models, and findings regarding the associated increases in statisti- cal power, constitute an important step forward and will be informative to researchers as they make use of available CoT and family data. It is also our desire that this article be of use to researchers interested in collecting new data on twins and their children. Our findings emphasise the ben- efits of collecting data on multiple children per parent. It is also worth considering other ways in which researchers might get the most out of CoT data. For example, the inclu- sion and retention of childless twins and incomplete pairs in CoT databases is advisable. In typical twin studies (i.e. twins without offspring), the inclusion of incomplete pairs means that twins without a co-twin in the sample can still contribute to the estimation of means and variances, even if they do not contribute information to the estimation of twin covariance and aetiological decomposition. In a CoT study, if only one member of a twin pair has a child, including data on the childless co-twin will also allow this family to contribute to avuncular correlations. Thus, such families can also inform on the nature of intergenerational associations. Discussion This is preferable to only including families in which both twins have offspring, and will increase the generalizability of the sample (although of course the inclusion of childless co- twins may not make sense/be possible for all phenotypes). causally influences parent phenotype above and beyond effects attributable to genetic relatedness and extended fam- ily effects. (3) Parent and offspring phenotype are each asso- ciated with a third variable, not included in the model and not captured by shared genetic or extended family effects, that has a causal influence on each, thus creating/inflating their statistical association. Note that these interpretations do not entirely preclude genetic effects from playing a role in parent–child associations—parent and child phenotype can each be under genetic influence, so e.g. the genetically influ- enced behaviour of a child could have an influence on their parent’s behaviour (and/or vice versa). Such effects would constitute an evocative rGE and in the models presented would load onto the phenotypic pathway. The phenotypic pathway suggests that there may be a role for exposure—that is, after accounting for genetic relatedness, the parent pheno- type may influence child phenotype, and/or vice versa. The p path has previously been described using several different terms including ‘non-genetic’, ‘environmental’, and ‘social’. We have chosen to use the term ‘phenotypic’, as this carries fewer implications about the origin of the association. In terms of the way that the SEMs in Figs. 1, 2 and 3 are specified, the phenotypic ‘p’ pathway is a causal path. That is, it is a single-headed arrow running from parent to child. However, it is important to note that no assumptions should be made regarding the direction of causation unless the data permits (e.g. by temporal precedence). Previously, these models have been used on cross-sectional data, so any association should be interpreted as correlational—not nec- essarily indicative of a causal influence of parent on child (or indeed vice versa). The path is modelled as causal (i.e. a single headed arrow) because it is not possible in SEM to model a path between two endogenous variables as corre- lational (i.e. a double headed arrow). It is however possible to re-specify these models such that the direction of causa- tion runs from child to parent. Doing so will result in the same conclusions as using the specifications and models we present here. Discussion It is also worth highlighting that the models we present are not only suitable for use with twin data but can eas- ily be adapted for use with population or family databases, where comparisons between MZ and DZ twin families can be accompanied or replaced by comparisons between sib- lings, half-siblings, cousins, and half-cousins (e.g. as in the ITOR sample in this article). That is, comparisons between any groups of differentially related families can be used in this way. Power will be lower where relatedness coefficients are smaller, but the potential for larger sample sizes will often mitigate this. Below we attempt to expand and clarify upon the inter- pretation of the CoT models we have presented, discuss some nuances, and highlight future directions: The shared environment in CoT models The shared environment is typically described as “environ- mental effects that make members of a family similar to one another” or “environmental effects that make siblings similar to one another”. Such definitions are straightfor- ward when working with standard twin datasets, but less so with CoT and CoS datasets, where several sib-ships can be nested within nuclear families, which in turn are nested within extended families. It is possible with extended family data to specify several types of shared environment. In the above models we have included C1—environmental effects that makes siblings in the parent generation similar to one another, and C2—environmental effects that make siblings in the offspring generation similar to one another (besides those going via the p path). We have also included a c1′ path, via which extended family effects can be transmitted that can explain some or all of the correlation between par- ent and child phenotypes. Other possible shared environ- mental effects are possible, and several have been modelled in Swedish population registry data using intergenerational models similar to those we present here (Chang et al. 2014; Kuja-Halkola et al. 2014; Latvala et al. 2015). y y g Previously, when presenting findings from CoT studies we have encountered questions from some reviewers and researchers along the lines of “if children-as-twin stud- ies report that the association between, for example, par- enting and behavioural problems is partially genetic (e.g. McAdams et al. 2013), then why is the A1′ pathway often non-significant in CoT models assessing similar associa- tions?” This is a common query based on a common mis- understanding, so we thought it worthwhile outlining a response. It is important here to consider the differences in information included in CaT vs. CoT studies. In a CaT sample, if a parenting measure is found to be heritable, then this indicates that child genes influence/correlate with the parenting that they receive. Often, in these studies, child genes associated with the parenting they receive are also associated with child behaviour (McAdams et al. 2013). In a CoT sample, if a parenting phenotype is heritable, then this indicates that parent genes influence parenting. In the While working with simulated data, we found that when parental shared environment (C1) was non-zero, power to detect A1′ reduced substantially, even when the shared envi- ronment played no role in explaining the intergenerational association. This finding aligns with reports by Narusyte et al. Interpreting CoT models: the phenotypic pathway The A1′ pathway in the CoT models presented captures covariance between the parent and child phenotypes that is attributable to their genetic relatedness. That is, this path- way is intended to estimate intergenerational genetic trans- mission. It is worth noting here that in recent years several papers have included reports of non-significant A1′ path- ways where some genetic transmission might have been expected. For example, if depression is heritable, why is none of the intergenerational association between parent and child depression attributable to genetic transmission (McAdams et al. 2015; Silberg et al. 2010; Singh et al. 2011)? There are several reasons that CoT models may find The path that we have labelled ‘p’ in Figs. 1, 2, and 3 is intended to capture any association between parent and child phenotype not attributable to genetic relatedness (i.e. not captured by the genetic route of transmission; a1′) or the extended family environment (c1′). We would interpret a significant phenotypic pathway as indicative that one or more of the following is true (conditional on the power considerations discussed earlier in this article): (1) Paren- tal phenotype causally influences the offspring phenotype above and beyond effects attributable to genetic related- ness and extended family effects. (2) Offspring phenotype 1 1 3 3 408 Behavior Genetics (2018) 48:397–412 CoT models presented in the preceding Figures, where off- spring phenotype is heritable, this indicates that child genes influence offspring phenotype. So in CaT models, genetic correlations between parenting and child behaviour indicate that children’s genes influence both. In the above CoT mod- els, genetic correlations would demonstrate an association between parent genes involved in parenting, and child genes involved in child phenotype. As such, these models are not directly comparable and tell us different things about the nature of parent–child associations. It is also worth noting here that in CaT studies, the aetiology of parenting vari- ables can be greatly affected by the reporter, with parents reporting that they treat their children very equally (result- ing in large C estimates), and children reporting otherwise (resulting in far smaller C estimates). In CaT studies the heritability of parenting is calculated based on how similarly individual parents parent their MZ vs. DZ twin children. In contrast, CoT studies involve comparing how similar MZ vs. DZ twins are in parenting their own children. Interpreting CoT models: the phenotypic pathway As such, estimates of the heritability of parenting derived from CoT samples are less susceptible to reporter effects. no genetic associations where they are expected: First, the models may be underpowered. The identification of inter- generational genetic transmission in a CoT model relies upon differences between MZ avuncular correlations, and DZ avuncular correlations. The genetic relatedness coefficients in these relationships are 0.50, and 0.25 respectively. This means that statistical power is lower than, for example, when conducting multivariate genetic analyses within a generation of twins, in which correlations are decomposed using dif- ferences between MZ and DZ correlations (where related- ness coefficients are 1.00 and 0.50 respectively). We have demonstrated in this article that this is a reason to include multiple children per parent wherever possible. Second, there is perhaps less reason to expect the inter- generational associations examined to be attributable to genetic relatedness than may commonly be assumed. For example, when studying the intergenerational transmission of psychopathology, datasets often comprise adult twins with child/adolescent offspring. Given that genetic innova- tion is a common finding in the literature (i.e. the genetic architecture of traits changes across the lifespan) (e.g. Han- nigan et al. 2017), it is perhaps not surprising that genetic factors involved in psychopathology in adults do not always correlate perfectly with those involved in child or adolescent psychopathology. It is also possible that cohort effects play a role—i.e. genetic factors involved in psychopathology in one generation may not be the same as those in subsequent generations. It is known that the genetic architecture of a trait can be dependent upon the environment in which it is expressed (Rutter et al. 2006). So, given the differences in environmental experiences of e.g. somebody who grew up in the 1960s and their offspring who grew up in the 1990s, it should perhaps not be too surprising if we find that genetic overlap between parent and child is not perfect, even if we measure ostensibly the same phenotype in each generation.i Limitations and considerations As with all models, CoT and MCoT models have limita- tions that should be taken into consideration. Many of these limitations have been discussed elsewhere. For example, a CoT-specific version of the equal environments assumption (EEA) is that the children of MZ twins are not exposed to their parent’s co-twin any more than are the children of DZ twins. Although, unlike the EEA in standard twin studies, in CoT studies it is quite easy to account for this by measuring the amount of avuncular contact and including in the model (Koenig et al. 2010). Eaves et al. (2005) have discussed the “dyadic variables issue”, whereby CoT models may not work when parental phenotypes are the product of the interaction between people (e.g. divorce) rather than the characteristic of a single parent. Dominance In the models presented in Figs. 1, 2 and 3 we have not included latent factors to account for the potential effects of non-additive genetic effects, or genetic dominance. In twin models, latent factors can be added to capture effects attributable to interactions between alleles at a locus (e.g. dominance; D). Such interactions increase the similarity between siblings whenever they share the same alleles at a given locus. That is, when siblings inherit the same genes as one another from both their mother and their father. MZ twins are genetically identical, so they share all dominance genetic effects with one another. Full siblings inherit the same genes from both their mother and their father at a given locus 25% of the time on average. Thus, for MZ twins the correlation for D (rD) is 1.00, whereas for DZ twins and full siblings rD = 0.25. For other family dyads (cousins, half- siblings, parent–child etc.) rD = 0.00 because they do not share both parents. In this paper we have focussed on the use of CoT/CoS data in understanding the nature of inter- generational associations. We have therefore omitted domi- nance effects from our models because such effects are not directly transmitted from one generation to the next. This is because children only inherit one copy of each gene from each parent. It is also generally not possible using twin data to estimate the effects of dominance and the shared environ- ment at the same time. However, in the scripts provided in Other considerations involve the fact that age differ- ences between cousins and siblings in the offspring genera- tion could lead to biases in estimates of offspring aetiology where age is associated with aetiology. To counter this, studies can be designed so that data is collected only when offspring reach a particular age. Alternatively, constraints can be applied to data included in models to minimise age differences (e.g. only including relatives within 5 years of each other). It would also be possible to regress the effects of age out of phenotypes before entering into SEMs, or to include age as a covariate. While we have not explored the possibility of modelling sex differences in our models, it would be possible to do so, although we would anticipate that power considerations would mean that very large sam- ples are required to accurately identify any differences. The shared environment in CoT models (2008), that their bidirectional model had problems converging on the correct solution when parental shared environment was present. Nonetheless, as highlighted by 1 3 3 3 409 Behavior Genetics (2018) 48:397–412 Narusyte et al. (2008), this is unlikely to be a major concern for most researchers using CoT/CoS data, as a majority of parental phenotypes likely to be included in these models (parenting, personality, psychopathology) are not under the influence of shared environmental influences in adulthood. That said, it is of course possible to think of exceptions to this rule (e.g. education, SES). the supplementary materials we do allow for parent (D1) and child (D2) dominance effects for use with phenotypes in which dominance is present. In simulations included in the supplementary materials we explored the consequences of omitting significant parental dominance effects from our models. Generally, the models we present appear to deal with unmodelled dominance well, and conclusions regard- ing the nature of intergenerational associations should not be biased except for instances in which the extended family environment also plays a role (see Supplementary Materials and Table S2 for more details). We also explored models designed for those situations in which the shared environment may play a role in explaining intergenerational associations via the pathway c1′. Where genetic and extended family environment effects explained equal portions of the intergenerational association, power to detect c1′ was higher than power to detect a1′. The pres- ence of c1′ also led to a slight reduction in power to detect a1′ relative to models in which c1′ was held at zero. Impor- tantly, failing to allow for a possible role of c1′ when it did in fact play a role in explaining intergenerational associations led to an overestimation of a1′ and underestimation of p. This is an important finding given that many CoT models to date have not allowed for the possibility of an effect of the extended family environment. To ensure accurate results we would encourage researchers to include c1′ paths (or similar) in their models. If clearly non-significant then these paths can be dropped from models in order to maximise power to detect a1′. Alternative methods The models that they have used are specified differently to those we have presented in this article, in that instead of estimating a1′, c1′ and p, they esti- mate correlations between parent A and child A, parent C and child C (additional shared environmental factors are often estimated as well), and parent E and child E. They interpret a significant E correlation as indicating that an association between parent and child phenotypes remains after accounting for shared genetic and familial environ- mental effects. We don’t see any reason why the conclu- sions regarding the nature of intergenerational associations should differ dependent upon whether researchers use the models presented by Kuja-Halkola and colleagues or those we present here. Besides incorporating assortative mating into CoT mod- els, future model developments should focus on the crea- tion of multivariate CoT models. At present, CoT models are focussed on the decomposition of associations between one parent phenotype and one child phenotype. However, for many research questions, it would be informative to include additional phenotypes. For example, when studying associa- tions between parent and child psychopathology, it would be of interest to incorporate parenting behaviours or relation- ship measures that are hypothesised to mediate or moder- ate associations. It is also known that many parental mental health phenotypes are predictive of multiple outcomes in off- spring. For example, parental depression is associated with a host of negative outcomes in children (Natsuaki et al. 2014). Likewise, most child outcomes are associated with multiple predictors in the parent generation. Being able to simulta- neously model such associations would be beneficial and may aid attempts to identify key intervention targets with which to maximise impact. While it is possible to assess the direction-of-effects between parent and child phenotypes using cross-sectional CoT data (Narusyte et al. 2008), being able to make use of longitudinal CoT data should also be a priority for future model development. While the focus of this article has been on SEM tech- niques, there are of course alternative approaches that can be used with MCoT/CoS data, including a range of multi- level regression models with modelled genetic relatedness. Previously such approaches have been taken with CoT/ CoS data to explore whether associations between parent and child persist after accounting for familial confounding (D’Onofrio et al. 2005; Jundong et al. 2012; Slutske et al. 2008). Alternative methods It may also be possible to decompose covariance into genetic and environmental components using such methods (i.e. Rabe-Hesketh et al. 2008), although to our knowledge this has not been done with CoT data. Another possible way to model multiple children per twin in CoT data would be to use multi-level SEM techniques (Rabe-Hesketh et al. 2007). Such techniques combine the advantages of SEM (ability to specify/identify latent variables, and to graphically represent and explore complex relationships between many variables) with those of multilevel modelling (ability to deal with com- plex data structures, and explore relationships at different levels within a dataset). To our knowledge, such models have yet to be developed or applied to CoT/CoS data, but it is pos- sible that this will happen in the future. In summary we have demonstrated that by incorporating multiple offspring per parent, the power of CoT and chil- dren-of-sibling models can be increased. Improving such models will aid in future efforts to disentangle potential social influences from genetic transmission in the study of intergenerational effects. Understanding the nature of inter- generational transmission has the potential to inform both on our understanding of genetics and on the influence that par- ents and children have on one another. For example, where Alternative methods In the present article we have followed others (e.g. D’Onofrio et al. 2003; Keller et al. 2009; Narusyte et al. 2008, 2011; Silberg and Eaves 2004; Silberg et al. 2010, 2012) in adapting well-established biometric SEMs that have previously been applied to extended twin family data. We are hopeful that by adapting this already well-established approach to intergenerational analyses, our models will 1 3 1 410 Behavior Genetics (2018) 48:397–412 example, several extended twin-family models include the spouses of twins (Keller et al. 2009), and spouses have been included in CoT models (Silberg and Eaves 2004; Silberg et al. 2010, 2012). In many instances, it may be of impor- tance to incorporate spouses into intergenerational mod- els, as aetiological estimates may be biased by assortative mating, and it is known that assortative mating is common across physical, behavioural, and cognitive phenotypes (Nor- dsletten et al. 2016; Plomin and Deary 2015; Stulp et al. 2017). In the nuclear twin family model and stealth model, twin-spouse correlations are modelled as ‘primary assort- ment’, meaning that twins choose spouses who are pheno- typically similar to them. However, alternative explanations for twin-spouse similarity exist, including social homogamy, whereby partners are chosen based on sociocultural similar- ity; and phenotypic convergence, whereby partners become more alike over time. Assortative mating is a complex issue, the implications of which have yet to be fully explored (Heath et al. 2014; Plomin et al. 2016). In the future, it will be important to consider the impact of assortative mating on the parameter estimates obtained using CoT models. prove useful to the field. For example, previously research- ers have demonstrated how to add spouses to these models to account for the effects of assortative mating (Keller et al. 2009; Silberg and Eaves 2004; Silberg et al. 2010, 2012) so it would be possible to include spouses in the models that we have presented. However, as is always the case with complex data, it is possible to model CoT/CoS data in many different ways. Kuja-Halkola et al. (2014) have previously applied biometric SEMs to intergenerational CoS data extracted from Swedish population registries (see also Chang et al. 2014; Latvala et al. 2015). Future directions Because our focus for this paper was on the benefits of including multiple offspring in CoT models, we did not include spouses in the models that we have presented. How- ever, it is possible to incorporate spousal information. For 1 3 Behavior Genetics (2018) 48:397–412 411 researchers establish that parent and offspring phenotypes are linked genetically, then we learn something about the genetic factors involved: They persist into adulthood (if off- spring are children), they are pleiotropic (if the phenotypes are distinct), they persist across time (i.e. they associate with the same phenotype in cohorts born in different eras), and they explain a portion of familial similarity. 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Acknowledgements The first author is supported by a Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and the Royal Society (107706/Z/15/Z). Future directions During the writing of this article, the sec- ond author was supported by a 1 + 3 Ph.D. studentship from the UK Economic and Social Research Council. The Norwegian Mother and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, NIH/ NIEHS (contract no. N01-ES-75558), NIH/NINDS (Grant Nos. 1 UO1 NS 047537-01, 2 UO1 NS 047537-06A1). We are grateful to all the participating families in Norway who take part in this on-going cohort study. We would also like to thank the anonymous reviewers of this manuscript, whose suggestions were a great help in improving the work that we present here. Gottesman II, Bertelsen A (1989) Confirming unexpressed genotypes for schizophrenia—risks in the offspring of Fischers Danish identical and fraternal discordant twins. Arch Gen Psychiatry 46(10):867–872 Hannigan LJ, McAdams T, Neiderhiser J, Eley T (2016) Shared genetic influences do not explain the association between parent-child relationship quality and offspring internalizing symptoms: results from a children-of-twins study. Behav Genet 46(6):786–786 Hannigan LJ, Walaker N, Waszczuk M, McAdams T, Eley T (2017) Aetiological influences on stability and change in emotional and behavioural problems across development: a systematic review. Psychopathol Rev 4(1):52 Heath AC, Waldron MC, Martin NG, Nelson EC, Bucholz KK, Mad- den PAF (2014) Human mate selection and addiction: a concep- tual critique. Behav Genet 44(5):419–426. https://doi.org/10.1007/ s10519-014-9669-3 Compliance with ethical standards Conflict of interest Tom A. McAdams, Laurie J. Hannigan, Espen Moen Eilertsen, Line C. Gjerde, Eivind Ystrom, and Fruhling V. Rijs- dijk declare that they have no conflict of interest. Jundong J, Kuja-Halkola R, Hultman C, Langstrom N, D’Onofrio BM, Lichtenstein P (2012) Poor school performance in offspring of patients with schizophrenia: what are the mechanisms? Psychol Med 42(1):111–123. https://doi.org/10.1017/s0033291711001127 d 42(1):111–123. https://doi.org/10.1017/s0033291711001 Ethical approval The MoBa study has been granted a license from the Norwegian Data Inspectorate, and the present study was approved by the Regional Committee for Medical Research Ethics. This study accords with the ethical standards of the 1964 Helsinki declaration. Keller MC, Medland SE, Duncan LE, Hatemi PK, Neale MC, Maes HHM, Eaves LJ (2009) Modeling extended twin family data I: description of the cascade model. Twin Res Hum Genet 12(1):8–18 Koenig LB, Jacob T, Haber JR, Xian H (2010) Testing the equal envi- ronments assumption in the children of twins design. Behav Genet 40(4):533–541. https://doi.org/10.1007/s10519-010-9345-1 Informed consent Informed consent was obtained from all individual participants included in the study. Kuja-Halkola R, D’Onofrio BM, Larsson H, Lichtenstein P (2014) Maternal smoking during pregnancy and adverse outcomes in off- spring: genetic and environmental sources of covariance. Behav Genet 44(5):456–467 Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creativeco mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Latvala A, Kuja-Halkola R, Långström N, Lichtenstein P (2015) Pater- nal antisocial behavior and sons’ cognitive ability: a population- based quasiexperimental study. Psychol Sci 26(1):78–88 Magnus P, Birke C, Vejrup K, Haugan A, Alsaker E, Daltveit AK et al (2016) Cohort profile update: the Norwegian Mother and Child Cohort Study (MoBa). Int J Epidemiol 45(2):382–388. https://doi. org/10.1093/ije/dyw029 Chang Z, Lichtenstein P, D’Onofrio BM, Almqvist C, Kuja-Halkola R, Sjölander A, Larsson H (2014) Maternal age at childbirth and risk for adhd in offspring: a population-based cohort study. Int J Epidemiol 43(6):1815–1824 References McAdams TA, Gregory AM, Eley TC (2013) Genes of experience: explaining the heritability of putative environmental variables through their association with behavioural and emotional traits. Behav Genet 43(4):314–328. https://doi.org/10.1007/s1051 9-013-9591-0 Chang Z, Lichtenstein P, D’Onofrio BM, Almqvist C, Kuja-Halkola R, Sjölander A, Larsson H (2014) Maternal age at childbirth and risk for adhd in offspring: a population-based cohort study. Int J Epidemiol 43(6):1815–1824 McAdams TA, Neiderhiser JM, Rijsdijk FV, Narusyte J, Lichtenstein P, Eley TC (2014). Accounting for genetic and environmental 1 3 412 Behavior Genetics (2018) 48:397–412 confounds in associations between parent and child characteris- tics: a systematic review of children-of-twins studies. https://doi. org/10.1037/a0036416 Rabe-Hesketh S, Skrondal A, Zheng X (2007) Multilevel structural equation modeling. In: Handbook of latent variable and related models. Elsevier, Amsterdam, pp 209–227 g McAdams TA, Rijsdijk F, Neiderhiser J, Narusyte J, Shaw D, Nat- suaki M et al (2015) The relationship between parental depres- sive symptoms and offspring psychopathology: evidence from a children-of-twins study and an adoption study. Psychol Med 45(12):1–12 Rabe-Hesketh S, Skrondal A, Gjessing HK (2008) Biometrical mode- ling of twin and family data using standard mixed model software. Biometrics 64(1):280–288fi Rutter M, Moffitt TE, Caspi A (2006) Gene-environment interplay and psychopathology: multiple varieties but real effects. J Child Psychol Psychiatry 47(3–4):226–261. https://doi.org/10.111 1/j.1469-7610.2005.01557.x McAdams TA, Rijsdijk FV, Narusyte J, Ganiban JM, Reiss D, Spotts E et al (2017) Associations between the parent–child relationship and adolescent self-worth: a genetically informed study of twin parents and their adolescent children. J Child Psychol Psychiatry 58(1):46–54 Silberg JL, Eaves LJ (2004) Analysing the contributions of genes and parent-child interaction to childhood behavioural and emo- tional problems: a model for the children of twins. Psychol Med 34(2):347–356. https://doi.org/10.1017/s0033291703008948l Narusyte J, Neiderhiser JM, Andershed AK, D’onofrio BM, Reiss D, Spotts E, Ganiban J, Lichtenstein P (2011) Parental criticism and externalizing behavior problems in adolescents: the role of environment and genotype–environment correlation. J Abnorm Psychol 120(2):365 Silberg JL, Maes H, Eaves LJ (2010) Genetic and environmental influ- ences on the transmission of parental depression to children’s depression and conduct disturbance: an extended children of twins study. J Child Psychol Psychiatry 51(6):734–744. https://doi.org/ 10.1111/j.1469-7610.2010.02205.xf y ( ) Narusyte J, Neiderhiser JM, D’Onofrio BM, Reiss D, Spotts EL, Gani- ban J, Lichtenstein P (2008) Testing different types of genotype- environment correlation: an extended children-of-twins model. Dev Psychol 44(6):1591–1603. References https://doi.org/10.1037/a0013911 Silberg JL, Maes H, Eaves LJ (2012) Unraveling the effect of genes and environment in the transmission of parental antisocial behavior to children’s conduct disturbance, depression and hyperactivity. J Child Psychol Psychiatry 53(6):668–677. https://doi.org/10.111 1/j.1469-7610.2011.02494.x y p g Natsuaki MN, Shaw DS, Neiderhiser JM, Ganiban JM, Harold GT, Reiss D, Leve LD (2014) Raised by depressed parents: is it an environmental risk? Clin Child Fam Psychol Rev 17(4):357–367f Singh AL, D’Onofrio BM, Slutske WS, Turkheimer E, Emery RE, Harden KP et al (2011) Parental depression and offspring psycho- pathology: a children of twins study. Psychol Med 41(7):1385– 1395. https://doi.org/10.1017/s0033291710002059 Neiderhiser JM, Lichtenstein P (2008) The twin and offspring study in Sweden: advancing our understanding of genotype-environment interplay by studying twins and their families. Acta Psychol Sin 40:1116–1123 Slutske WS, D’Onofrio BM, Turkheimer E, Emery RE, Harden KP, Heath AC, Martin NG (2008) Searching for an environmental effect of parental alcoholism on offspring alcohol use disorder: a genetically informed study of children of alcoholics. J Abnorm Psychol 117(3):534–551. https://doi.org/10.1037/a0012907 Nordsletten AE, Larsson H, Crowley JJ, Almqvist C, Lichtenstein P, Mataix-Cols D (2016) Patterns of nonrandom mating within and across 11 major psychiatric disorders. JAMA Psychiatry 73(4):354–361. https://doi.org/10.1001/jamapsychiatry.2015.3192f Stulp G, Simons MJP, Grasman S, Pollet TV (2017). Assortative mat- ing for human height: a meta-analysis. Am J Human Biol 29(1). https://doi.org/10.1002/ajhb.22917 Plomin R, Deary IJ (2015) Genetics and intelligence differences: five special findings. 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https://openalex.org/W4234828241	https://zenodo.org/records/2479011/files/article.pdf	English	null	Mechanical Flight	Science	1,906	public-domain	1,772	.MESSRS. ORVILLE WRIGHT and Wilbur Wright, of Dayton, Ohio, under date of March 12, 1906, have addressed the following state- ment to the Aero- Club of America : "lt will be seen that an average speed of a little more than 38 miles an hour was maintained in the last flight. All of the flights were made over a circular course of about three fourths of a mile to the lap, whioh reduced the speed some- what. The maohine increased its velocity on the straight parts of the course and slowed down on the curves. It is believed that in straight flight the normal speed is more than 40 miles an hour. In the earlier of the flights named above less than 6 pounds of gasoline was carried. In the later ones a tank was fitted large enough to hold fuel for an hour, but, by oversight it was not completely filled before the flight of October 5. Though America, through the labors of Pro- fessor Lmgley, Mr. Chanute, and others, had ac- quired not less than ten years ago the recognized leadership in that branch of aeronautics which pertains to bird-like flight, it has not heretofore been possible for American workers to present a summary of each year's experiments to a society of their own country devoted exclusively to the promotion of aeronautical studies and sports. It is with great pleasure, therefore, that we now find ourselves able to make a report to such a society. "Previous to the year 1905 we had experi- mented a t Kitty Hawk, North Carolina, with man- carrying gliding machines in the years 1900, 1901, 1902 and 1903; andt with a man-carrying motor flyer, whioh, on the 17th day of December, 1903, sustained itself in the air for 59 seconds, during which time it advanced against a 20-mile wind a distance of .852 feet. Flights to the number of more than 100 had also been made a t Dayton, Ohio, in 1904, with a second motor flyer. 'NCE. It is believed that in straight flight the normal speed is more than 40 miles an hour. In the earlier of the flights named above less than 6 pounds of gasoline was carried. In the later ones a tank was fitted large enough to hold fuel for an hour, but, by oversight it was not completely filled before the flight of October 5. "Owing to frequent experimental changes in the machine and the resulting differences in its management, the earlier flights were short; but, towards the middle of September, means of cor- recting the obscure troubles were found, and the flyer was a t last brought under satisfactory con- trol. From this time forward almost every flight established a new record. In the following schedule the duration, distance and cause of stopping are given for some of the later flights. E.B. w. 'NCE. 'NCE. tion to zoology was, however, a n earlier paper (published as his doctor's dissertation) on the Spermatogenesis of A w a tristis, which formed one of the first careful studies of the history of the ' accessory chromosome ' since its discovery by Henking, and which gave impor- tant data for the general study of the repro- duction problem i n animals. H e was a good observer, a n enthusiastic field naturalist, and a master of the finer laboratory technique. H e bore with cheerful courage a malady that for many years formed an obstacle to his scientific activity and a t length caused his death. H e had many interests outside the field of his special work and was a generous and helpfnl friend. - time on the 20th of September, and two flights of 3 miles each made on the 9th of November and the 1st of December, respectively, were the more notable performances. "The object of the 1905 experiments was to de- termine the cause and discover remedies for sev- eral obscure and somewhat rare difficulties whioh had been encountered in some of the 1904 flights, and which it was necessary to oyercome before it would be safe to employ flyers for practical pur- poses. The experiments were made in a swampy meadow about 8 miles east of Dayton, Ohio, and continued from June until the early days of Oc- tober, when the impossibility of longer maintain- ing privacy necessitated, their discontinuance. "Owing to frequent experimental changes in the machine and the resulting differences in its management, the earlier flights were short; but, towards the middle of September, means of cor- recting the obscure troubles were found, and the flyer was a t last brought under satisfactory con- trol. From this time forward almost every flight established a new record. In the following schedule the duration, distance and cause of stopping are given for some of the later flights. "lt will be seen that an average speed of a little more than 38 miles an hour was maintained in the last flight. All of the flights were made over a circular course of about three fourths of a mile to the lap, whioh reduced the speed some- what. The maohine increased its velocity on the straight parts of the course and slowed down on the curves. Of these flights, a complete circle made for the first "In the past three years a total of 160 flights have been made with our motor-driven flyers, and a total distance of almost exactly 160 miles cov- ered, an average of a mile to each flight, but until the maohine had received its final improvements the flights were mostly short, as is evidenced by the fact that the flight of October 5th was longer than the 105 flights of the year 1904 together. 18 min. 9 sec. Exhaustion of fuel. 19 min. 65 sec. Exhaustion of fuel. 17 min. 15 sec. Hot bearing. 25 min. 5 sec. Hot bearing. 33 min. 17 see. Hot bearing. 38 min. 3 sec. Exhaustion of fuel. Sept. 26 17,961 meters (11% miles) Sept. 29 19;570 metera ( 12 miles) Sept. 30 ........................ Oct. 3 24,535 meters (15% miles) Oct. 4 33,456 meters (2074 miles) Oct. 5 38,956 meters (24% miles) SCIENCE. [N. S. VOL.XXIII. No. 688. "The lengths of the flights were measured by a Richard anemometer which was attached to the machine. The records were found to agree closely with the distance measured over the ground when the flights were made in calm air over a straight course; but when the flights were made in circles a close comparison was impossible because it was not practicable to accurately trace the course over the ground. In the flight of October 5th a total of 29.7 circuits of the field was made. The times were taken with stop-watches. In operating the machine it has been our custom for many years to alternate in making flights, and such care has been observed that neither of us has suflered any serious injury, though in the earlier flights our ignorance and the inadequacy of the means of control made the work exceedingly dangerous. hundredth anniversary of the birth of Ben- jamin Franklin, its founder. DR. HEINRICH professor astron- BRUNS, of omy at Leipzig, and Dr. Hugo von Seeliger, professor of astronomy at Munich, have been elected corresponding members of tho Berlin Academy of Sciences. PROFESSOR KOCH,of has ROBERT Berlin, been elected a foreign member of the Brussels Academy of Sciences. PROFESSOR director the J. M. PERNTEB, of Vienna Meteorological Bureau, has been elected a n honorary member of the London Meteorological Society. PROFESSOR A. KELLERMAN,the WILLIAM of Ohio State University, has returned from Guatemala where for three months he has been studying and collecting parasitic fungi. H e reports a very interesting and satisfactory trip, and brings from several sections, especially from the higher altitudes including three ,vol- canoes, a very large quantity of material for critical study. N o mycologist has traversed these regions before, and it is expected that in- teresting results will be secured. "The 1905 flyer had a total weight of about 925 pounds, including the operator, and was of such substantial construction as to be able to make landings a t high speed without being strained or broken. From the beginning the prime object was to devise a machine of practical utility, rather than a useless and extravagant toy. For this rea- son extreme lightness of construction has always been resolutely rejected. On the other hand, every effort has been made to increase the scien- tific eaciency of the wings and screws in order that even heavily built machines may be carried with a moderate expenditure of power. The favorable results which have been obtained have been due to improvements in flying quality result- ing from more scientific design and to improved methods of balancing and steering. The motor and machinery possess no extraordinary qualities. The best dividends on the labor invested have in- variably come from seeking more knowledge rather than more power." DR. PAULKUCKUCK, curator of the Biolog- ical Institute of IIeligoland, has been granted the title of professor by the Gelxian govern- ment. M. BOUQUET has been appointed astrononier in the Paris Observatory. DR. F. W. CLARKE,professor of niineral chemistry, George Washington University, will give a special course of lectures i n chem- ical geology on Mondays at 4 5 0 P.M. as .follows : Very respectfully, (Signed) ORVILLE WRIGHT (Signed) ORVILLE WRIGHT (Signed) ORVILLE WRIGHT ( Signed) WILBUR WRIGIIT April 2.-' Introductory: The Elements and the Atmosphere.' SCIEArTI#'IC NOTES AND NEWLY. 9.' April The I-Iydrosphere.' DR.WALTIIERNERNST,professor of physical chemistry in the University of Berlin, will give this year the Silliman lectures a t Yale University. April 16.-' The Magma and the Igneous Rocks.' April 16.-' The Magma and the Igneous Rocks.' April 23.-'The Sedimentary Rocks.' April 30.-' Ore Deposits.' May 7.-' Coal, Petroleum and Natural Gas.' May 7.-' Coal, Petroleum and Natural Gas.' MR. WILLIAM for years SOWERBY, many secretary of the Royal Botanic Society, Re- gent's Park, died in Hertfordshire, on March 9. MR. WILLIAM for years SOWERBY, many secretary of the Royal Botanic Society, Re- gent's Park, died in Hertfordshire, on March 9. SIRGEORGE arrived in New York on DARWIN March 23. H e will represent the Royal So- ciety, the British Association, the Royal In- stitution and the University of Cambridge a t the anniversary meeting of the American Philosophical Society to commeniorate the two TIIE death is also announced of Dr. Her- mann Lorberg, associate professor of physics in the University of Bonn; of Albert Nilsson, TIIE death is also announced of Dr. Her- mann Lorberg, associate professor of physics in the University of Bonn; of Albert Nilsson,
https://openalex.org/W3207791663	https://hal.archives-ouvertes.fr/hal-03409291/document	English	null	Field-induced vortex-like textures as a probe of the critical line in reentrant spin glasses	Scientific reports	2,021	cc-by	16,904	Field-induced vortex-like textures as a probe of the critical line in reentrant spin glasses N Martin, L J Bannenberg, M Deutsch, C Pappas, G Chaboussant, R Cubitt, I Mirebeau N Martin, L J Bannenberg, M Deutsch, C Pappas, G Chaboussant, R Cubitt, I Mirebeau I Mirebeau To cite this version: N Martin, L J Bannenberg, M Deutsch, C Pappas, G Chaboussant, et al.. Field-induced vortex-like textures as a probe of the critical line in reentrant spin glasses. Scientific Reports, 2021, 11 (1), pp.20753. 10.1038/s41598-021-99860-2. hal-03409291 Distributed under a Creative Commons Attribution 4.0 International License N. Martin1, L. J. Bannenberg2, M. Deutsch3, C. Pappas2, G. Chaboussant1, R. Cubitt4 & I. Mirebeau1 N. Martin1, L. J. Bannenberg2, M. Deutsch3, C. Pappas2, G. Chaboussant1, R. Cubitt4 & I. Mirebeau1 We study the evolution of the low-temperature field-induced magnetic defects observed under an applied magnetic field in a series of frustrated amorphous ferromagnets (Fe1−xMnx)75P16B3Al3 (“a- Fe1−xMnx”). Combining small-angle neutron scattering and Monte Carlo simulations, we show that the morphology of these defects resemble that of quasi-bidimensional spin vortices. They are observed in the so-called “reentrant” spin-glass (RSG) phase, up to the critical concentration xC ≈0.36 which separates the RSG and “true” spin glass (SG) within the low temperature part of the magnetic phase diagram of a-Fe1−xMnx. These textures systematically decrease in size with increasing magnetic field or decreasing the average exchange interaction, and they finally disappear in the SG sample ( x = 0.41 ), being replaced by field-induced correlations over finite length scales. We argue that the study of these nanoscopic defects could be used to probe the critical line between the RSG and SG phases. The role of disorder is central in condensed matter physics, as it favors the nucleation of defects which play a crucial role in the evolution and functionalities of a large variety of systems. Examples are magnetic vortices in type-II superconductors, skyrmions in helical magnets, Taylor cells in liquid flows, or twist grain boundary phases in cholesteric liquid crystals. Quite generally, defects allow new properties to penetrate in the system by forming intermediate states of matter, precursors of a phase transitions. In this context, we study here the influence of nm-size magnetic defects on the evolution from ferromagnetic (FM) and spin glass (SG) ground states. SG are archetypal disordered magnetic systems that have mobilized a large and continuous attention for decades. Their physics is mainly driven by atomic disorder and random sign interactions. In this work, we focus on the so-called “reentrant” spin glasses (RSG), where a mixture of FM and antiferromagnetic (AFM) couplings (usually tuned by the concentration of magnetic ions and, in some cases, heat treatment) leads to SG-like irreversibilities at low temperatures, deep inside the FM ordering region. In RSGs, the nature of the coexistence between SG behavior and ferromagnetism has been much debated. HAL Id: hal-03409291 https://hal.science/hal-03409291v1 Submitted on 29 Oct 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License www.nature.com/scientificreports www.nature.com/scientificreports Samples and their macroscopic magnetic propertiesh p p g p p The a-Fe1−xMnx system is perfectly suitable for our study. Its macroscopic properties and transition temperatures are well known and almost independent of the sample synthesis, while the amorphous character guarantees the absence of structural defects which could otherwise act as pinning centers12. Frustration is monitored by the Mn concentration x which controls the relative amounts of AFM Mn-Mn nearest neighbor (NN) bonds with respect to the FM Mn–Fe and Fe–Fe ones. Here, we study seven RSG samples of concentrations ranging from x = 0.22 to x = 0.35 and a SG sample with x = 0.41 , previously studied by Mössbauer spectroscopy, neutron depolarization and muon spin rotation13,14 ( µSR). Samples were prepared using the “wheelbarrow” technique and their amorphous nature was checked using neutron diffraction (see Supplementary Information with Sup- plementary Figures S1 and S2). The resulting ribbons, of typical thickness ≈ 30–70 µ m and ≈ 8–10 mm width, can be easily cut or piled-up to perform magnetic and neutron scattering experiments.hi y p p p g g p The zero-field magnetic phase diagram of a-Fe1−xMnx is shown in Fig. 1a. In the RSG regime, i.e. for small x, a transition from the high temperature paramagnet to the low temperature FM state occurs at the Curie tempera- ture TC . The latter decreases upon increasing x, up to a tricritical point located at x = xC ≈0.36 (as determined by previous magnetic susceptibility15 and neutron depolarization14 measurements). At this point, the TC line meets another transition line, corresponding to a spin glass freezing of the system upon cooling below TF . The low-temperature part of the phase diagram is then ruled out by strong irreversibilities of the magnetization, in both the M2 phase (for x < xC ) and the “pure” SG state (for x > xC ). Although they present similar low-field macroscopic magnetic properties, it is important to note that the M2 and SG phases are fundamentally differ- ent. Indeed, M2 is a “mixed” phase, within which FM and SG orderings coexist at a microscopic scale, while the FM order is lost in the “pure” SG state. Samples and their macroscopic magnetic propertiesh We inferred the TC and TF of our samples from sharp increases and decreases of the real part of the ac-susceptibility (see Supplementary Information with Supplementary Table S1 and Supplementary Figures S4, S5 and S6) and compared them with the results of Yeshurun et al.15 in Fig. 1a. and Supplementary Figures S4, S5 and S6) and compared them with the results of Yeshurun et al. in Fig. 1a. For x ≤xC , an additional transition takes place at the so-called “canting” temperature TK , situated between TC and TF as predicted by the MF model of Gabay and Toulouse1. It corresponds to the freezing of the transverse spin components and the onset of weak irreversibilities throughout the intermediate mixed M1 phase16. TK is however not visible in our zero-field ac-susceptibility data, since its observation requires distinguishing between transverse and longitudinal components of the magnetic moments. Signatures of TK have been reported in a-Fe1−x Mnx by comparing magnetization with either Mössbauer spectroscopy13 or µSR14 data (see Fig. 1a). This canting temperature has been identified as the temperature border, above which the vortex-like textures described in the present work vanish7,10 and below which dynamical anomalies, such as damping of the FM spin waves, occur17. We also performed dc-magnetization measurements in the 0-5 T field range in order to verify that all samples with x < xC retain a ferromagnetic character, while the field at which technical saturation takes place increases with x under the effect of increasing magnetic frustration (Fig. 1b). From the corresponding Arrott plots (Fig. 1c), we also deduced the x-dependence of the spontaneous moment M0 = M(µ0Hint →0) . As shown in the inset of Fig. 1c, this leads to an extrapolated zero moment for x = 0.38(3) , which is consistent with the literature value of the critical concentration xC where long-range FM order is lost. pp y g p g For x ≤xC , an additional transition takes place at the so-called “canting” temperature TK , situated between TC and TF as predicted by the MF model of Gabay and Toulouse1. It corresponds to the freezing of the transverse spin components and the onset of weak irreversibilities throughout the intermediate mixed M1 phase16. TK is however not visible in our zero-field ac-susceptibility data, since its observation requires distinguishing between transverse and longitudinal components of the magnetic moments. www.nature.com/scientificreports/ interactions and sample form (single crystal, polycrystal or amorphous samples)7–10. In all cases, SANS experi- ments show that the transverse spin components rotate over a finite length scale which defines the average size of the vortex-like spin textures. These data, supported by MC simulations11, also indicate that they shrink with increasing the applied field, but their behaviour at strong frustration and across the RSG-SG threshold has not been studied so far. In order to address this point, we focus here on the series of frustrated amorphous ferromagnets (Fe1−xMnx)75 P16B6Al3 (“a-Fe1−xMnx”). a-Fe1−xMnx maps a case of 3d disordered Heisenberg spins, where frustration can be chemically tuned through the competition of FM and AFM interactions. Using SANS, we follow the evolution of the field-induced vortex-like spin textures with increasing frustration, as the magnetic ground state evolves from RSG to SG. We show that these non-singular defects are characteristic of the RSG ground state. Their average size obeys scaling laws up to the critical concentration and maximum applied field. They eventually disappear above the RSG-SG threshold, showing that a non-zero average exchange is needed for their stabilization. Our results may open a route to reconcile the MF and RF pictures of the RSG state, opposed for decades. Indeed, such nm-size magnetic defects can probe the nature of the frustrated medium and be the fingerprints of a quantum phase transition at a microscopic level. Our experimental study is also supported by Monte Carlo simulations, which show that the occurrence of these defects, as well as their evolution as a function of magnetic field, can be globally reproduced using a very limited amount of ingredients. These results suggest that frustration and disorder can be used to engineer the average size of individual defects in a controlled and reproducible way in disordered frustrated ferromagnets. Samples and their macroscopic magnetic propertiesh Signatures of TK have been reported in a-Fe1−x Mnx by comparing magnetization with either Mössbauer spectroscopy13 or µSR14 data (see Fig. 1a). This canting temperature has been identified as the temperature border, above which the vortex-like textures described in the present work vanish7,10 and below which dynamical anomalies, such as damping of the FM spin waves, occur17.i p y p g p We also performed dc-magnetization measurements in the 0-5 T field range in order to verify that all samples with x < xC retain a ferromagnetic character, while the field at which technical saturation takes place increases with x under the effect of increasing magnetic frustration (Fig. 1b). From the corresponding Arrott plots (Fig. 1c), we also deduced the x-dependence of the spontaneous moment M0 = M(µ0Hint →0) . As shown in the inset of Fig. 1c, this leads to an extrapolated zero moment for x = 0.38(3) , which is consistent with the literature value of the critical concentration xC where long-range FM order is lost. N. Martin1, L. J. Bannenberg2, M. Deutsch3, C. Pappas2, G. Chaboussant1, R. Cubitt4 & I. Mirebeau1 Here, we show that the observation of magnetic field-induced vortex-like textures, although not predicted by current theories, could be a key point to distinguish between RSG and SG ground states at a microscopic level.h Historically, the RSG and SG ground states have been described by two concurrent theoretical approaches. The infinite range mean field (MF) picture yields a phase diagram with a tricritical point and a vertical line between RSG and SG phases1. Below this line, i.e. in the weakly frustrated case, lowering temperature leads to the occur- rence of mixed RSG phases, where SG and FM order parameters coexist microscopically. At each magnetic site, “longitudinal” spin component, forming a long-range magnetic order (LRMO), coexist with a “transverse” one, randomly oriented in the perpendicular plane. Alternatively, random field (RF) arguments predict the break- down of LRMO for an arbitrarily small amount of disorder in dimensions d ≤4 , as formalized by the Imry-Ma (IM) argument2. This argument was used together with percolation approaches to describe the RSG phase as randomly oriented clusters spatially separated from the “infinite” FM one. The latter would break due to RFs at the RSG-SG threshold, in a cross-over transition3,4. The IM argument was recently complemented by a series of Monte-Carlo (MC) simulations suggesting that, in the case of ferromagnets exposed to a RF, the IM domains are protected against a full collapse of the magnetization by the nucleation of topological defects, such as pinned hedgedhogs in 3 dimensions5 or a “skyrmion-antiskyrmion glass” in 2 dimensions6. g g y y g On the experimental side, magnetic defects -akin to nm-size vortices- have been observed by small-angle neutron scattering (SANS) in weakly frustrated RSGs under an applied magnetic field. The family of studied compounds (Ni1−xMnx , AuxFe1−x , Fe1−xAlx , or a-Fe1−xMnx ) includes different types of disorder, magnetic 1Université Paris‑Saclay, CNRS, CEA, Laboratoire Léon Brillouin, 91191 Gif‑sur‑Yvette, France. 2Faculty of Applied Science, Delft University of Technology, 2629 JB Delft, The Netherlands. 3CNRS, CRM2, Université de Lorraine, Nancy, France. 4Institut Laue Langevin, BP156, 38042 Grenoble, France. email: nicolas.martin@cea.fr \| https://doi.org/10.1038/s41598-021-99860-2 Scientific Reports \| (2021) 11:20753 www.nature.com/scientificreports/ Small‑angle neutron scattering (SANS) We have carried out a small-angle neutron scattering (SANS) experiment at the PAXY beamline (Orphée Reactor, Saclay, France). A horizontal magnetic field µ0H up to 4 T was applied transverse to the beam direction (i.e., in the detector plane). All data were obtained in the zero field cooled (ZFC) state at T = 3 K. This temperature was chosen because (i) it is well-below TC and TF for all samples (Fig. 1), and (ii) allows neglecting the contribution of phonons and magnetic excitations (spin waves) to the SANS patterns. Data were corrected and calibrated as described in the Supplementary Information with Supplementary Tables S2 and S3. y y Typical SANS patterns are shown in Fig. 2, for compositions x respectively below (Fig. 2a,b) and above (Fig. 2c) xC . One can immediately note differences between these two cases. On the one hand, maxima of intensity appear parallel to the applied field at a finite value of the momentum transfer Q when x < xC . On the other hand, https://doi.org/10.1038/s41598-021-99860-2 Scientific Reports \| (2021) 11:20753 \| www.nature.com/scientificreports/ 300 250 200 150 100 50 0 T (K) 0.5 0.4 0.3 0.2 x PM FM SG M2 M1 TC TF TK (Fe1-xMnx)75P16B6Al3 Zero-field / This work / Yeshurun et al. Mirebeau et al. (a) 0.8 0.6 0.4 0.2 0.0 M 2 (µB 2.f.u. -2) 25 20 15 10 5 0 µ0Hint/M (T.f.u.µB -1) (c) 1.0 0.5 0.0 M0 (µB / f.u.) 0.40 0.30 0.20 x 1.0 0.8 0.6 0.4 0.2 0.0 M (µB / f.u.) 5 4 3 2 1 0 µ0Hint (T) (b) T = 5 K x = 0.22 x = 0.235 x = 0.247 x = 0.3 x = 0.32 x = 0.35 x = 0.41 Figure 1. Phase diagram and magnetization of a-Fe1−xMnx—(a) Zero-field magnetic phase diagram of a-Fe1−x Mnx inferred from magnetic ac-susceptibility. For 0.2 ≲x ≲xC , cooling from the high-temperature FM state leads to a sequence of two mixed states: M1, involving the freezing of transverse spin components18 and M2, where replica symmetry is spontaneously broken (i.e., an analog of the SG state). Above x = xC ≈0.36 , the ferromagnetic (FM) phase is suppressed and replaced with a “canonical” spin-glass (SG) state at low temperature. Open symbols are data from Mirebeau et al.13,14 and Yeshurun et al.15 which are added to our results for TC and TF for the sake of completeness. Small‑angle neutron scattering (SANS) The green vertical line indicates a putatively vertical critical line between the RSG (M2 phase) and SG regimes1. (b) Low-temperature magnetization curves of a-Fe1−xMnx . (c) Arrot plots computed from the data of panel (b). Dashed lines are linear fits to the high-field data. Inset shows the x-dependence of the spontaneous magnetization M0 = M(µ0Hint →0). 300 250 200 150 100 50 0 T (K) 0.5 0.4 0.3 0.2 x PM FM SG M2 M1 TC TF TK (Fe1-xMnx)75P16B6Al3 Zero-field / This work / Yeshurun et al. Mirebeau et al. (a) 1.0 0.8 0.6 0.4 0.2 0.0 M (µB / f.u.) 5 4 3 2 1 0 µ0Hi (T) (b) T = 5 K x = 0.22 x = 0.235 x = 0.247 x = 0.3 x = 0.32 x = 0.35 x = 0.41 3 (a) x Figure 1. Phase diagram and magnetization of a-Fe1−xMnx—(a) Zero-field magnetic phase diagram of a-Fe1−x Mnx inferred from magnetic ac-susceptibility. For 0.2 ≲x ≲xC , cooling from the high-temperature FM state leads to a sequence of two mixed states: M1, involving the freezing of transverse spin components18 and M2, where replica symmetry is spontaneously broken (i.e., an analog of the SG state). Above x = xC ≈0.36 , the ferromagnetic (FM) phase is suppressed and replaced with a “canonical” spin-glass (SG) state at low temperature. Open symbols are data from Mirebeau et al.13,14 and Yeshurun et al.15 which are added to our results for TC and TF for the sake of completeness. The green vertical line indicates a putatively vertical critical line between the RSG (M2 phase) and SG regimes1. (b) Low-temperature magnetization curves of a-Fe1−xMnx . (c) Arrot plots computed from the data of panel (b). Dashed lines are linear fits to the high-field data. Inset shows the x-dependence of the spontaneous magnetization M0 = M(µ0Hint →0). Figure 1. Phase diagram and magnetization of a-Fe1−xMnx—(a) Zero-field magnetic phase diagram of a-Fe1−x Mnx inferred from magnetic ac-susceptibility. For 0.2 ≲x ≲xC , cooling from the high-temperature FM state leads to a sequence of two mixed states: M1, involving the freezing of transverse spin components18 and M2, where replica symmetry is spontaneously broken (i.e., an analog of the SG state). Above x = xC ≈0.36 , the ferromagnetic (FM) phase is suppressed and replaced with a “canonical” spin-glass (SG) state at low temperature. Small‑angle neutron scattering (SANS) Open symbols are data from Mirebeau et al.13,14 and Yeshurun et al.15 which are added to our results for TC and TF for the sake of completeness. The green vertical line indicates a putatively vertical critical line between the RSG (M2 phase) and SG regimes1. (b) Low-temperature magnetization curves of a-Fe1−xMnx . (c) Arrot plots computed from the data of panel (b). Dashed lines are linear fits to the high-field data. Inset shows the x-dependence of the spontaneous magnetization M0 = M(µ0Hint →0). SANS from the x = 0.41 sample is typical of field-induced ferromagnetic-like correlations centered at Q = 0 . In order to separate contributions of magnetic moments transverse (T) and longitudinal (L) to the applied field, we make use of the neutron selection rule which states that only components perpendicular to the scattering vector Q contribute to the observed scattering cross section σ(Q) . This translates into the following relations (1) σT(Q) + σbg(Q)/2 = ˜σ(Q H)/2 and σL(Q) + σbg(Q)/2 = ˜σ(Q ⊥H) −˜σ(Q H)/2, (1) σT(Q) + σbg(Q)/2 = ˜σ(Q H)/2 and σL(Q) + σbg(Q)/2 = ˜σ(Q ⊥H) −˜σ(Q H)/2, σT(Q) + σbg(Q)/2 = ˜σ(Q H)/2 and σL(Q) + σbg(Q)/2 = ˜σ(Q ⊥H) −˜σ(Q (1) where σbg denote the background contributions from the sample (e.g., nuclear scattering) and ˜σ the full observed scattering within sectors of 60◦ opening angle, parallel or perpendicular to H . Therefore, a radial integration of the SANS data along the horizontal and vertical direction allows retrieving the Q-dependences of σT and σL independently, assuming an isotropic σbg.h g The result of such procedure is shown in Fig. 2d–i. In the weakly frustrated x = 0.22 RSG sample, the intensity is clearly enhanced along the field direction, i.e. for Q ∥H , showing that the contribution of spin components transverse to the magnetic field are dominant in the explored Q-range, whereas the opposite behavior is observed in the x = 0.41 SG sample. As a general feature, we observe field-induced peaks in σT(Q) at Q = Qmax for all compositions x < xC . Qmax moves to higher values when the field increases at constant x, and also shows a systematic stiffening as x increases towards xC . Small‑angle neutron scattering (SANS) σL(Q) shows a broad maximum, but it is more difficult to point because its intensity is much smaller.i s on the transverse cross section σT(Q) , which is defined in the most general case as y In what follows, we focus on the transverse cross section σT(Q) , which is defined in the most general case as (2) σT(Q) ∝F2 T(Q) −FT(Q)2[1 −Sint(Q)], (2) where FT(Q) is the form factor of the transverse defects and Sint(Q) is an interference function that expresses the local correlations between two such defects. Assuming that the latter are organized in a liquid-like order ( Sint(Q) →1 ) and noting that the form factor of a “regular” bidimensional vortex is null ( FT(Q) = 0)19,20, σT(Q) is simply proportional to ⟨F2 T(Q)⟩. where FT(Q) is the form factor of the transverse defects and Sint(Q) is an interference function that expresses the local correlations between two such defects. Assuming that the latter are organized in a liquid-like order ( Sint(Q) →1 ) and noting that the form factor of a “regular” bidimensional vortex is null ( FT(Q) = 0)19,20, σT(Q) is simply proportional to ⟨F2 T(Q)⟩.i T( ) p y p p ⟨T( )⟩ In the whole x < xC-range, the field-dependence of Qmax obeys a scaling law of the form T( )⟩ field-dependence of Qmax obeys a scaling law of the for (3) Qmax(µ0 Hint, x) = κ(x) [µ0 (Hint −H0(x))]γ , (3) https://doi.org/10.1038/s41598-021-99860-2 https://doi.org/10.1038/s41598-021-99860-2 Scientific Reports \| (2021) 11:20753 \| www.nature.com/scientificreports/ -0.2 -0.1 0.0 0.1 0.2 z (m) -0.2 0.0 0.2 x (m) x = 0.22 (a) -0.2 0.0 0.2 x (m) x = 0.35 (b) -0.2 0.0 0.2 x (m) 4.0 3.5 3.0 2.5 2.0 1.5 Log. intensity x = 0.41 (c) 6 4 2 0 σT(Q) (barn.sr -1.at -1) 0.12 0.08 0.04 (d) 4 3 2 1 0 0.20 0.10 (e) 100 80 60 40 20 02 3 4 5 6 0.1 2 (f) 0.5 T 0.65 T 0.8 T 1 T 1.5 T 2 T 4 3 2 1 0 σL(Q) (barn.sr -1.at -1) 0.12 0.08 0.04 Q (Å -1) (g) 4 3 2 1 0 0.20 0.10 Q (Å -1) (h) 200 150 100 50 02 3 4 5 6 0.1 2 Q (Å -1) (i) 0.5 T 0.65 T 0.8 T 1 T 1.5 T 2 T H H H Figure 2. Small‑angle neutron scattering (SANS) SANS data on a-Fe1−xMnx in the M2 and SG phases—(a–c) Scattering maps recorded at T = 5 K under an applied magnetic field of 1.5 T. (d–f) Field-dependence of the transverse magnetic cross section σT(Q) (g-i) Field-dependence of the longitudinal magnetic cross section σL(Q) . The values of the applied magnetic field are indicated for panels (d–i). -0.2 0.0 0.2 x (m) 4.0 3.5 3.0 2.5 2.0 1.5 Log. intensity x = 0.41 (c) H -0.2 -0.1 0.0 0.1 0.2 z (m) -0.2 0.0 0.2 x (m) x = 0.22 (a) H -0.2 0.0 0.2 x (m) x = 0.35 (b) H 4 ( ) 4 3 2 1 0 0.2 0.10 (e) ( ) ( ) ( 6 4 2 0 σT(Q) (barn.sr -1.at -1) 0.12 0.08 0.04 (d) 4 3 2 1 0 0.20 0.10 (e) 100 80 60 40 20 02 3 4 5 6 ( ) 0 100 80 60 40 20 02 3 4 5 6 0.1 2 (f) 0.5 T 0.65 T 0.8 T 1 T 1.5 T 2 T (e) 2 4 3 2 1 0 0.20 0.10 Q (Å -1) (h) 2 1 1 4 3 2 1 0 σL(Q) (barn.sr -1.at -1) 0.12 0.08 0.04 Q (Å -1) (g) 0 200 150 100 50 02 3 4 5 6 0.1 2 Q (Å -1) (i) 0.5 T 0.65 T 0.8 T 1 T 1.5 T 2 T (i) (g) 0.1 1 0.1 Å -1) Q (Å -1) Figure 2. SANS data on a-Fe1−xMnx in the M2 and SG phases—(a–c) Scattering maps recorded at T = 5 K under an applied magnetic field of 1.5 T. (d–f) Field-dependence of the transverse magnetic cross section σT(Q) (g-i) Field-dependence of the longitudinal magnetic cross section σL(Q) . The values of the applied magnetic field are indicated for panels (d–i). where H0(x) is a composition-dependent saturation field, increasing with x. A global fit of Eq. 3 to the data yields a unique exponent γ = 0.39(1) and x-dependent scaling parameters κ(x) (Fig. 3a,b). Results previously obtained on Ni0.81Mn0.19 are also reported in Fig. 3a. In this case, a fit of Eq. 3 to the data yields an exponent γ = 0.34(2) , quite close to the value derived for the a-Fe1−xMnx series. In order to account for this scaling law, we propose a simple picture and provide a physical meaning for its parameters. Small‑angle neutron scattering (SANS) We can compare this dependence with that observed in ferromagnets close to satura- tion, where the magnetic field suppresses micro-structural defects. Here the magnetostatic exchange length which controls the defect size is defined as24 (4) = 2A µ0M2 1/2 , (4) where A is the exchange stiffness and M the bulk magnetization. Identifying A with the average exchange term J⟩ and with ⟨rd⟩ leads to the following dependence for Qmax where A is the exchange stiffness and M the bulk magnetization. Identifying A with the average exchange term ⟨J⟩ and with ⟨rd⟩ leads to the following dependence for Qmax (5) Qmax ≈(µ0 [Hint −H0])1/3 J1/2 . (5) which is quite close to the dependence found experimentally (Eq. 3), noticing that the experimental value of the exponent γ = 0.39(1) is slightly above the value 1/3 from macroscopic magnetization. This comparison however confirms that the average exchange interaction and the applied magnetic field are the main ingredients needed to control the behaviour of the observed magnetic defects, although additional anisotropic exchange terms (such as the Dzyaloshinskii-Moriya interaction) could play a minor role. A perfect mapping of the two cases is in fact not expected, especially for the strongly frustrated RSGs, where the magnetization does not show any clear saturation plateau. Taking this analysis into account, one can however tentatively evaluate the average exchange constant from the scaling parameter κ by J = κ−1/γeﬀ , with 1/3 ≤γeﬀ≤0.39.i which is quite close to the dependence found experimentally (Eq. 3), noticing that the experimental value of the exponent γ = 0.39(1) is slightly above the value 1/3 from macroscopic magnetization. This comparison however confirms that the average exchange interaction and the applied magnetic field are the main ingredients needed to control the behaviour of the observed magnetic defects, although additional anisotropic exchange terms (such as the Dzyaloshinskii-Moriya interaction) could play a minor role. A perfect mapping of the two cases is in fact not expected, especially for the strongly frustrated RSGs, where the magnetization does not show any clear saturation plateau. Small‑angle neutron scattering (SANS) First, we note band structure calculations of dilute FeMn alloys21–23 where Mn-Mn NN interactions are AFM whereas Fe-Fe and and Fe-Mn NN interactions are FM. Based on previous SANS results on a crystalline Ni0.81Mn0.19 sample, as well as toy models and MC simulations20, we interpret σT(Q) by assuming uncorrelated defects, akin to nm-size vortex-like textures, nucleated around Mn-Mn first neighbor pairs. In the simplest pic- ture, the spin components MT are ferromagnetically correlated and rotate over an average radius rd = π/Qmax to compensate the transverse magnetization inside the vortex-like textures20.i p g Using this picture, we can readily interpret the evolution of the SANS patterns with magnetic field and Mn concentration x. At a given x, ⟨rd⟩ decreases with increasing the magnetic field (Fig. 3a), as the spins composing the defects progressively align along the field, albeit not necessarily in a uniform fashion. Their gradual suppres- sion yields a slow increase of the magnetization, taking place in the magnetization quasi-plateau (Fig. 1b). At a given field, ⟨rd⟩ also decreases with an increase of x, which governs the concentration of AFM NN pairs within the samples. As we will show below, these features translate the weakening of the average exchange interaction ⟨J⟩. Altogether, such scaling law suggests that the characteristic size of the defect is governed by the ratio between the magnetic field and the average exchange interaction. To check this picture in more details, we have searched for a common law governing the bulk magnetization curves M(H) at 5 K in the a-Fe1−xMnx system. From the experimental curves, we find a M ≈(µ0 [Hint −H0])1/3-dependence above a threshold field value H0 , which scales with the saturation field deduced from the magnetization curves (see Supplementary Information with Scientific Reports \| (2021) 11:20753 \| https://doi.org/10.1038/s41598-021-99860-2 www.nature.com/scientificreports/ 0.16 0.14 0.12 0.10 0.08 0.06 Scaling parameter κ 0.36 0.32 0.28 0.24 0.20 x (b) 0.25 0.20 0.15 0.10 0.05 0.00 4.0 3.0 2.0 1.0 0.0 µ0Hint (T) 0.22 0.3 0.235 0.32 0.247 0.35 Ni0.81Mn0.19 (a) 120 100 80 60 40 20 <rd> (Å) 65 60 55 50 45 40 <ϕC> (°) (c) a-FeMn Ni0.81Mn0.19 Figure 3. Scaling of the properties of the vortex-like textures—(a) Field-dependence of the position Qmax of the maximum in σT(Q) for each studied compositions. Solid lines are results of a global fit of Eq. 3 to the data. Small‑angle neutron scattering (SANS) Taking this analysis into account, one can however tentatively evaluate the average exchange constant from the scaling parameter κ by J = κ−1/γeﬀ , with 1/3 ≤γeﬀ≤0.39.i We can further define the canting angle ⟨ϕC⟩ , averaged over the size of the vortex-like textures, by the expression (6) ϕC = arctan M2 T/M2 L rd ≈arctan σT(qmax) σL(qmax) · qmax. (6) The result is shown in Fig. 3c, which illustrates the correlation between the average defect radius ⟨rd⟩ and ⟨ϕC⟩ . ⟨ϕC⟩ is maximum at low fields when ⟨rd⟩ is the largest (around 120 Å), and reaches values of 55-60 degs. As for comparison, the canting angle deduced from the Mössbauer measurements of the 57 Fe hyperfine field in the x = 0.235 sample, is around 35(7) degs, with a large distribution13. As the field increases, ⟨rd⟩ decreases and the canting angles decreases as well, reaching values around 45 degrees at the smallest ⟨rd⟩ ≈ 20 Å. To summarize this point, the average canting angle ⟨ϕC⟩ increases as the transverse spin components and magnetic disorder in the transverse plane increase. Corresponding data, extracted from our previous SANS experiment on Ni0.81 Mn0.19 fall within the same range and follows a very similar trend20. This suggests that the observed vortex-like textures are relatively independent on the sample form (single crystalline or amorphous) and could represent an immanent feature of the large family of RSGs. Small‑angle neutron scattering (SANS) (b) x-dependence of the scaling parameter κ , extracted from a fit of Eq. 3 to the data of panel (a). (c) Average canting angle ⟨ϕC⟩ as a function of the average radius of the vortex-like textures ⟨rd⟩ . In panel (a, c), data for Ni0.81Mn0.19 is shown for comparison (pink crosses)20. Dashed pink line is the result of a fit of Eq. 3 to the data. 0.16 0.14 0.12 0.10 0.08 0.06 Scaling parameter κ 0.36 0.32 0.28 0.24 0.20 x (b) <ϕC> (°) 6 120 100 80 60 40 20 <rd> (Å) 65 60 55 50 45 40 <ϕC> (°) (c) a-FeMn Ni0.81Mn0.19 0.25 0.20 0.15 0.10 0.05 0.00 Qmax (Å -1) 4.0 3.0 2.0 1.0 0.0 µ0Hint (T) 0.22 0.3 0.235 0.32 0.247 0.35 Ni0.81Mn0.19 (a) Figure 3. Scaling of the properties of the vortex-like textures—(a) Field-dependence of the position Qmax of Figure 3. Scaling of the properties of the vortex-like textures—(a) Field-dependence of the position Qmax of the maximum in σT(Q) for each studied compositions. Solid lines are results of a global fit of Eq. 3 to the data. (b) x-dependence of the scaling parameter κ , extracted from a fit of Eq. 3 to the data of panel (a). (c) Average canting angle ⟨ϕC⟩ as a function of the average radius of the vortex-like textures ⟨rd⟩ . In panel (a, c), data for Ni0.81Mn0.19 is shown for comparison (pink crosses)20. Dashed pink line is the result of a fit of Eq. 3 to the data. Figure 3. Scaling of the properties of the vortex-like textures—(a) Field-dependence of the position Qmax of the maximum in σT(Q) for each studied compositions. Solid lines are results of a global fit of Eq. 3 to the data. (b) x-dependence of the scaling parameter κ , extracted from a fit of Eq. 3 to the data of panel (a). (c) Average canting angle ⟨ϕC⟩ as a function of the average radius of the vortex-like textures ⟨rd⟩ . In panel (a, c), data for Ni0.81Mn0.19 is shown for comparison (pink crosses)20. Dashed pink line is the result of a fit of Eq. 3 to the data. Supplementary Figure S3). We can compare this dependence with that observed in ferromagnets close to satura- tion, where the magnetic field suppresses micro-structural defects. Here the magnetostatic exchange length which controls the defect size is defined as24 Supplementary Figure S3). Monte Carlo simulations Yellow lines border the regions where impurity spins are located, anchoring the field-induced localized vortex-like textures (see text). (d) Field-dependence of the average longitudinal magnetization \|MZ\| . (e, f) Squared Fourier transforms of the transverse (e) and longitudinal (f) spin correlations for different values of H/J, here averaged over 40 realizations (see Methods and Supplementary Information with Supplementary Figures S9 and S10). pairs. All simulations started by generating spin configurations at a temperature T/J = 2 , where a concentration x of “impurities” (i.e., analogs of Mn ions) are scattered randomly within an otherwise ferromagnetic matrix (i.e., analogs of Fe ions). The following rule is then applied to calculate the sign of the nearest-neighbors exchange terms: two nearest-neighbor impurities will be coupled antiferromagnetically ( Jij = −1 ) while all other pairs will be coupled ferromagnetically ( Jij = 1 ). This situation is meant to map quite closely the one expected from band structure calculations21–23, let alone the actual atomic connectivities. The key quantity describing the MC sample is therefore the concentration cAFM of AFM bonds (with cAFM = x2 ). In order to stick even more to the experimental situation, the system is slowly cooled down to T/J = 0.01 at H = 0 (in steps H/J = 0.1 ) and the field is further raised in steps H/J = 0.01 . We shall show in the following that such a simple scheme allows for a “zero-order” simulation of the properties of the RSGs, and a reasonable description of the experimental observations reported in this paper. p p p We now focus on the case of a weakly frustrated RSG sample ( x = 0.23 , leading to cAFM ≈0.05 ) to investigate the spin configurations and their corresponding Fourier transforms as the magnetic field increases (additional cases, displaying essentially similar behaviors, are addressed in the Supplementary Information with Supple- mentary Figures S9 and S10). In the zero-field or low field region, vortex-like structures are observed around AFM NN pairs, coexisting with domains walls of large length scale which separate the magnetic domains. As shown in Fig. 4a, these domain walls can involve transverse chiral components as well as local defects. However, a Fourier transform of the spin maps show that they do not yield a maximum of the scattered intensity as for the vortex-like structures, but rather a huge increase of the intensity at low Q values. Monte Carlo simulations x H = 1.65 (b) H/J = 0.2 y x H = 1.65 (a) H/J = 0 x H = 1.65 (b) H/J = 0.2 x -1.0 -0.5 0.0 0.5 1.0 Mz H = 1.65 (c) H/J = 1 1.0 d) 2.5 2.0 1.5 1.0 0.5 0.0 \|FT(Q)\| 2 (x 10 3) 0.3 0.2 0.1 0.0 Q (r.l.u.) (e) 1.5 1.0 0.5 0.0 \|FL(Q)\| 2 (x 10 3) 0.3 0.2 0.1 0.0 Q (r.l.u.) H/J 0 0.2 0.25 0.3 0.4 0.6 1.0 (f) H = 1.65 x x 1.0 0.8 0.6 J (d) 2.5 2.0 1.5 1.0 0.5 0.0 \|FT(Q)\| 2 (x 10 3) 0.3 0.2 0.1 0.0 Q (r.l.u.) (e) 1.5 1.0 0.5 0.0 \|FL(Q)\| 2 (x 10 3) 0.3 0.2 0.1 0.0 Q (r.l.u.) H/J 0 0.2 0.25 0.3 0.4 0.6 1.0 (f) x 1.0 0.8 0.6 0.4 0.2 0.0 \|Mz\| 1.0 0.8 0.6 0.4 0.2 0.0 H/J (d) \|FT(Q)\| 2 (x 10 3) Figure 4. Spin configurations and Fourier analysis of the MC simulations—(a–c) Snapshot of a spin configuration ( 15 × 15 spins) obtained on a x = 0.23 sample ( cAFM ≈0.05 ) at T/J = 0.01 for (a) H/J = 0 , (b) H/J = 0.2 (slightly above technical saturation) and (c) H/J = 1 . Arrows represent the in-plane magnetization while color map shows the out-of-plane (i.e., parallel to the applied magnetic field) component MZ . Yellow lines border the regions where impurity spins are located, anchoring the field-induced localized vortex-like textures (see text). (d) Field-dependence of the average longitudinal magnetization \|MZ\| . (e, f) Squared Fourier transforms of the transverse (e) and longitudinal (f) spin correlations for different values of H/J, here averaged over 40 realizations (see Methods and Supplementary Information with Supplementary Figures S9 and S10). Figure 4. Spin configurations and Fourier analysis of the MC simulations—(a–c) Snapshot of a spin i Figure 4. Spin configurations and Fourier analysis of the MC simulations—(a–c) Snapshot of a spin configuration ( 15 × 15 spins) obtained on a x = 0.23 sample ( cAFM ≈0.05 ) at T/J = 0.01 for (a) H/J = 0 , (b) H/J = 0.2 (slightly above technical saturation) and (c) H/J = 1 . Arrows represent the in-plane magnetization while color map shows the out-of-plane (i.e., parallel to the applied magnetic field) component MZ . Monte Carlo simulations In order to get a deeper insight onto the properties of the magnetic defects evidenced in our SANS experiments, we have carried out a series of Monte Carlo simulations on 2d square lattices containing 104 spins. The model is described by the following classical Hamiltonian: (7) H = − ij Jij Si · Sj −αH i Sz i , (7) where Si,j are Heisenberg spins with \|Si,j\| = 1 , Jij are NN exchange constants with \|Jij\| = 1 , α = µB/kB ≈0.672 is a coupling constant and the magnetic field H is applied along the z direction. The first sum in Eq. 7 runs over NN where Si,j are Heisenberg spins with \|Si,j\| = 1 , Jij are NN exchange constants with \|Jij\| = 1 , α = µB/kB ≈0.672 is a coupling constant and the magnetic field H is applied along the z direction. The first sum in Eq. 7 runs over NN https://doi.org/10.1038/s41598-021-99860-2 Scientific Reports \| (2021) 11:20753 \| www.nature.com/scientificreports/ y x H = 1.65 (a) H/J = 0 x H = 1.65 (b) H/J = 0.2 x -1.0 -0.5 0.0 0.5 1.0 Mz H = 1.65 (c) H/J = 1 1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 H/J (d) 2.5 2.0 1.5 1.0 0.5 0.0 \|FT(Q)\| 2 (x 10 3) 0.3 0.2 0.1 0.0 Q (r.l.u.) (e) 1.5 1.0 0.5 0.0 \|FL(Q)\| 2 (x 10 3) 0.3 0.2 0.1 0.0 Q (r.l.u.) H/J 0 0.2 0.25 0.3 0.4 0.6 1.0 (f) Figure 4. Spin configurations and Fourier analysis of the MC simulations—(a–c) Snapshot of a spin configuration ( 15 × 15 spins) obtained on a x = 0.23 sample ( cAFM ≈0.05 ) at T/J = 0.01 for (a) H/J = 0 , (b) H/J = 0.2 (slightly above technical saturation) and (c) H/J = 1 . Arrows represent the in-plane magnetization while color map shows the out-of-plane (i.e., parallel to the applied magnetic field) component MZ . Yellow lines border the regions where impurity spins are located, anchoring the field-induced localized vortex-like textures (see text). (d) Field-dependence of the average longitudinal magnetization \|MZ\| . (e, f) Squared Fourier transforms of the transverse (e) and longitudinal (f) spin correlations for different values of H/J, here averaged over 40 realizations (see Methods and Supplementary Information with Supplementary Figures S9 and S10). Discussion k The MC calculations strongly suggest that the defects are nucleated around AFM NN pairs. In “real” samples, these pairs likely arise from Mn–Mn first neighbours, as suggested by band structure calculations in Fe–Mn21–23. i g gg y In the weakly frustrated RSGs, isolated nm-size vortex-like textures can be clearly distinguished from domain walls by combining magnetization, SANS and MC simulations. The domain walls recall those observed in non- frustrated ferromagnets, but they involve AFM bonds, which induce magnetic defects where the spin canting is locally enhanced, and which act as pinning centers at low temperature. Below TF , this process leads to a strong decrease of the susceptibility χ(T) and to strong irreversibilities of the magnetization M(H). This picture is sup- ported by electron microscopy25, neutron depolarisation18 and recent acoustic absorption measurements26, which clearly show that LRMO and µm-sized domains are preserved in the ground state of the weakly frustrated RSGs. As the field increases, the domain walls are rapidly washed out, whereas isolated vortex-like textures persist up to much higher fields, where their contribution to the SANS can be clearly identified. In the highly frustrated RSGs, the distinction between these objects and the domain walls smears, since the average domain size decreases (becoming comparable to the domain wall thickness) and the magnetization plateau disappears. The vortex contribution is still clearly observed in the SANS data, in sharp contrast with the SG sample. l l fi h b d l d d l fi ld y p p MC simulations are in turn crucial to refine the above picture, and already extend our results to a field range (or more , a H/J range) inaccessible to experiment. The good agreement between the Fourier transform of the MC spin maps and our experimental results should be noticed, considering that the simulated case is over-simplified with respect to the experimental one. We outline here that the MC spin maps show a huge amount of disorder around the local defects which nucleate the vortices. Considering the chemical disorder, many different types of vortices could a priori exist in the sample and they are indeed observed in the MC spin maps. Discussion k As our key experimental result, we have shown that in a frustrated ferromagnetic system, vortex-like defects are a characteristic feature of the RSG which are observable when the system approaches magnetic saturation. However, unlike flux lines in type-II superconductors or skyrmions in helical magnets, these objects do not form a lattice made of infinite length “tubes”. Instead, they resemble “pancakes” made of locally twisted spin configurations and are independent one another, as consistently shown by our SANS data and MC simulations. Their quasi-2d character is also supported by previous simulations performed on 3d spin maps20.h h q pp y p p p p Their average size ⟨rd⟩ decreases with decreasing the average exchange interaction, and the vortex-like tex- tures disappear in the true SG, while ⟨rd⟩ also decreases with increasing the applied magnetic field. The whole behaviour is captured by a scaling law governing ⟨rd⟩ , where the only ingredient is the ratio of the internal magnetic field to the average exchange interaction. Similar laws derived from magnetostatics govern the field behaviour of different macroscopic quantities. For example, one can quote the quasi saturated magnetization of ferromagnets with microstructral defects, the magnetization of type II superconductors, or the thickness of Bloch walls in ferromagnets. g In the RSGs, the presence of vortex-like defects up to the critical concentration, and their collapse in the SG phase when the average exchange interaction becomes smaller than the width of its distribution, strongly supports the existence of a critical line between RSG and SG regions. Our observations therefore support a MF description of the RSG phase diagram, rather than the crossover evolution towards FM breakdown predicted by random field arguments. We however recall that the original MF model of Gabay–Toulouse1, although being able to correctly describe the experimental (x, T)-phase diagram, cannot predict any defect, since the transverse spin component is randomly distributed in the transverse plane. The present observation can therefore help refining the current models for the RSG problem, by considering the observed magnetic microstructure. Altogether, our SANS results combined with MC simulations suggest two complementary phenomena: (1) the vortex-like textures observed in both cases emerge from an average ferromagnetic medium acting as a vacuum field, required for their stabilization (and, hence, their experimental observation using SANS) and (2) they protect the ferromagnetic domains from breaking down under the influence of magnetic fustration. www.nature.com/scientificreports/ very similar to the experimental ones, both for the transverse and longitudinal contributions to the cross sec- tion (compare Figs. 2d,e,g,h and 4e,f). Finally, we note that while being resilient to very large applied fields, the vortex-like textures obtained in the simulations have a vanishing topological charge, most likely due to the ill- defined FM vacuum endowing them with irregular shapes. They are however characterized by a finite vorticity. This confirms the picture obtained using SANS, namely that of finite-size objects within which the transverse magnetization is compensated. Monte Carlo simulations As the field increases, these walls are rapidly suppressed, leading to a strong increase of the magnetization, and to the observation of isolated vortex-like defects. Such textures are nucleated randomly in the sample around AFM NN pairs (Fig. 4a–c), so that in zero (or small) applied field, they could form both in the ferromagnetic domains and in the domain walls (Fig. 4a). However, they are observed in the spin maps only when the field is high enough to suppress the con- tribution of the domain walls. In this regime, vortex-like textures emerge from the ferromagnetic vacuum while magnetization shows a quasi-plateau (Fig. 4d). This leads to a clear maximum in the squared Fourier transforms of the transverse magnetization \|FT(Q)\|2 at a finite Qvalue for intermediate and large applied magnetic fields (Fig. 4e). The defects shrink as the field increases further (and so, the peak in \|FT(Q)\|2 shifts towards larger Q values), and they slowly disappear together with the AFM pairs which nucleate them. The complete destruction of all AFM pairs should only occur at very high fields, much larger than the exchange interaction ( H ≫J ). To summarize, Fourier transforms of the spin maps in the region of the magnetization plateau shows features https://doi.org/10.1038/s41598-021-99860-2 Scientific Reports \| (2021) 11:20753 \| www.nature.com/scientificreports/ Materials and methodsh Materials. The amorphous samples of (Fe1−xMnx)75P16B3Al3 (0.22 ≤ x ≤ 0.41) used in this study were pre- pared using the “wheelbarrow” technique, which consists in casting molten alloy with the desired composition on a spinning wheel. Being a strong neutron absorber, 10 B was replaced with isotopic 11 B. Samples were cut in foils of about 1 cm2 surface with thicknesses varying from 30 to 70 µ m. These foils were piled up in order to increase the total sample thickness and yield a large enough sample mass for the small-angle neutron scattering experiments. Conversely, individual foils were cut into rectangular pieces, having a height to width ratio close to 2, for the magnetic measurements. Magnetic measurements. The ac-susceptibility of the a-Fe1−xMnx samples have been obtained using a Quantum Design Physical Properties Measurement System (PPMS, Dynacool 9 T, Laboratoire Léon Brillouin, France). Magnetization curves were measured using a Quantum Design Superconducting Quantum Interfer- ence Device magnetometer (SQUID, MPMS-XL 5 T, Technische Universiteit Delft, The Netherlands). Small‑angle neutron scattering (SANS). SANS experiments were performed on the PAXY instrument at the Orphée reactor (Laboratoire Léon Brillouin, Gif-sur-Yvette, France), operated in a standard pinhole geom- etry. Neutron wavelength was set to 4 and 6 Å, while keeping the sample-to-detector distance to 2.8 m. An horizontal magnetic field was applied using a cryomagnet (Oxford SM4000), allowing to reach fields of 10 T while cooling the sample down to 2 K. Additional SANS measurements were performed on the D33 instrument (Institut Laue Langevin, Grenoble, France)31, as described in the Supplementary Information with Supplemen- tary Figures S7 and S8. Monte Carlo simulations. Monte Carlo simulations were carried out using the “adaptative” algorithm described by Alzate-Cardona et al. 32. 40 maps, containing 104 spins sitting on the vertices of a square lattice were generated at high temperature. In this model, each spin interacts with its nearest neighbors only (see Eq. 7). A concentration x of “impurity” spins was randomly scattered across the matrix in order to introduce AFM couplings ( J = −1 ) within the FM matrix ( J = 1 ). Each sample was cooled down to T/J = 0.01 in zero-applied field (in steps of T/J = 0.1 ), and the field was then increased in small steps ( H/J = 0.01 ) to study the evolution of the spin configurations and their Fourier transforms. Materials and methodsh In order to properly equilibrate the system, 500 Monte Carlo steps were performed at each stage of the simulation (both during the zero-field cooling and the low- temperature field ramping). Note that Fig. 4a–c) refer to a “snapshot” of a single configuration, while Fig. 4e,f shows averages over the 40 realizations. References 1. Gabay, M. & Toulouse, G. Coexistence of Spin-Glass and Ferromagnetic Orderings. Phys. Rev. Lett. 47, 201–204. https://doi.org/ 10.1103/PhysRevLett.47.201 (1981). y 2. Imry, Y. & Ma, S.-K. Random-Field Instability of the Ordered State of Continuous Symmetry. Phys. Rev. Lett. 35, 1399–1401 https://doi.org/10.1103/PhysRevLett.35.1399 (1975). p g y ( ) 3. Aeppli, G., Shapiro, S. M., Birgeneau, R. J. & Chen, H. S. Spin correlations and reentrant spin-glass behavior in amorphous Fe-Mn alloys: Statics. Phys. Rev. B 28, 5160–5172. https://doi.org/10.1103/PhysRevB.28.5160 (1983). p g y 3. Aeppli, G., Shapiro, S. M., Birgeneau, R. J. & Chen, H. S. Spin correlations and reentrant spin-glass behavior in amorphous Fe-Mn alloys: Statics. Phys. Rev. B 28, 5160–5172. https://doi.org/10.1103/PhysRevB.28.5160 (1983). 4. Niidera, S. & Matsubara, F. Fluctuating clusters in a reentrant spin-glass system. Phys. Rev. B 75, 144413, 2007, DOI: https://doi org/10.1103/PhysRevB.75.144413. g p g y y p org/10.1103/PhysRevB.75.144413. 5 Proctor T C Garanin D A & Chudnovsky E M Random Fields Topology and the Imry Ma Argument Phys Rev Lett 112 g y 5. Proctor, T. C., Garanin, D. A. & Chudnovsky, E. M. Random Fields, Topology, and the Imry-Ma Argument. Phys. Rev. Lett. 112 097201. https://doi.org/10.1103/PhysRevLett.112.097201 (2014). p g y ( ) 6. Chudnovsky, E. M. & Garanin, D. A. Topological Order Generated by a Random Field in a 2D Exchange Model. Phys. Rev. Lett 121, 017201. https://doi.org/10.1103/PhysRevLett.121.017201 (2018).i p g y 7. Hennion, M., Mirebeau, I., Hennion, B., Lequien, S. & Hippert, F. Magnetic structure observed in an applied field in two kinds o re-entrant spin glasses. EPL 2, 393. 10.1209/0295-5075/2/5/008 (1986). p g ( ) 8. Böni, P., Shapiro, S. & Motoya, K. Field induced modulated structure in the re-entrant spin glass Fe704Al296 in applied magnetic fields. Solid State Commun. 60, 881–884. 10.1016/0038-1098(86)90828-8 (1986).i i 9. Lequien, S. et al. Magnetic structure observed in an applied field in reentrant Au081Fe019 and Ni081Mn019 single crystals. Phys. Rev. B 35, 7279–7282. 10.1103/PhysRevB.35.7279 (1987). y 10. 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Received: 18 May 2021; Accepted: 29 September 2021 Received: 18 May 2021; Accepted: 29 September 2021 Received: 18 May 2021; Accepted: 29 September 2021 Discussion k However, their average size can be determined without ambiguity, as it leads to a maximum in Q-space, the position Qmax of which is tuned by the H/J ratio.l y To conclude, we briefly compare the above vortex-like textures with the topological defects observed in fer- romagnets submitted to weak random fields. Quite generally, topological defects are expected when the number of spin components n is such that n ≤d + 1 where d is the dimension of space27, namely in all experimental cases. For instance, non-singular skyrmions with a finite topological charge are observed in the ( n = 3 , d = 2 ) case5. Their existence is a consequence of crossing points between lines where all RF field components cancel at the same time. This leads to the very interesting concept of “skyrmion glass”, composed of regions with oscil- lating positive and negative topological charges, and sizes scaling that of the IM domains6. Importantly, these defects, which prevent the magnetization from collapsing, should lead to a measurable topological Hall effect (THE). Conversely, the vortex-like defects stabilized by magnetic frustration (induced by competing interac- tions and bare interaction randomness) have a very small topological charge due to their very irregular shape, but they could also yield a peculiar Hall signal. In this context, it is worth noting that an anomalous Hall effect was actually predicted28 and observed in AuFe RSG or SG alloys29,30, as a probe of non coplanar (chiral) spin configurations. A quantitative study of the field-dependent Hall response of a-Fe1−xMnx above and below xC Scientific Reports \| (2021) 11:20753 \| https://doi.org/10.1038/s41598-021-99860-2 www.nature.com/scientificreports/ could refine the description of the RSG ground state, given that the defects involved in the two regimes likely have different natures. could refine the description of the RSG ground state, given that the defects involved in the two regimes likely have different natures. Acknowledgements g We thank S. Gautrot (LLB) and M. Bonnaud (ILL) for their assistance during the small-angle neutron scattering experiments at the PAXY and D33 instruments, respectively. We are indebted to P. Durand (CRM2, PMD2 X platform, Jean Barriol Institut, Lorraine University) for his characterization of our samples using X-ray powder diffraction. g We thank S. Gautrot (LLB) and M. Bonnaud (ILL) for their assistance during the small-angle neutron scattering experiments at the PAXY and D33 instruments, respectively. We are indebted to P. Durand (CRM2, PMD2 X platform, Jean Barriol Institut, Lorraine University) for his characterization of our samples using X-ray powder diffraction. g We thank S. Gautrot (LLB) and M. Bonnaud (ILL) for their assistance during the small-angle neutron scattering experiments at the PAXY and D33 instruments, respectively. We are indebted to P. Durand (CRM2, PMD2 X platform, Jean Barriol Institut, Lorraine University) for his characterization of our samples using X-ray powder diffraction. www.nature.com/scientificreports/ Magnetic structures in reentrant spin-glasses observed by transmission electron microscopy. Phys. Rev. Lett. 61, 1013–1016, 1988, DOI: 10.1103/PhysRevLett.61.1013. py y y 26. Kustov, S., Torrens-Serra, J., Salje, E. K. H. & Beshers, D. N. Re-entrant spin glass transitions: new insights from acoustic absorp by domain walls. Scientific Reports 7, 16846 (2017).i y fi p 7. Toulouse, G. & Kléman, M. Principles of a classification of defects in ordered media. J. Physique Lett. 37, 149–151, 1976, DOI 10.1051/jphyslet:01976003706014900.f j y 28. Kawamura, H. Anomalous Hall Effect as a Probe of the Chiral Order in Spin Glasses. Phys. Rev. Lett. 90, 047202. https://doi.org/ 10.1103/PhysRevLett.90.047202 (2003). 28. Kawamura, H. Anomalous Hall Effect as a Probe of the Chiral Order in Spin Glasses. Phys. Rev. Lett. 90, 047202. https://doi.org/ 10.1103/PhysRevLett.90.047202 (2003). 29. Pureur, P., Fabris, F. W., Schaf, J. & Campbell, I. A. Chiral susceptibility in canonical spin glass and re-entrant alloys from Hall effect measurements. EPL 67, 123 (2004).f 29. Pureur, P., Fabris, F. W., Schaf, J. & Campbell, I. A. Chiral susceptibility in canonical spin glass and re-entrant alloys from Hall effect measurements. EPL 67, 123 (2004).f f ( ) 30. Fabris, F. W., Pureur, P., Schaf, J., Vieira, V. N. & Campbell, I. A. Chiral anomalous Hall effect in reentrant AuFe alloys. Phys. B 74, 214201, 2006, DOI: https://doi.org/10.1103/PhysRevB.74.214201. p g y 1. Mirebeau, I. et al. Frustrated skyrmions in reentrant spin glasses: checking the Skyrmion lattice by SANS. https://doi.org/10.5291/ ILL-DATA.5-42-409 (2015). 31. Mirebeau, I. et al. Frustrated skyrmions in reentrant spin glasses: checking the Skyrmion lattice by SANS. https://doi.org/10.5291/ ILL-DATA.5-42-409 (2015). 2. Alzate-Cardona, J. D., Sabogal-Suárez, D., Evans, R. F. L. & Restrepo-Parra, E. Optimal phase space sampling for Monte Carlo simulations of Heisenberg spin systems. J. Phys.: Condens. Matter 31, 095802. https://doi.org/10.1088/1361-648x/aaf852 (2019). 32. Alzate-Cardona, J. D., Sabogal-Suárez, D., Evans, R. F. L. & Restrepo-Parra, E. Optimal phase space sampling for Monte Carlo simulations of Heisenberg spin systems. J. 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ABSTRACT In this supplement, we provide information concerning the synthesis and structural characterization of the (Fe1−xMnx)75P16B6Al3 samples used in this study (Sec. 1). Composition-dependence of the magnetization and AC susceptibility data is presented in Sec. 2. In Sec. 3, we describe the strategy used to scale and analyze the small-angle neutron scattering (SANS) data. Finally, the details of our Monte Carlo simulations on systems with varying antiferromagnetic bond concentration are given in Sec. 4. N. Martin1,, L.J. Bannenberg2, M. Deutsch3, C. Pappas2, G. Chaboussant1, R. Cubitt4, and I. Mirebeau1 1Universit´e Paris-Saclay, CEA, CNRS, Laboratoire L´eon Brillouin, CEA Saclay 91191 Gif-sur-Yvette, France 2Faculty of Applied Science, Delft University of Technology, 2629 JB Delft, the Netherlands 3Universit´e de Lorraine, CNRS, CRM2, Nancy, France 4Institut Laue Langevin, BP156, F-38042 Grenoble, France nicolas.martin@cea.fr 1Universit´e Paris-Saclay, CEA, CNRS, Laboratoire L´eon Brillouin, CEA Saclay 91191 Gif-sur-Yvette, France 2Faculty of Applied Science, Delft University of Technology, 2629 JB Delft, the Netherlands 3Universit´e de Lorraine, CNRS, CRM2, Nancy, France 4Institut Laue Langevin, BP156, F-38042 Grenoble, France nicolas.martin@cea.fr 1Universit´e Paris-Saclay, CEA, CNRS, Laboratoire L´eon Brillouin, CEA Saclay 91191 Gif-sur-Yvette, France 2Faculty of Applied Science, Delft University of Technology, 2629 JB Delft, the Netherlands 3Universit´e de Lorraine, CNRS, CRM2, Nancy, France 4Institut Laue Langevin, BP156, F-38042 Grenoble, France nicolas.martin@cea.fr Competing interests h p g The authors declare no competing interests. p g The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-99860-2. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-99860-2. Correspondence and requests for materials should be addressed to N.M. Correspondence and requests for materials should be addressed to N.M. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. 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To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2021 https://doi.org/10.1038/s41598-021-99860-2 Scientific Reports \| (2021) 11:20753 \| Supplementary Information for "Field-induced vortex-like textures as a probe of the critical line in reentrant spin glasses" N. Martin1,*, L.J. Bannenberg2, M. Deutsch3, C. Pappas2, G. Chabouss and I. Mirebeau1 1 Samples synthesis and structural characterization Amorphous samples of (Fe1−xMnx)75P16B6A3 (0.22 ≤x ≤0.41), herafter named "a-Fe1−xMnx", were prepared using the "wheelbarrow" technique, which consists in casting molten alloy with the desired composition on a spinning wheel, by J. Bigot (Centre d’Études de Chimie—Metallurgie, Vitry sur Seine). Being a strong neutron absorber, 10B (σabs = 3835 barn) was replaced with isotopic 11B (σabs = 0.0055 barn). Samples were cut in foils of about 1 cm2 surface with thicknesses varying from 30-70 µm. These foils were then piled up in order to increase the total sample thickness and yield a large enough sample mass for the small-angle neutron scattering (SANS) experiments (see Sec. 3). Density – The density da−Fe1−xMnx of these materials is an important value, allowing to calibrate the magnetization and SANS data. However, it is rather difﬁcult to measure it directly, given the small thickness of the foils. It can nevertheless be estimated using the random close packing approximation in a hard-sphere model. The maximum density is 64 % of that of the densest crystalline arrangement, namely fcc, with a compacity of 0.74. Taking the atomic masses mn (with n = {Fe, Mn, P, B, Al}) into account, one gets: da−Fe1−xMnx = 0.75·[(1−x) mFe +xmMn]+0.16mP +0.06mB +0.03mAl mFe · 0.64 0.74 ·dfccFe , (1) = 0.75·[(1−x) mFe +xmMn]+0.16mP +0.06mB +0.03mAl mFe · 0.64 0.74 ·dfccFe , (1) where dfccFe = 8.879 g·cm−3 is the density of fcc Fe. Whenever relevant, Eq. 1 is used to scale the data presented in the main text and this supplementary material. Amorphous nature of the samples – The amorphous nature of the samples can be assessed using neutron diffraction. As shown in Fig. 1a in the case of a-Fe0.765Mn0.235, the normalized structure factor S(Q)/S(Qmax) (where Qmax is the largest momentum transfer reached in the experiment) lacks Bragg reﬂections and is characteristic of an amorphous (liquid-like) compound. The small-Q region of the pattern (see Fig. 1b) is marked by an upturn, which is well-described by a power law of the form ap/Qp with an exponent p = 2.97(12) (typical of surface roughness, yielding p = 3). The presence of a prepeak, before the main structural one, indicates a possible clustering of the main chemical specie. Its intensity is however magniﬁed due to the fact that the scattering lengths of the main constituents (i.e., Fe and Mn) have opposite signs. 1 Samples synthesis and structural characterization 1 3.0 2.5 2.0 1.5 1.0 0.5 S(Q)/S(Qmax) 10 8 6 4 2 0 Q (Å -1) (Fe0.765Mn0.235)75P16B6Al3 T = 300 K (a) 1.6 1.4 1.2 1.0 0.8 S(Q)/S(Qmax) 2.0 1.0 Q (Å -1) (b) Figure 1. (a) Diffraction pattern of a-Fe0.765Mn0.235 (taken from Mirebeau1). Data is normalized such that S(Q) →1 in the limit of large Qs. (b) Zoom into the low Q part of the diffraction pattern, showing the prepeak at Q ≈1.7 Å −1. Line is a ﬁt of a power law to the data (see text). 3.0 2.5 2.0 1.5 1.0 0.5 S(Q)/S(Qmax) 10 8 6 4 2 0 Q (Å -1) (Fe0.765Mn0.235)75P16B6Al3 T = 300 K (a) 1.6 1.4 1.2 1.0 0.8 S(Q)/S(Qmax) 2.0 1.0 Q (Å -1) (b) Figure 1. (a) Diffraction pattern of a-Fe0.765Mn0.235 (taken from Mirebeau1). Data is normalized such that S(Q) →1 in the limit of large Qs. (b) Zoom into the low Q part of the diffraction pattern, showing the prepeak at Q ≈1.7 Å −1. Line is a ﬁt of a power law to the data (see text). The pair distribution function (PDF) g(r) is obtained from S(Q)/S(Qmax) using The pair distribution function (PDF) g(r) is obtained from S(Q)/S(Qmax) using The pair distribution function (PDF) g(r) is obtained from S(Q)/S(Qmax) using Qmax 0 Q S(Q) S(Qmax) −1 sin(Qr) dQ , (2) g(r) = 1+ 1 2π2 ρ0 r Z Qmax 0 Q S(Q) S(Qmax) −1 sin(Qr) dQ , (2) (2) where ρ0 is the atomic number density. Finally, one obtains the radial distribution function (RDF) ρ(r) from the PDF via ρ(r) = 4π ρ0 r2 g(r) (3) ρ(r) = 4π ρ0 r2 g(r) ρ(r) = 4π ρ0 r2 g(r) (3) This procedure allows determining the coordination numbers zn, i.e. the number of atoms in the nth shell surrounding any central atom, by integrating ρ(r) within the r-range bounded by its ﬁrst two minima (shaded region in Fig. 2b). For n = 1, we ﬁnd z1 ≈10.6 and r1 ≈2.6Å, in good agreement with results obtained on amorphous Fe powder2 and liquid Fe3 This procedure allows determining the coordination numbers zn, i.e. the number of atoms in the nth shell surrounding any central atom, by integrating ρ(r) within the r-range bounded by its ﬁrst two minima (shaded region in Fig. 2b). 2.1 Magnetization g The ﬁeld-dependences of the magnetization M of the a-Fe1−xMnx samples were measured using a MPMS-XL 5T Quantum Design SQUID magnetometer. The samples were zero-ﬁeld cooled from T ≫TC,TF down to 5 K, and speciﬁc care was taken to avoid the presence of a residual ﬁeld. Subsequently, the measurements were performed by stepwise increasing the magnetic ﬁeld. Samples masses of the order of several mg were used in order to be able to accurately scale the magnetic moment in Bohr magneton per formula unit (µB/f.u.). As shown in Fig. 3a, the ﬁeld value at which magnetization reaches quasi-saturation (H0) increases with increasing x, underscoring the increasing magnetic frustation. In all cases, M retains a ﬁnite slope up to the largest ﬁelds, as a result of the gradual collapse of the vortex-like textures located around the AFM pairs (see main text). In this regime, M is well-described by a law of the form M ≈(µ0Hint)1/3 (Fig. 3b). This property is used in the main text to discuss the possible scaling law governing the ﬁeld-evolution of the observed nanoscopic magnetic textures. We can also deﬁne the saturation ﬁeld H0, at which M acquires the (µ0Hint)1/3-dependence, and compare it with the H0 extracted from the SANS scaling laws, i.e. the ﬁeld above which maxima in the transverse scattering cross section σT can be deﬁned (Fig. 3a of main text). The good correlation between these values is illustrated by Fig. 3c. ) g y g The Arrott plots computed from the data of Fig. 3a are shown in Fig. 3d. All studied samples with x < xC display a non-zero spontaneous magnetization M0. Theses values are plotted in Fig. 3e. The extrapolated value of M0 for x →0 compares well with literature values for crystalline Fe (Shull4), and amorphous Fe75B25 (Cowlam & Carr5), Fe (Grinstaff et al.6) and Fe75P12.5B12.5 (Durand & Yung7), see Fig. 3. 1.0 0.8 0.6 0.4 0.2 0.0 M (µB / f.u.) 5 4 3 2 1 0 µ0Hint (T) (a) T = 5 K x = 0.22 x = 0.235 x = 0.247 x = 0.3 x = 0.32 x = 0.35 x = 0.41 1.0 0.8 0.6 0.4 0.2 0.0 1.5 1.0 0.5 0.0 (µ0Hint) 1/3 (T 1/3) (b) 0.5 0.4 0.3 0.2 0.1 0.0 H0 from SANS (T) 0.6 0.4 0.2 0.0 H0 from magnetization (T) (c) 0.8 0.6 0.4 0.2 0.0 M 2 (µB 2.f.u. 1 Samples synthesis and structural characterization For n = 1, we ﬁnd z1 ≈10.6 and r1 ≈2.6Å, in good agreement with results obtained on amorphous Fe powder2 and liquid Fe3 2.5 2.0 1.5 1.0 0.5 0.0 g(r) 14 12 10 8 6 4 2 r (Å) (Fe0.765Mn0.235)75P16B6Al3 T = 300 K (a) 50 40 30 20 10 0 ρ(r) 8 7 6 5 4 3 2 r (Å) (b) Figure 2. (a) Pair distribution function g(r) and (b) radial distribution function ρ(r) calculated from the data of Fig. 1. 2.5 2.0 1.5 1.0 0.5 0.0 g(r) 14 12 10 8 6 4 2 r (Å) (Fe0.765Mn0.235)75P16B6Al3 T = 300 K (a) ρ(r) 50 40 30 20 10 0 8 7 6 5 4 3 2 r (Å) (b) r (Å) r (Å) Figure 2. (a) Pair distribution function g(r) and (b) radial distribution function ρ(r) calculated from the data of Fig. 1. 2/13 2.1 Magnetization -2) 25 20 15 10 5 0 µ0Hint/M (T.f.u.µB -1) (d) 2.5 2.0 1.5 1.0 0.5 0.0 M0 (µB / f.u.) 0.40 0.30 0.20 0.10 0.00 x (e) Cryst. Fe Amorph. Fe75B25 Amorph. Fe Amorph. Fe75P12.5B12.5 This work Figure 3. Macroscopic magnetization M of the studied a-FeMn samples as a function of (a) µ0Hint and (b) (µ0Hint)1/2. (c) Saturation ﬁeld value H0 deduced from SANS data as a function of H0 deduced from magnetization curves. (d) Low temperature Arrott plots (T = 5 K). (e) x-dependence of the saturated moment, inferred from the Arrott plots. It vanishes for x ≈0.38 and extrapolates to a value of ≈1.86µB·f.u.1 for x →0. This value is compared with previous results for crystalline and amorphous Fe. 1.0 0.8 0.6 0.4 0.2 0.0 M (µB / f.u.) 5 4 3 2 1 0 µ0Hint (T) (a) T = 5 K x = 0.22 x = 0.235 x = 0.247 x = 0.3 x = 0.32 x = 0.35 x = 0.41 1.0 0.8 0.6 0.4 0.2 0.0 1.5 1.0 0.5 0.0 (µ0Hint) 1/3 (T 1/3) (b) 0.5 0.4 0.3 0.2 0.1 0.0 H0 from SANS (T) 0.6 0.4 0.2 0.0 H0 from magnetization (T) (c) 2.5 2.0 1.5 1.0 0.5 0.0 M0 (µB / f.u.) 0.40 0.30 0.20 0.10 0.00 x (e) Cryst. Fe Amorph. Fe75B25 Amorph. Fe Amorph. Fe75P12.5B12.5 This work 0.8 0.6 0.4 0.2 0.0 M 2 (µB 2.f.u. -2) 25 20 15 10 5 0 µ0Hint/M (T.f.u.µB -1) (d) x Figure 3. Macroscopic magnetization M of the studied a-FeMn samples as a function of (a) µ0Hint and (b) (µ0Hint)1/2. (c) Saturation ﬁeld value H0 deduced from SANS data as a function of H0 deduced from magnetization curves. (d) Low temperature Arrott plots (T = 5 K). (e) x-dependence of the saturated moment, inferred from the Arrott plots. It vanishes for x ≈0.38 and extrapolates to a value of ≈1.86µB·f.u.1 for x →0. This value is compared with previous results for crystalline and amorphous Fe. 3/13 2.2 ac-susceptibility p y The magnetic phase diagram presented in the main text was infered through AC susceptibility measurements, performed using a Quantum Design Dynacool 9 T Physical Properties Measurement Systems (PPMS) at the Laboratoire Léon Brillouin. Unless otherwise stated, data presented in this section were measured under an AC ﬁeld of 1 kHz frequency and 10 Oe amplitude, in zero-applied static ﬁeld. In order to suppress demagnetizing ﬁeld effects, we have cut the individual foils into rectangular pieces, having a height to width ratio close to 2 in each case. This however lead to very small samples masses (< 100 µg) and therefore to relatively weak signals. The AC ﬁeld was applied in the sample plane, along its larger dimension. In what follows, we show how phase boundaries are deduced from maxima in dχ′(T)/dT curves. 1. Dimensions of the a-Fe1−xMnx samples used for the AC susceptibility measurements. Table 1. Dimensions of the a-Fe1−xMnx samples used for the AC susceptibility measurements. x Height (mm) Width (mm) 0.22 4.7 2.3 0.235 4.4 2.4 0.247 4.2 2.2 0.3 4.6 2.2 0.32 5.0 2.2 0.35 4.3 1.8 0.41 4.0 2.1 The temperature-dependence of the real (χ′) and imaginary (χ′′) part of the AC susceptibility of a-Fe1−xMnx samples with 0.22 ≤x ≤0.41 is shown in Figs. 4-6. For compositions x ≤0.32, the Curie (TC) and spin freezing (TF) temperatures are easily evidenced by well-separated extrema in the ﬁrst temperature derivative of χ′ (Figs. 4 and 5), (i.e. using the same procedure as used by Yeshurun8). The x = 0.35 case, located very close to the RSG-SG thershold composition xC ≈0.36, is more difﬁcult to anlyze. At ﬁrst glance, its ac-susceptibility is very close to that of the pure SG with x = 0.41 (Fig. 6). However, a modest ﬁeld has a large impact on the dχ′/dT of the x = 0.35 sample as opposed to the x = 0.41 one. This suggests a remanence of ferromagnetism in the former, disappearing in the latter case. The temperature-dependence of the real (χ′) and imaginary (χ′′) part of the AC susceptibility of a-Fe1−xMnx samples with 0.22 ≤x ≤0.41 is shown in Figs. 4-6. For compositions x ≤0.32, the Curie (TC) and spin freezing (TF) temperatures are easily evidenced by well-separated extrema in the ﬁrst temperature derivative of χ′ (Figs. 4 and 5), (i.e. using the same procedure as used by Yeshurun8). 2.2 ac-susceptibility The x = 0.35 case, located very close to the RSG-SG thershold composition xC ≈0.36, is more difﬁcult to anlyze. At ﬁrst glance, its ac-susceptibility is very close to that of the pure SG with x = 0.41 (Fig. 6). However, a modest ﬁeld has a large impact on the dχ′/dT of the x = 0.35 sample as opposed to the x = 0.41 one. This suggests a remanence of ferromagnetism in the former, disappearing in the latter case. In all cases, χ′′ peaks at temperatures slightly higher than TF, while it falls off to ≈0 around TC. Since TF is known to depend on the ac-ﬁeld frequency fac, we have measured the temperature-dependence of the ac-susceptibility of all samples for 100 Hz ≤fac ≤10 kHz (not shown). The various TF shown in Fig. 1a of the main text are the results of extrapolations to fac = 1 Hz. In all cases, χ′′ peaks at temperatures slightly higher than TF, while it falls off to ≈0 around TC. Since TF is known to depend on the ac-ﬁeld frequency fac, we have measured the temperature-dependence of the ac-susceptibility of all samples for 100 Hz ≤fac ≤10 kHz (not shown). The various TF shown in Fig. 1a of the main text are the results of extrapolations to fac = 1 Hz. 4/13 4 3 2 1 0 -1 χ', dχ'/dT (10 -4) 300 200 100 T (K) x = 0.22 4 3 2 1 0 -1 χ', dχ'/dT (10 -4) 250 200 150 100 50 0 T (K) x = 0.235 6 5 4 3 2 1 0 χ'' (10 -5) 300 200 100 0 T (K) x = 0.22 7 6 5 4 3 2 1 0 χ'' (10 -5) 250 200 150 100 50 0 T (K) x = 0.235 3 2 1 0 -1 χ', dχ'/dT (10 -4) 250 200 150 100 50 0 T (K) x = 0.247 5 4 3 2 1 0 χ'' (10 -5) 250 200 150 100 50 0 T (K) x = 0.247 4. Zero-ﬁeld AC suceptibility of a-Fe1−xMnx samples with 0.22 ≤x ≤0.247 in zero applied ﬁeld – (Lef n) Real part of the AC susceptibility χ′ (dots) and its ﬁrst temperature derivative dχ′/dT (solid lines). (Right ary part of the AC susceptibility χ′′. Data is normalized to samples’ surfaces, such that χ′ and χ′′ are expresse 1.Oe−1.mm−2, and dχ′/dT in emu.g−1.Oe−1.mm−2.K−1. 2.2 ac-susceptibility 4 3 2 1 0 -1 χ', dχ'/dT (10 -4) 300 200 100 x = 0.22 250 200 150 100 50 0 T (K) 5 4 3 2 1 0 χ'' (10 -5) 250 200 150 100 50 0 T (K) x = 0.247 T (K) 3 2 1 0 -1 χ', dχ'/dT (10 -4) 250 200 150 100 50 0 T (K) x = 0.247 5 T (K) T (K) Figure 4. Zero-ﬁeld AC suceptibility of a-Fe1−xMnx samples with 0.22 ≤x ≤0.247 in zero applied ﬁeld – (Left column) Real part of the AC susceptibility χ′ (dots) and its ﬁrst temperature derivative dχ′/dT (solid lines). (Right column) Imaginary part of the AC susceptibility χ′′. Data is normalized to samples’ surfaces, such that χ′ and χ′′ are expressed in emu.g−1.Oe−1.mm−2, and dχ′/dT in emu.g−1.Oe−1.mm−2.K−1. 5/13 3 2 1 0 χ', dχ'/dT (10 -4) 160 120 80 40 0 T (K) x = 0.3 6 4 2 0 χ'' (10 -5) 160 120 80 40 0 T (K) x = 0.3 6 4 2 0 χ', dχ'/dT (10 -5) 100 80 60 40 20 0 T (K) x = 0.32 5 4 3 2 1 0 χ'' (10 -6) 100 80 60 40 20 0 T (K) x = 0.32 Figure 5. AC suceptibility of a-Fe1−xMnx samples with x = 0.3 and 0.32 in zero applied ﬁeld – (Left column) Real part of the AC susceptibility χ′ (dots) and its ﬁrst temperature derivative dχ′/dT (solid lines). (Right column) Imaginary part of the AC susceptibility χ′′. Data is normalized to samples’ surfaces, such that χ′ and χ′′ are expressed in emu.g−1.Oe−1.mm−2, and dχ′/dT in emu.g−1.Oe−1.mm−2.K−1. χ', dχ'/dT (10 -4) 0 160 120 80 40 0 T (K) 5 4 3 2 1 0 χ'' (10 ) 100 80 60 40 20 0 T (K) x = 0.32 T (K) T (K) Figure 5. AC suceptibility of a-Fe1−xMnx samples with x = 0.3 and 0.32 in zero applied ﬁeld – (Left column) Real part of the AC susceptibility χ′ (dots) and its ﬁrst temperature derivative dχ′/dT (solid lines). (Right column) Imaginary part of the AC susceptibility χ′′. Data is normalized to samples’ surfaces, such that χ′ and χ′′ are expressed in emu.g−1.Oe−1.mm−2, and dχ′/dT in emu.g−1.Oe−1.mm−2.K−1. 2.2 ac-susceptibility 6/13 3 2 1 0 χ' (10 -6) 70 60 50 40 30 20 T (K) (a) x = 0.35 0 Oe 20 Oe 40 Oe 8 6 4 2 0 χ' (10 -7) 70 60 50 40 30 20 T (K) (c) x = 0.41 0 Oe 20 Oe 40 Oe 1.5 1.0 0.5 0.0 χ'' (10 -7) 70 60 50 40 30 20 T (K) x = 0.35 0 Oe 20 Oe 40 Oe (b) 3.0 2.5 2.0 1.5 1.0 0.5 0.0 χ'' (10 -8) 70 60 50 40 30 20 T (K) x = 0.41 0 Oe 20 Oe 40 Oe (d) 3 2 1 0 -1 dχ'/dT (10 -7) 70 60 50 40 30 20 T (K) (e) x = 0.35 0 Oe 20 Oe 40 Oe 4 3 2 1 0 -1 -2 dχ'/dT (10 -8) 70 60 50 40 30 20 T (K) (f) x = 0.41 0 Oe 20 Oe 40 Oe Figure 6. AC suceptibility of a-Fe1−xMnx samples with x = 0.35 and 0.41 in zero and small applied ﬁeld – (a,b) Real (χ′) and imaginary (χ′′) part of the AC susceptibility of a-Fe0.65Mn0.35. (c,d) Real (χ′) and imaginary (χ′′) part of the AC susceptibility of a-Fe0.59Mn0.41. (e,f) First temperature-derivative of χ′. While a weak applied ﬁeld has a substantial effect of the high-temperature minimum in dχ′/dT for the x = 0.35 sample, it remains unchanged in the x = 0.41 case. In all panels, data is normalized to samples’ surfaces, such that χ′ and χ′′ are expressed in emu.g−1.Oe−1.mm−2, and dχ′/dT in emu.g−1.Oe−1.mm−2.K−1. T (K) T (K) Figure 6. AC suceptibility of a-Fe1−xMnx samples with x = 0.35 and 0.41 in zero and small applied ﬁeld – (a,b) Real (χ′) and imaginary (χ′′) part of the AC susceptibility of a-Fe0.65Mn0.35. (c,d) Real (χ′) and imaginary (χ′′) part of the AC susceptibility of a-Fe0.59Mn0.41. (e,f) First temperature-derivative of χ′. While a weak applied ﬁeld has a substantial effect of the high-temperature minimum in dχ′/dT for the x = 0.35 sample, it remains unchanged in the x = 0.41 case. In all panels, data is normalized to samples’ surfaces, such that χ′ and χ′′ are expressed in emu.g−1.Oe−1.mm−2, and dχ′/dT in emu.g−1.Oe−1.mm−2.K−1. 7/13 3.2 Data correction 3.2 Data correction 3.2 Data correction In order to get scattering cross sections in absolute units, we follow the usual data reduction procedure. First, the contribution from the environment and direct beam are removed using In order to get scattering cross sections in absolute units, we follow the usual data reduction procedure. First, the contribution from the environment and direct beam are removed using Isub(Q) = I(Q)/t(Q)− t(Q) tempty cell(0) ·Iempty cell(Q)/tempty cell(Q) Ω(Q) , (4) (4) Ω(Q) where I(Q), t(Q) and Ω(Q) are the Q-dependent raw intensities, sample transmissions and solid angles subtended by the corresponding detector pixels10. In Eq. 4, the subscript "empty cell" denotes a measurement performed using the same sample holder assembly as for the sample (including the Al foil). This subtraction procedure is applied to the SANS from the samples and from a Ni single crystal. The latter is used to transform the observed intensities in absolute cross sections, according to σ(Q) = Isub a−FeMn(Q)·tNi(0)·dNi ·eNi ⟨Isub Ni (Q)⟩·ta−FeMn(0)·da−FeMn ·ea−FeMn ·σinc Ni , (5) (5) where t, d and e denote transmission, atomic density (see Sec. 1) and sample thickness (see Tab. 3), respectively, while σinc Ni = 5.2/4π barn.sr−1 is the incoherent scattering cross section of Ni (⟨...⟩denotes the average over the detector surface, where incoherent scattering of Ni is expected to be ﬂat). 3.1 Experimental geometry x Thickness (mm) Width (mm) Height (mm) 0.22 3.0 9.1 30.9 0.235 2.1 8.0 36.6 0.247 2.3 8.9 38.0 0.3 1.6 8.6 37.7 0.32 1.5 9.0 21.6 0.35 1.9 9.2 33.8 0.41 4.9 8.7 33.0 3.1 Experimental geometry 3.1 Experimental geometry The small-angle neutron scattering (SANS) experiment described in the main text was performed on the PAXY instrument at the Orphée reactor (LLB, Gif-sur-Yvette, France). We have used a standard pinhole geometry, with parameters given in Tab. 2. p g y The small-angle neutron scattering (SANS) experiment described in the main text was performed the Orphée reactor (LLB, Gif-sur-Yvette, France). We have used a standard pinhole geometry, with Table 2. Parameters used for the SANS experiment on the PAXY instrument. Neutron wavelength → 4 and 6 Å Source aperture (diameter) → 16 mm Collimation length → 2.25 m Sample aperture (diameter) → 5 mm Sample-to-detector distance → 2.8 m Neutron wavelength → 4 and 6 Å Source aperture (diameter) → 16 mm Collimation length → 2.25 m Sample aperture (diameter) → 5 mm Sample-to-detector distance → 2.8 m A horizontal magnetic ﬁeld H = 0−4T was supplied using an Oxford 10 T cryomagnet (Spectromag SM4000). Throughout the experiment, ﬁeld was applied perpendicular to the beam direction in order to optimally resolve the azimuthal asymmetry of the magnetic scattering (see Eq. 1 of main text). Samples were wrapped into a thin Al foil, sandwiched between two Cd slabs (to suppress background and get a well-deﬁned sample surface with 5 mm diameter) and stuck on a Cu frame (to insure a good thermal conduction with the thermometer). 0.00 emptywave 0.00 emptywavex Figure 7. Experimental sample geometry used during the SANS experiment. The horizontal magnetic ﬁeld ⃗H was applied transverse to the largest sample dimension. Figure 7. Experimental sample geometry used during the SANS experiment. The horizontal magnetic ﬁeld ⃗H was applied transverse to the largest sample dimension. For practical reasons, the magnetic ﬁeld could not be applied along the largest dimensions of the samples (see Fig. 7). In order to obtain scaling laws depending on the internal magnetic ﬁeld µ0Hint experienced by the samples, we have calculated the demagnetizing ﬁeld using magnetization data presented in Sec. 2 (for which demagnetizing ﬁeld was negligible) and the demagnetization factor for a very ﬂat ellipsoid given by Osborn9 (Eq. 2.24). The latter approximation is justiﬁed in view of the effective sample dimensions (Tab. 3). 8/13 Table 3. Dimensions of the a-FeMn samples used in the SANS study. Table 3. Dimensions of the a FeMn samples used in the SANS study. 3.3 Effect of a cooling ﬁeld on the SANS of the x = 0.22 sample As noted in seminal experimental11 and theoretical12 studies of spin glasses, the anisotropy ﬁeld maintaining the remanent magnetization in the direction of an initial applied ﬁeld strongly depends of the elements composing the studied material. This comes from additional terms in the Ruderman-Kittel-Kasuya-Yosida (RKKY) interaction for atoms with large spin-orbit coupling (such as Au, Pt, etc.), leading to Dzyaloshinskii-Moriya (DM) anisotropy of unidirectional type. In SG and RSG, this DM anisotropy modiﬁes the torque magnetization, the shape and position of the hysteresis cycle, and the effect of a cooling ﬁeld. When the DM term is small, an applied ﬁeld causes the magnetization to rotate "rigidly" against the DM-induced domain anisotropy. On the other hand, when the DM term is strong, domain conﬁgurations may relax, yielding a distribution in the strength of the resulting frictional torque. In the former case, this leads to displaced narrow hysteresis loops under ﬁeld cooling (FC) conditions while, in the latter case, one observe undisplaced broadened loops, independent of the cooling conditions. Rotational magnetization measurements in a-FeMn (x = 0.235) by Goeckner & Kouvel13 have revealed that this system is much less "rigid" than e.g. Ni1−xMnx, where the anisotropy ﬁeld is much smaller14. To test the inﬂuence of the DM anisotropy on the vortex size, we have checked the effect of a cooling ﬁeld on the scaling law of their average size vs ﬁeld Qmax = f(µ0Hint) (see Eq. 3 of main text) in both systems15. The result is shown on Fig. 8, clearly demonstrating that Qmax is progressively shifted upwards by a cooling ﬁeld in Ni0.81Mn0.19 while it remains unchanged in the case of a-Fe0.78Mn0.22. In other words, FC induces an extra magnetic ﬁeld which reduces the vortex size in NiMn, whereas it has no effect in a-FeMn. This shows that the DM anisotropy indeed plays a role on the vortex landscape, as it does on the magnetization, and underscores the strong relation between the spin vortices and the underlying ferromagnetic vacuum. To test the inﬂuence of the DM anisotropy on the vortex size, we have checked the effect of a cooling ﬁeld on the scaling law of their average size vs ﬁeld Qmax = f(µ0Hint) (see Eq. 3 of main text) in both systems15. The result is shown on Fig. 8, clearly demonstrating that Qmax is progressively shifted upwards by a cooling ﬁeld in Ni0.81Mn0.19 while it remains unchanged in the case of a-Fe0.78Mn0.22. 3.3 Effect of a cooling ﬁeld on the SANS of the x = 0.22 sample In other words, FC induces an extra magnetic ﬁeld which reduces the vortex size in NiMn, whereas it has no effect in a-FeMn. This shows that the DM anisotropy indeed plays a role on the vortex landscape, as it does on the magnetization, and underscores the strong relation between the spin vortices and the underlying ferromagnetic vacuum. 9/13 0 0.02 0.04 0.06 0.08 0.1 Qmax (Å -1) 2.0 1.6 1.2 0.8 0.4 Applied field (T) (a) Ni0.81Mn0.19, T = 3 K ZFC FC 1 T FC 0.5 T FC 2 T 0 0.02 0.04 0.06 0.08 0.1 Qmax (Å -1) 2.0 1.5 1.0 0.5 0.0 Applied field H (T) a-Fe0.78Mn0.22, T = 3 K ZFC FC 2 T (b) Figure 8. Field-dependence of the position Qmax of the maximum in transverse magnetic cross section σT(Q) for (a) Ni0.81Mn0.19 single crystal16 and (b) a-Fe0.78Mn0.22. 0 0.02 0.04 0.06 0.08 0.1 Qmax (Å -1) 2.0 1.6 1.2 0.8 0.4 Applied field (T) (a) Ni0.81Mn0.19, T = 3 K ZFC FC 1 T FC 0.5 T FC 2 T Å 1 0 0.02 0.04 0.06 0.08 0.1 Qmax (Å ) 2.0 1.5 1.0 0.5 0.0 a-Fe0.78Mn0.22, T = 3 K ZFC FC 2 T (b) Figure 8. Field-dependence of the position Qmax of the maximum in transverse magnetic cross section σT(Q) for (a) Ni0.81Mn0.19 single crystal16 and (b) a-Fe0.78Mn0.22. 10/13 4.1 Model and parameters of the simulations p Monte Carlo simulations presented in the main text have been performed using a standard local update algorithm on a square lattice containing L×L = 104 spins, allowed to point in all directions of the 3d space (i.e. we work out a 2d Heisenberg model). "Impurity" spins are randomly spread over the matrix with the desired concentration x to form a binary alloy with composition A1−xBx. These impurities emulate the Mn ions and are antiferromagnetically (AFM) coupled with other ﬁrst neighbor impurity spins (J = −1), while all other couplings are ferromagnetic (FM, J = +1). In order to obtain reasonable convergence times, we have used the "adaptative" algorithm proposed by Alzate-Cardona et al.17. Trial moves of individual spin orientations are performed within a cone having an opening angle ν = 60◦with respect to the initial spin orientation. After each Monte Carlo step (MCS, corresponding to 104 moves), ν is modiﬁed according to νnew →νold × f with f = 0.5 1−Rold , (6) where Rold is the acceptance rate observed during the previous MCS. This procedure allows keeping the average acceptance rate of the algorithm close to 50 %, thereby leading to a relatively quick convergence. For each move, the classical energy H = −∑ ij Ji j Si ·Sj −0.672H∑ i Sz i (7) is calculated, where Si,j are classical Heisenberg spins with \|Si,j\| = 1, Jij are nearest neighbor (NN) exchange constants with \|Jij\| = 1 and the magnetic ﬁeld H is applied along the z direction. The ﬁrst sum in Eq. 7 runs over NN pairs. The factor 0.672 (= µB/kB) appearing in Eq. 7 allows getting energies in K for magnetic ﬁelds expressed in T. The acceptance of each trial move is tested against the Maxwell-Boltzmann statistics exp(−∆E/T), where ∆E is the energy difference between the "old" and "new" conﬁgurations). For each studied composition, we performed between 24 and 40 realizations of the following sequence: 1. Random generation of the spin matrix at T/J = 2 and H = 0 with a chosen "impurity" concent 2. Slow cooling down to T/J = 0.01 at H = 0 (500 MCS per step), in steps of T/J = 0.1, 3. Field sweep in the H/J = 0−1 range, in small steps of ∆H/J = 0.01 (500 MCS per step). 4.2 Data analysis & x-dependences In order to compare the result of our MC simulations with that of the SANS experiment, we compute the square of the Fourier transform of the spin matrices which is formally equivalent to the scattering cross section in the absence of correlation between defects (see Eq. 2 of main text). As in the experimental case, one can separate correlation functions for the transverse (T) and longitudinal (L) magnetization. Illustrative examples are shown in Fig. 4 of main text. Note that the explored momentum range is bounded downwards by the size of the spin maps (100×100) and upwards by the nearest neighbor (NN) distance, but remains much larger than the one covered by SANS (see below). In addition to the x = 0.23 (cAFM ≈0.05)-case discussed in the main text, we have also explored different concentrations in order to check the applicability of our primitive simulations when x (and, hence, cAFM = x2) changes within the weakly frustrated side of the phase diagram (i.e., for ≈0.05 ≲cAFM ≲0.26). The obtained magnetization curves are plotted in Fig. 9a, showing the same behavior as a function of increasing frustration as the experimental ones (Fig. 1b of main text and Fig. 3a of this supplement). We have also determined the scaling laws of Qmax as a function of the applied magnetic ﬁeld (Fig. 9b). A global ﬁt of Eq. 3 of main text to the data yields an exponent γ = 0.49(1) that is slightly different from the experimental value (≈0.39). Of course, our simulations are only taking NN interactions on a square lattice into account. It is not surprising that the scaling laws are renormalized by the effect of an increased number of ﬁrst neighbors (see Sec. 1) and longer-ranged interactions in the real amorphous metallic samples. However, the simulated scaling laws are very similar to the experimental ones in the sense that the scaling paramter κ, monitoring the "stiffness" of these curves, increases linearly with x in agreement with the experiment (Fig. 9c). Taking these results together, we ﬁnd that such a simpliﬁed model already captures many experimental features. Moreover, the MC simulations show that the scaling laws are indeed veriﬁed up to large concentrations of AF interactions, and to high values of the H/J ratio. / This simple model could of course be extended to account e.g. / This simple model could of course be extended to account e.g. for the behavior at the RSG-SG threshold, taking more realistic values for the moments, exchange constants and atomic connectivities. 4.2 Data analysis & x-dependences for the behavior at the RSG-SG threshold, taking more realistic values for the moments, exchange constants and atomic connectivities. 11/13 1.0 0.8 0.6 0.4 0.2 0.0 Mz = <Sz> 1.0 0.8 0.6 0.4 0.2 0.0 H/J (a) 0.25 0.20 0.15 0.10 0.05 0.00 Qmax (r.l.u.) 1.0 0.8 0.6 0.4 0.2 0.0 H/J x = 0.50, cAF = 0.26 x = 0.45, cAF = 0.20 x = 0.39, cAF = 0.15 x = 0.32, cAF = 0.11 x = 0.23, cAF = 0.05 (b) 1.0 0.8 0.6 M0 0.5 0.4 0.3 0.2 x 0.24 0.22 0.20 0.18 0.16 0.14 κ 0.5 0.4 0.3 0.2 x (c) Figure 9. (a) x-dependence of the magnetization curves derived from the MC simulations. Inset shows the x-dependence of the spontaneous magnetization M0, determined from Arrot plots of the ﬁeld-dependent magnetization Mz(H). (b) Field-dependence of the position Qmax of the maximum in \|FT(Q)\|2 for each studied compositions. Solid lines are results of a global ﬁt of Eq. 3 of main text to the data. (c) x-dependence of the scaling parameter κ, extracted from a ﬁt of Eq. 3 of main text to the data of panel (b). 0.25 0.20 0.15 0.10 0.05 0.00 1.0 0.8 0.6 0.4 0.2 0.0 H/J (b) κ 1.0 0.8 0.6 0.4 0.2 0.0 Mz = <Sz> 1.0 0.8 0.6 0.4 0.2 0.0 H/J (a) Q (r l u ) 1.0 0.8 0.6 M0 0.5 0.4 0.3 0.2 x 0.25 0.20 0.15 0.10 0.05 0.00 1.0 0.8 0.6 0.4 0.2 0.0 H/J x = 0.50, cAF = 0.26 x = 0.45, cAF = 0.20 x = 0.39, cAF = 0.15 x = 0.32, cAF = 0.11 x = 0.23, cAF = 0.05 (b) 0.24 0.22 0.20 0.18 0.16 0.14 κ 0.5 0.4 0.3 0.2 x (c) H/J Figure 9. (a) x-dependence of the magnetization curves derived from the MC simulations. Inset shows the x-dependence of the spontaneous magnetization M0, determined from Arrot plots of the ﬁeld-dependent magnetization Mz(H). (b) Field-dependence of the position Qmax of the maximum in \|FT(Q)\|2 for each studied compositions. Solid lines are results of a global ﬁt of Eq. 3 of main text to the data. (c) x-dependence of the scaling parameter κ, extracted from a ﬁt of Eq. 3 of main text to the data of panel (b). 4.2 Data analysis & x-dependences As discussed above, the MC simulations allow extending the explored Q-range to values Q ≈1, sensitive to the smallest interatomic distance. This is an interesting asset, since one can expect in this Q-range (which naturally evades the SANS window), a growing AFM contribution to the scattering pattern as x increases. As shown in Fig. 10, where we have selected ﬁeld values such that Qmax stays constant for different x, this is indeed the case. 10 2 2 4 10 3 2 4 10 4 2 4 10 5 \|FT(Q)\| 2 0.6 0.4 0.2 0.0 Q (r.l.u.) x = 0.23, H = 0.5 x = 0.32, H = 0.34 x = 0.39, H = 0.28 x = 0.45, H = 0.23 x = 0.5, H = 0.2 (a) 10 2 2 4 6 8 10 3 2 4 6 8 10 4 \|FL(Q)\| 2 0.6 0.4 0.2 0.0 Q (r.l.u.) (b) Figure 10. Squared Fourier transforms of the transverse (a) and longitudinal (b) spin correlations for different x and magnetic ﬁeld values, chosen to yield a constant Qmax (see text). x = 0.23, H = 0.5 x = 0.32, H = 0.34 x = 0.39, H = 0.28 x = 0.45, H = 0.23 x = 0.5, H = 0.2 10 2 2 4 6 8 10 3 2 4 6 8 10 4 0.6 0.4 0.2 0.0 Q (r.l.u.) (b) 10 2 2 4 10 3 2 4 10 4 2 4 10 5 \|FT(Q)\| 2 0.6 0.4 0.2 0.0 Q (r.l.u.) (a) \|FL(Q)\| 2 Figure 10. Squared Fourier transforms of the transverse (a) and longitudinal (b) spin correlations for different x and magnetic ﬁeld values, chosen to yield a constant Qmax (see text). 12/13 References 1. Mirebeau, I. Contribution à l’Etude des Systèmes Ferromagnétiques Réentrants à Tendance Verre de Spin. Ph.D. thesis, Université de Paris-Sud (1987). 2. Bellissent, R., Galli, G., Grinstaff, M. W., Migliardo, P. & Suslick, K. S. Neutron diffraction on amorphous iron powder. Phys. Rev. B 48, 15797–15800, DOI: 10.1103/PhysRevB.48.15797 (1993). 3. Waseda, Y. & Suzuki, K. Atomic distribution and magnetic moment in liquid iron by neutron diffraction. physica status solidi (b) 39, 669–678, DOI: https://doi.org/10.1002/pssb.19700390235 (1970). https://onlinelibrary.wiley.com/doi/pdf/10. 1002/pssb.19700390235. 4. Shull, C. G. & Wilkinson, M. K. Neutron diffraction studies of the magnetic structure of alloys of transition elements. Phys. Rev. 97, 304–310, DOI: 10.1103/PhysRev.97.304 (1955). 5. Cowlam, N. & Carr, G. E. Magnetic and structural properties of Fe-B binary metallic glasses. I. Variation of magnetic moment with composition. J. Phys. F: Met. Phys. 15, 1109–1116, DOI: 10.1088/0305-4608/15/5/016 (1985). 6. Grinstaff, M. W., Salamon, M. B. & Suslick, K. S. Magnetic properties of amorphous iron. Phys. Rev. B 48, 269–273, DOI: 10.1103/PhysRevB.48.269 (1993). 7. Durand, J. & Yung, M. Electronic and Magnetic Properties of Amorphous Fe-P-B Alloys, 275–288 (Springer US, Boston, MA, 1977). 8. Yeshurun, Y., Salamon, M. B., Rao, K. V. & Chen, H. S. Critical phenomena in amorphous ferromagnetic and spin-glass alloys. Phys. Rev. B 24, 1536–1549, DOI: 10.1103/PhysRevB.24.1536 (1981). 9. Osborn, J. A. Demagnetizing factors of the general ellipsoid. Phys. Rev. 67, 351–357, DOI: 10.1103/PhysRev.67.351 (1945). 10. Brûlet, A., Lairez, D., Lapp, A. & Cotton, J.-P. Improvement of data treatment in small-angle neutron scattering. J. Appl. Crystallogr. 40, 165–177, DOI: 10.1107/S0021889806051442 (2007). 11. Préjean, J., Joliclerc, M. & Monod, P. Hysteresis in cumn : The effect of spin orbit scattering on the anisotropy in the spin glass state. J. Phys. France 41, 427–435, DOI: 10.1051/jphys:01980004105042700 (1980). 12. Fert, A. & Levy, P. M. Role of anisotropic exchange interactions in determining the properties of spin-glasses. Phys. Rev. Lett. 44, 1538–1541, DOI: 10.1103/PhysRevLett.44.1538 (1980). 13. Goeckner, H. & Kouvel, J. Rotational spin-glass properties of amorphous (Fe1−xMnx)75P16B6Al3. J. Appl. Phys. 70, 6089–6091, DOI: 10.1063/1.350055 (1991). https://doi.org/10.1063/1.350055. 14. Kouvel, J. S., Abdul-Razzaq, W. & Ziq, K. Ferro-spin-glass domain model for disordered Ni-Mn. Phys. Rev. B 35, 1768–1775, DOI: 10.1103/PhysRevB.35.1768 (1987). 15. Mirebeau, I. et al. Frustrated skyrmions in reentrant spin glasses: checking the Skyrmion lattice by SANS (2015). DOI: 10.5291/ILL-DATA.5-42-409. 16. Mirebeau, I. et al. Spin textures induced by quenched disorder in a reentrant spin glass: Vortices versus “frustrated” skyrmions. Phys. Rev. References B 98, 014420, DOI: 10.1103/PhysRevB.98.014420 (2018). 17. Alzate-Cardona, J. D., Sabogal-Suárez, D., Evans, R. F. L. & Restrepo-Parra, E. Optimal phase space sampling for Monte Carlo simulations of Heisenberg spin systems. J. Physics: Condens. Matter 31, 095802, DOI: 10.1088/1361-648x/aaf852 (2019). 13/13
https://openalex.org/W2328708830	https://zenodo.org/record/2071623/files/article.pdf	English	null	The English Land System.	The economic journal/Economic journal	1,915	public-domain	1,041	This content downloaded from 131.247.112.3 on Mon, 27 Jun 2016 04:07:18 UTC All use subject to http://about.jstor.org/terms Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://about.jstor.org/terms Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://about.jstor.org/terms JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Royal Economic Society, Wiley are collaborating with JSTOR to digitize, preserve and extend access to The Economic Journal This content downloaded from 131.247.112.3 on Mon, 27 Jun 2016 04:07:18 UTC All use subject to http://about.jstor.org/terms ARRIOTT: THE ENGLISH LAND SY 69 1915 Prof. Levy. The chief defect of the historical sections lies in the author's acceptance of the old division of the historv into three critical periods-the fourteenth and sixteenth centuries and the age of parliamentary enclosures. This is now hardly adequate, for it is clear that much enclosure took place before 1485, and the researches of Prof. Gonner and Miss Leonard have revealed its prevalence in the seventeenth century. The treatment of the medieval history is the least satisfactory. The uninitiated reader would not gather from this book that Seebohm's theory of manorial origins is now generally discredited. Mr. Marriott describes it as "logical," and though he criticises it, he does not tell us that an essential part of Seebohm's argument was based on a thoroughly uncritical "diplomatic," and is vitiated by its author's readiness to generalise from most insufficient evidence. Again, one may question the prominence which Mr. Marriott assigns to the Black Death as a solvent of the manorial economy. In this connection he quotes Dr. Page's figures of commutation, without taking notice of the weighty criticism passed upon them by Prof. Vinogradoff. Apart from the neglect of intermediate periods and the omission of geographical particulars, the chapters on enclosures are drawn in excellent perspective and are very judicial in spirit. But the best and most novel thing in the book is the discussion of the reaction of changes in the value of the precious metals upon agriculture since 1873 (page 130). In passing to current controversies, Mr. Marriott somewhat changes his key, and the modulation is made rather harsh by a misleading slip on page 133. This content downloaded from 131.247.112.3 on Mon, 27 Jun 2016 04:07:18 UTC All use subject to http://about.jstor.org/terms After quoting some remarks of Mr. A. D. Hall as to the prosperity noticeable in agriculture when he made his "Pilgrimage of British Farming," Mr. Marriott adds: "Then just as things were beginning to look distinctly more hopeful, we have another set-back," and he tells us that " the prevailing confusion " dates from the land-taxes of 1909-1910. But Mr. Hall's pilgrimage was made in 1910, 1911, and 1912. g g The last chapter, though lively and humorous, is spoilt by the restriction of its subject to questions directly affecting the tenure of land. The Liberal land policy is denounced without any mention being made of the minimum wage which Mr. Lloyd George has declared to be the foundation of the whole scheme. In the discussion of Unionist schemes, agricultural protection and imperial preference are passed over in silence. But surely it is a little unkind to devote four pages to the "single-taxers" and not give one word to poor Mr. Chaplin? The treatment of land purchase in this chapter is vague : the finance of the policy is ONOMIC JOURN 7 ARCH not considered in any detail. Though Mr. Marriott appears to approve of the scheme, he says nothing to quell the doubts which he raises in earlier chapters by the statement that tenant farmers, if they purchase their farms, "must, under any circumstances, make inroads upon capital vitally necessary to the efficient working of their farms" (page 9), aind by showing that small occupying owners felt the stress of agricultural depression more severely than the tenant farmers (page 131). REGINALD LENNARD Political Economy. By CHARLES GIDE. Translated (under the direction of Professor Smart) by Constance. H. M. Archibald. (London: George G. Harrap & Co. 1914. Pp. xiii+762. lOs. 6d. net.) THE French are supreme in the art of exposition; and in the front rank of the supreme, so far as economics goes, stands Professor Gide. Happily, in this translation of his Cours d'lconomie politique his enchanting lucidity is not lost. As to its contents, the book contains much on the human side and in reference to social questions that is apt to be left out of the books of English experts; but, on the other hand, it omits much that the dominant English school would deem of fundamental scientific importance and place even in an introductory work. This content downloaded from 131.247.112.3 on Mon, 27 Jun 2016 04:07:18 UTC All use subject to http://about.jstor.org/terms It is remarkable that the French generally have made so little use of Marshall. Gide quotes him twice, and on both occasions on merely incidental points. Of course, there are notable excep- tions to the French inclination to take little account of pure theory-notably Colson and Landry, and Pareto if writing in French makes a Frenchman-but, as Gide says in a note, of the theory of marginal utility, "It is rarely taught in France, even in books." Consequently on price-determination, inter- national trade, wages, and so forth, we find our author coming to a stop just where the English and certain American theorists would begin. Gide does not seem to finish, judged in the light of a theorist's prejudices; but, on the other hand, after reading this treatise, one cannot escape the suspicion that theorists seldom begin. The truth of the matter probably is that the science is best served when each authority sticks to the last that he knows best; and that, to get your economics complete, you must read all. One could not dispense with the conception of a system of economic forces like that of cosmic physics; nor could one dispense with the board room and trade-union committee room point of
https://openalex.org/W2804743943	https://researchonline.ljmu.ac.uk/id/eprint/18783/1/s00417-018-4010-0.pdf	English	null	Outcomes of viscocanalostomy and phaco-viscocanalostomy in patients with advanced glaucoma	Graefe's archive for clinical and experimental ophthalmology	2,018	cc-by	5,628	LJMU Research Online Abstract Purpose To determine the medium-term outcomes for patients with advanced glaucoma undergoing viscocanalostomy. Methods All patients with advanced glaucoma (mean deviation (MD) −12.00 dB or above) and patients with poor visual acuity secondary to advanced glaucoma which precluded formal visual field assessment undergoing viscocanalostomy (VC) and phaco- viscocanalostomy between 2010 and 2014 under the care of a single surgical team were included. Intraocular pressure (IOP), visual acuity (VA) and visual field outcomes were assessed from data prospectively collected into a surgical outcome database. Success was defined at two IOP cut-off points: IOP ≤21 and ≤16 mmHg with (qualified) or without (complete) medications. Results One hundred thirty-five patients were included. Mean IOP changed from 23.6 ± 6.4 mmHg pre-operatively to 15.3, 15.8 and 14.8 mmHg at 1, 2 and 3 years, a change of 35, 33.5 and 39% respectively. Qualified success for an IOP ≤21 mmHg was achieved in 95.66, 90.6 and 80% and complete success in 52.5, 48.6 and 30.6% at year 1, 2 and 3. Qualified success for an IOP ≤ 16 mmHg was achieved in 66.6, 66.05 and 60% and complete success in 44.8, 37.6 and 30.6% at year 1, 2 and 3. The cumulative probability for achieving an IOP ≤21 mmHg with or without drops was 86.1, 81.4 and 81.4% at 12, 24 and 36 months. Eleven patients (8.1%) failed to achieve adequate IOP control and needed further surgical intervention. Eleven (8.1%) patients needed an intervention (Yag goniopuncture) following VC. Four patients (2.9%) had some post-operative complications, which resolved within 2 weeks following surgery. Nine patients (6.7%) lost more than 2 Snellen lines. There was no significant change in the MD across time points. Conclusion Viscocanalostomy and viscocanalostomy combined with phacoemulsification is a safe and effective method of controlling IOP in the medium term in patients with advanced glaucoma. Keywords Viscocanalostomy . Phaco-viscocanalostomy . Advanced glaucoma . Non-penetrating glaucoma surgery . Glaucoma Outcomes of viscocanalostomy and phaco-viscocanalostomy in patients with advanced glaucoma M. Tsagkataki1 & T. M. Bampouras2 & A. Choudhary1 Received: 15 March 2018 /Revised: 2 May 2018 /Accepted: 7 May 2018 /Published online: 22 May 2018 # The Author(s) 2018 Article Citation (please note it is advisable to refer to the publisher’s version if you intend to cite from this work) Tsagkataki, M, Bampouras, TM and Choudhary, A (2018) Outcomes of viscocanalostomy and phaco-viscocanalostomy in patients with advanced glaucoma. Graefe's Archive for Clinical and Experimental Ophthalmology, 256 (8). pp. 1481-1487. ISSN 0721-832X LJMU has developed LJMU Research Online for users to access the research output of the University more effectively. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LJMU Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain. The version presented here may differ from the published version or from the version of the record. Please see the repository URL above for details on accessing the published version and note that access may require a subscription. For more information please contact researchonline@ljmu.ac.uk http://researchonline.ljmu.ac.uk/ Graefe's Archive for Clinical and Experimental Ophthalmology (2018) 256:1481–1487 https://doi.org/10.1007/s00417-018-4010-0 Graefe's Archive for Clinical and Experimental Ophthalmology (2018) 256:1481–1487 https://doi.org/10.1007/s00417-018-4010-0 GLAUCOMA GLAUCOMA * A. Choudhary a.choudhary@liverpool.ac.uk 1 Royal Liverpool University Hospital, Liverpool L7 8XP, UK 2 Active Ageing Research Group, Department of Medical and Sport Sciences, University of Cumbria, Lancaster, England * A. Choudhary a.choudhary@liverpool.ac.uk Introduction specialists in the UK is to start with primary medical therapy, citing surgical risk as the primary reason behind it (23 and 22% respectively) [5]. Patients most at risk of blindness during their lifetime, due to glaucoma, are those who present with advanced disease. Almost 60% of patients progressing to statutory blindness have one eye with an MD worse than −14 dB at baseline [1]. Ten to thirty-nine percent of glaucoma patients present with advanced disease in at least one eye in the UK [2–4]. The preferred option for most glaucoma and non-glaucoma National Institute for Health and Clinical Excellence (NICE) guidelines recommend primary surgery for patients presenting with advanced disease [6]. However, there is lim- ited evidence supporting this recommendation and the type of surgery to be offered. Stead and King [7] have reported medium-term results for trabeculectomy combined with mitomycin C (MMC) in pa- tients with advanced glaucoma (MD ≤20 dB). Although trabeculectomy was successful at controlling intraocular pres- sure (IOP) in this group, a quarter of patients experienced a significant reduction in acuity, with the pre-operative MD cit- ed as the only determinant for it. * A. Choudhary a.choudhary@liverpool.ac.uk * A. Choudhary a.choudhary@liverpool.ac.uk 1 Royal Liverpool University Hospital, Liverpool L7 8XP, UK 2 Active Ageing Research Group, Department of Medical and Sport Sciences, University of Cumbria, Lancaster, England The treatment for advanced glaucoma study (TAGS) will report the outcomes of primary trabeculectomy augmented 1482 Graefes Arch Clin Exp Ophthalmol (2018) 256:1481–1487 with MMC compared with medical management for advanced glaucoma [8]. Non-penetrating glaucoma surgery (NPGS) has been shown to provide comparable long-term qualified suc- cess rates to trabeculectomy, with reduced post-operative complications [9–11] but there is limited information on its success in patients with advanced glaucoma. Subgroup analysis was performed to look at confounding factors such as age, race, combined cataract surgery and pre- vious glaucoma surgery or laser. Differences in outcomes be- tween primary open-angle glaucoma (POAG) and secondary glaucoma were also examined. with MMC compared with medical management for advanced glaucoma [8]. Non-penetrating glaucoma surgery (NPGS) has been shown to provide comparable long-term qualified suc- cess rates to trabeculectomy, with reduced post-operative complications [9–11] but there is limited information on its success in patients with advanced glaucoma. SPSS v22 (SPSS, Chicago, IL) was used to perform statis- tical analysis. Normality of data (IOP, Drops and visual field MD) was examined using the Kolmogorov-Smirnov test. Introduction A linear mixed model was used to examine for differences in IOP and MD between pre-operative (pre-op) and year 1 (Y1), year 2 (Y2), year 3 (Y3) post-operatively and to com- pare the two MD groups at these time points. Drops were examined with Friedman’s test and if a difference was found, pairwise comparisons were conducted using the Wilcoxon test. Associations between presenting IOP, cataract surgery post-VC and combined phaco-VC were examined with point biserial correlation. In this study, we assessed the outcomes of viscocanalostomy (VC) and phaco-viscocanalostomy (phaco-VC) in patients with advanced glaucoma to document success in terms of IOP and VA and the post-operative interventions undertaken to achieve these. Success definition All patients with advanced glaucoma and patients with poor VA secondary to advanced glaucoma which precluded formal visual field (VF) assessment undergoing VC and phaco-VC between 2010 and 2014 under the care of a single surgical team were included. Success was defined at two IOP cut-off points (IOP ≤ 21 mmHg and IOP ≤16 mmHg). Complete surgical success was IOP ≤21 or ≤16 mmHg with no additional medications and qualified surgical success with additional glaucoma med- ications. Failure was defined as IOP > 21 mmHg on two con- secutive visits, IOP ≤5 mmHg on two consecutive visits after 3 months, reoperation for glaucoma or loss of light perception. VA was measured on a Snellen chart, and a reduction of ≥2 lines was considered clinically significant. The technique involved superior corneal traction with 7-0 vicryl. A fornix-based conjunctival flap was raised superiorly and haemostasis was achieved using wet field cautery. A two- third scleral thickness limbus-based flap (5 × 5 mm) was raised and advanced 1 mm into clear cornea. A 4 × 4-mm deep scleral flap was dissected to the level of Schlemm’s canal (SC), de-roofing it, and extended into corneal stroma to the level of Descemet’s membrane to create the trabeculo- Descemet’s membrane (TDM). Viscoat™(sodium hyaluronate and sodium chondroitin sulphate) was injected into the two surgically created ostia of SC, aiming at dilating both the ostia and the canal, and was also placed in the scleral bed. The deep flap was excised close to the TDW. The super- ficial scleral flap and conjunctiva were closed tightly with 10- 0 vicryl sutures. The formation of a bleb was not intended. No antimetabolite was used. Results One hundred thirty-five eyes of 133 patients were included in the study. Patient demographics for all variables over time can be seen in Table 1. Sixty-three (46.7%) patients had phaco- VC. The majority of eyes (132) were on topical glaucoma drops pre-operatively. IOP at diagnosis was not known in 55 patients, as these were referred from other units, and this in- formation was not provided. Twenty-five patients had had some previous intervention for glaucoma including trabeculectomy, cyclodiode laser, argon laser trabeculoplasty (ALT) and selective laser trabeculoplasty (SLT) (Table 1). There were no specific exclusion criteria. Data on all pa- tients were included until the last recorded appointment, which was considered the end of their follow-up. Post- operative time points analysed were day 1, week 1, month 3, month 6 and then every 6 months or closest to that point. Minimum follow-up was 1 year. An intervention was defined as any procedure or process undertaken after VC aimed at enhancing the success of the surgical outcome. This was Nd:YAG laser goniopuncture (Yag GP). Methods Advanced glaucoma was classified as MD between −12.00 and −20.00 dB and severe glaucoma MD −20.01 dB or worse [12]. Advanced glaucoma was classified as MD between −12.00 and −20.00 dB and severe glaucoma MD −20.01 dB or worse [12]. All patients with advanced glaucoma and patients with poor VA secondary to advanced glaucoma which precluded formal visual field (VF) assessment undergoing VC and phaco-VC between 2010 and 2014 under the care of a single surgical team were included. IOP outcome Drops and MD scores between the two MD groups, no signif- icant interaction was seen between groups and time points for IOP (p = 0.999), Drops (p = 0.384) or MD (p = 0.061). Descriptive statistics of all of the above can be seen in Table 2, while Fig. 1 displays IOP and Drops data plotted over the time points. IOP outcome IOP was significantly lower at all examined time points (Y1 by 35.0%; Y2 by 33.5%; Y3 by 39.2%, p < 0.001 at all time points) compared to the pre-op value. Glaucoma drops were significantly different across time points (p = 0.001) with a significantly lower median at all examined time points com- pared to pre-op. Visual field MD was not significantly differ- ent across time points (p = 0.289). When comparing IOP, Primary outcomes were changes in IOP and visual field (MD). These were assessed from data prospectively collected into a surgical outcome database. Secondary outcomes were change in VA, post-operative complications and interventions and number of glaucoma drops (Drops) used. Graefes Arch Clin Exp Ophthalmol (2018) 256:1481–1487 Graefes Arch Clin Exp Ophthalmol (2018) 256:1481–1487 1483 Table 1 Patient demographics N (%) Mean SD Median Range Age (years) 135 (100) 69.8 13.9 73 25 to 92 Duration of glaucoma (years) 101 (74.8) 10.8 7.1 10 1 to 30 Race Caucasian 125 (92.6) African/Afro-Caribbean 4 (3) Asian 6 (4.4) Presenting intraocular pressure (mm Hg) 78 (57.8) 32.7 12.0 29 17 to 64 Pre-operative intraocular pressure (mm Hg) 135 (100) 23.6 6.4 22 14 to 44 Pre-operative visual acuity 111 (82.2) 6/6–6/9 6/6–6/9 to hand movement Pre-operative drops (number) 135 (100) 3.1 1.1 3 0 to 5 Pre-operative MD (dB) 115 (85.2) −19.6 5.5 −19.4 −12 to −32.7 Previous procedures None 110 (81.4) Trabeculectomy 11 (8.1) Retinal detachment surgery 7 (5.2) SLT, ALT 4 (3.0) Cyclodiode 3 (2.2) Glaucoma type Primary open-angle glaucoma 92 (68.1) Chronic angle closure glaucoma 11 (8.1) Pseudoexfoliation 10 (7.4) Uveitic 8 (5.9) Pigment dispersion 6 (4.4) Normal tension glaucoma 5 (3.7) Fuchs’ heterochromic cyclitis 3 (2.2) Patient numbers (and as percentage of whole sample), mean ± SD or median and range (minimum to maximum values), as appropriate, are presented for each variable Patient numbers (and as percentage of whole sample), mean ± SD or median and range (minimum to maximum values), as appropriate, are presented for h i bl Patient numbers (and as percentage of whole sample), mean ± SD or median and range (minimum to maximum values), as appropriate, are presented for each variable (complete (p = 0.912) and qualified (p = 0.541)) or between MD groups (complete (p = 0.512) and qualified (p = 0.079)). Visual acuity outcome VA was stable for the vast majority of patients (126 patients, 93.3%). Nine patients lost > 2 Snellen lines. This was from glaucoma and high myopia in seven and proliferative diabetic retinopathy in two patients. Majority of these patients (eight out of nine) had MD worse than −20 dB. In two patients with MD < −20 dB, VA dropped significantly from 6/36 at pre-op to hand movement and perception of light in the early post- operative period (presumed wipe out). Qualified success for an IOP ≤21 mmHg was achieved in 95.66, 90.6 and 80% and complete success in 52.5, 48.6 and 30.6% at year 1, 2 and 3. Qualified success for an IOP ≤ 16 mmHg was achieved in 66.6, 66.05 and 60% and complete success in 44.8, 37.6 and 30.6% at year 1, 2 and 3 (Table 3). Eleven patients (8%) failed (4 in Y1, 4 in Y2 and 3 in Y3) to reach any of the above success criteria and needed further surgical intervention (Table 4). Of these, four had uveitic glau- coma, two were pseudoexfoliative glaucoma (PXFG) and five were POAG. Qualified success for an IOP ≤21 mmHg was achieved in 95.66, 90.6 and 80% and complete success in 52.5, 48.6 and 30.6% at year 1, 2 and 3. Qualified success for an IOP ≤ 16 mmHg was achieved in 66.6, 66.05 and 60% and complete success in 44.8, 37.6 and 30.6% at year 1, 2 and 3 (Table 3). Eleven patients (8%) failed (4 in Y1, 4 in Y2 and 3 in Y3) to reach any of the above success criteria and needed further surgical intervention (Table 4). Of these, four had uveitic glau- coma, two were pseudoexfoliative glaucoma (PXFG) and five were POAG. Post-operative intervention Eleven (8.1%) patients needed Yag GP following their opera- tion. The time frame for this varied between 2 and 18 months. Visual field changes Kaplan-Meier survival curves were significantly different between complete and qualified success with IOP ≤21 (p = 0.001, Fig. 2) but not between different glaucoma types One hundred fifteen patients were able to perform a reliable VF (24-2 Humphrey’s visual field) prior to surgery. The num- ber of patients that were able to perform a reliable VF Graefes Arch Clin Exp Ophthalmol (2018) 256:1481–1487 1484 Table 2 Descriptive statistics of IOP, Drops and MD at all time points including number of patients per year Table 2 Descriptive statistics of IOP, Drops and MD at all time points including number of patients per year Pre-op (135) Y1 (135) Y2 (109) Y3 (75) IOP (mm Hg) 23.6 ± 6.4 15.3 ± 3.2* 15.8 ± 4.1* 14.8 ± 3.4* For MD between −12.01 and −20.00 dB 22.1 ± 5.2 15.3 ± 3.1 15.9 ± 3.3 15.0 ± 2.7 For MD −20.01 dB and worse 23.9 ± 6.5 15.5 ± 3.6 15.2 ± 3.1 14.8 ± 3.8 Drops (number of) 3, 0–5 0, 0–3* 1, 0–4* 1, 0–4* Mean deviation (dB) −19.6 ± 5.5 −18.8 ± 5.6 −18.0 ± 10.5 −19.8 ± 4.4 IOP and MD data is presented as mean ± SD, while Drops data as median and range. Asterisk denotes significant difference with pre-op IOP intraocular pressure, Drops number of medications, MD mean deviation, Y1 year 1 post-operation, Y2 year 2 post-operation, Y3 year 3 post-operation by their final follow-up. Twenty-three (17%) were pseudophakic pre-viscocanalostomy. by their final follow-up. Twenty-three (17%) were pseudophakic pre-viscocanalostomy. gradually decreased over the follow-up period. Fifty-two pa- tients were able to complete a reliable VF by final follow-up. In these patients, MD was not significantly different across time points (p = 0.105) compared to pre-op as a group and for individual patients. Complications Four patients (2.9%) had some post-operative complication following VC, which resolved within 2 weeks and did not cause any visual loss (Table 5). Confounding factors Presenting IOP, age, glaucoma type and glaucoma duration and having previous surgery did not comprise a sufficiently good model that could predict the intervention (Yag GP, p = 0.128) or intra- and post-operative complications following surgery (p = 0.175). There was no significant difference noted in the out- come between VC and phaco-VC (p = 0.313). All descriptive statistics of the above variables can be found in Table 1. Cataract surgery The risk of loss of central vision in patients with advanced VF loss ranges from rare to as high as 14% [16, 17]. This may be attributable to readily identifiable compli- cations including cataract, cystoid macular edema, suprachoroidal and vitreous haemorrhage, endophthalmitis and uveitis or be unexplained (wipe out). The exact mech- anism of the Bwipe out^ phenomenon remains elusive, but has been linked to sudden intra-operative hypotony resulting in optic nerve haemorrhage and decreased perfu- sion pressure to an already compromised nerve [18]. The lower rates of drop in vision in the present study reflect the benefit of avoiding sudden decompression with VC in eyes with end-stage glaucoma. Trabeculectomy is still considered the gold standard and achieves better control of IOP than VC [14]. The benefits of NPGS however are potential gains for the patient in terms of their quality of life and reduced likelihood for post-operative interventions and sight-threatening compli- cations [14]. Kirwan et al. [15] in a recent multicenter analysis of current trabeculectomy practice reported the requirement for frequent post-operative interventions in the majority of patients concluding that completion of trabeculectomy is just the beginning of a process that takes several months to complete. There are no like-for-like trials and limited published data to compare our results to those for trabeculectomy or NPGS in a similar patient cohort. Stead and King’s [7] results for trabeculectomy augmented with MMC in advanced glaucoma fare better in terms of IOP control compared to our group. However, with regard to post-operative interventions, 79.8% patients had some form of bleb manipulation [7] compared to only 8.1% in our study that had Yag GP. Reduced VA is a well-recognised complication of glau- coma surgery and might be due to glaucoma progression, Cataract formation is a reported complication after trabeculectomy and can be in the order of 78% [19]. Cataract surgery Pre-op, pre-operative; M6, month 6 post-operative (not consid- ered in statistical analysis); Y1, year 1 post-operation; Y2, year 2 post- operation; Y3, year 3 post-operation Graefes Arch Clin Exp Ophthalmol (2018) 256:1481–1487 1485 Table 3 Complete and qualified success for IOP ≤16 mmHg and IOP ≤21 mmHg (number and percentage) Y1 Y2 Y3 IOP ≤16 mmHg with no medication 60/135 (44.8%) 41/109 (37.6%) 23/75 (30.6%) IOP ≤16 mmHg with additional medication 90/135(66.6%) 72/109 (66.05%) 45/75 (60.0%) IOP ≤21 mmHg with no medication 71/135 (52.5%) 53/109 (48.6%) 23/75 (30.6%) IOP ≤21 mmHg with additional medication 129/135(95.66%) 96/109 (90.6%) 60/75 (80%) IOP intraocular pressure, Y1 year 1 post-operation, Y2 year 2 post-operation, Y3 year 3 post-operation Graefes Arch Clin Exp Ophthalmol (2018) 256:1481–1487 1485 Table 3 Complete and qualified success for IOP ≤16 mmHg and IOP ≤21 mmHg (number and percentage) Y1 Y2 Y3 IOP ≤16 mmHg with no medication 60/135 (44.8%) 41/109 (37.6%) 23/75 (30.6%) IOP ≤16 mmHg with additional medication 90/135(66.6%) 72/109 (66.05%) 45/75 (60.0%) IOP ≤21 mmHg with no medication 71/135 (52.5%) 53/109 (48.6%) 23/75 (30.6%) IOP ≤21 mmHg with additional medication 129/135(95.66%) 96/109 (90.6%) 60/75 (80%) IOP intraocular pressure, Y1 year 1 post-operation, Y2 year 2 post-operation, Y3 year 3 post-operation IOP intraocular pressure, Y1 year 1 post-operation, Y2 year 2 post-operation, Y3 year 3 post-operation success rates for VC in the present study. We have previ- ously augmented VC with MMC in high-risk eyes [9] but did not find a difference in outcome when compared to un- augmented VC [9, 10], which suggests a possible bleb- independent mechanism for the success of VC. comorbidity or the procedure itself. Kirwan et al. [15] re- ported the outcomes of 428 trabeculectomies. Fifteen per- cent had lost > 1 Snellen line at 1 year and 6% had lost > 2 Snellen lines by 2 years post-trabeculectomy (13% with advanced visual field loss). Twenty-seven percent of pa- tients in Stead and King’s study experienced a loss of two or more lines of Snellen acuity [7]. Nine patients (6.7%) in our study experienced a loss of > 2 Snellen lines. Eight of these nine patients had a MD worse than 20 dB. The drop in vision was attributed to glaucomatous progression in seven eyes (5.2%), two (1.5%) of which were presumed to be a wipe out. Cataract surgery We report our results with un-augmented VC in a cohort of patients with advanced glaucoma. VC was able to achieve an IOP ≤21 mmHg in 80 (3 years) to 95% (1 year) patients with a 35–39% drop in IOP from baseline with a good safety profile. To our knowledge, our study provides the largest number of eyes with the longest follow-up yet reported for VC in patients with advanced glaucoma. Three (5%) out of the 59 patients, who underwent VC and were phakic at the time of surgery, underwent cataract surgery Fig. 1 Mean intraocular pressure (IOP) and median number of drops (Drops) plotted against the time points. Error bars have been excluded for clarity. Asterisk denotes significant difference with pre-operative values. Pre-op, pre-operative; M6, month 6 post-operative (not consid- ered in statistical analysis); Y1, year 1 post-operation; Y2, year 2 post- operation; Y3, year 3 post-operation There is limited evidence for the outcomes of glaucoma surgery for advanced glaucoma and no recent studies reporting the outcomes of NPGS for this cohort of patients. Ates et al. [13], in 1999, reported their experience of deep sclerectomy with collagen implant in 54 eyes with ad- vanced glaucoma with 96.2% patients maintaining an IOP < 18 mmHg over 2 years. In our cohort, IOP reduction was maintained significantly below pre-operation levels up to 3 years after surgery. We have previously reported qual- ified (87.5–90.2%) and complete success (78–90%) rates in a cohort of patients with POAG [10]. Shaarawy et al. [11] have previously reported a 90% qualified and 60% complete success rate at 5 years with VC. It is likely that Schlemm’s canal sclerosis and collapse with advanced dis- ease is the most likely explanation for the lower complete Fig. 1 Mean intraocular pressure (IOP) and median number of drops (Drops) plotted against the time points. Error bars have been excluded for clarity. Asterisk denotes significant difference with pre-operative values. U/C unable to perform reliable fields, POAG primary open-angle glaucoma, FHC Fuchs’ heterochromic cyclitis, PXFG p U/C unable to perform reliable fields, POAG primary open-angle glaucoma, FHC Fuchs’ heterochromic cyclitis, PXFG pseudoexfoliation glaucoma Cataract surgery More Table 4 Details of failures No Type of glaucoma Previous surgery/laser Pre-op IOP (mmHg) Pre-op MD (dB) Pre-op BCVA Pre-op drops (No) BCVA at final follow-up MD (dB) at final follow-up Time of failure post-op (months) 1 FHC Nil 20 U/C 6/60 3 6/60 U/C 24 2 FHC Nil 32 −18.59 6/12 3 6/18 −21.54 36 3 POAG Nil 28 −25.7 6/6 1 6/9 −29.23 36 4 PXFG Nil 39 −27.48 6/36 5 6/36 U/C 24 5 POAG Previous VC 30 −12.87 6/6 4 6/9 −14.85 24 6 FHC Nil 28 −23.92 6/12 4 6/18 −25.48 24 7 POAG Nil 24 −21.73 6/6 4 6/9 −22.68 12 8 POAG Nil 24 U/C 6/60 4 6/60 U/C 24 9 Uveitic glaucoma Cyclodiode 28 −14.73 6/24 4 6/36 −16.73 24 10 POAG Nil 25 −16.73 6/6 4 6/6 −17.75 12 11 PXFG Trabeculectomy 16 −21.07 6/12 4 6/12 −22.78 24 U/C unable to perform reliable fields, POAG primary open-angle glaucoma, FHC Fuchs’ heterochromic cyclitis, PXFG pseudoexfoliation glaucoma Graefes Arch Clin Exp Ophthalmol (2018) 256:1481–1487 1486 Fig. 2 Kaplan-Meier survival plot for complete (defined as IOP ≤ 21 mmHg with no medication) and qualified (defined as IOP ≤ 21 mmHg with additional glaucoma medications) success NPGS techniques have greater safety with a lower risk of complications when compared to trabeculectomy [22–25]. A Cochrane review reported relatively fewer complications with NPGS (17%) compared to trabeculectomy (65%) [14]. The UK national trabeculectomy surgery survey [26] reported ear- ly complications in 46.6% and late complications in 42.3% cases. Only four patients (2.9%) had some post-operative complication in our study, which resolved within 2 weeks with no long-term complications. Visual field MD was not significantly different across time points (p = 0.105) compared to pre-op in our study. However, the number of patients completing a VF declined year on year, with 45.2% of patients finally completing a reliable VF. This may be expected with time [1] and is comparable to previous reports where only 39% of patients with advanced glaucoma were able to complete a reliable VF at 1 year, 35% at 3 years and 17% at 5 years post- trabeculectomy [7, 27]. Fig. Cataract surgery 2 Kaplan-Meier survival plot for complete (defined as IOP ≤ 21 mmHg with no medication) and qualified (defined as IOP ≤ 21 mmHg with additional glaucoma medications) success Of the 11 eyes that failed, 4 had uveitic glaucoma (3 Fuchs’ heterochromic cyclitis (FHC)) and 2 PXFG) both known to have an aggressive course [28]. We have previously reported good outcomes for NPGS in uveitic glaucoma [10]. In this study, eight patients had uveitic glaucoma of which three had FHC, all of which failed. This could be related to the increased likelihood of subclinical neovascularisation and Schlemm’s canal sclerosis in FHC [29]. recent data suggest this to be in the order of 30% [15]. King et al. [7] reported a 63% incidence of cataract requiring surgery in 27% cases. In comparison, only 5% patients in our group required cataract surgery in the study period. Cataract surgery can decrease the success of a trabeculectomy with an in- creased likelihood of post-operative interventions and require- ment for glaucoma medication in 30–39% cases [20, 21]. These risks are avoided with VC due to its bleb-independent mechanism of action. Limitations of this study include the loss of numbers, particularly those able to complete a reliable VF test during the follow-up period. However, to our knowledge, this is the first study to report the effectiveness of un-augmented VC for advanced glaucoma. IOP remained controlled over a period of up to 3 years, albeit with the requirement of increasing medications with time and VA remained stable in the majority of patients. VC had similar qualified suc- cess rate to MMC trabeculectomy with a good safety pro- file, avoidance of MMC and its attendant complications and minimal post-operative interventions. The benefits can also be extrapolated to the wider context of economic and quality of life benefits to be achieved with NPGS [30]. NICE recommends primary surgery in this group of pa- tients [6]. TAGS will address the outcomes of primary trabeculectomy with MMC for advanced glaucoma [8]. Our study supports the extension of the trial to include the use of primary VC for advanced glaucoma. NICE recommends primary surgery should be offered to patients presenting with advanced loss [6]. Stead and King recommend this to be trabeculectomy augmented with MMC [7]. Cataract surgery The use of antimetabolites is a recognised risk factor for bleb-related infection and endophthalmitis [22] and reflects the opinion of UK Consultants’ for not advocating primary surgery for this group [2]. Avoidance of antimetabo- lite use and bleb-related complications with VC is an impor- tant consideration in this group of patients. An advantage of VC is retention of the TDW, which appears to serve as a barrier to infection [22] and allows titrated aqueous flow, thus avoiding hypotony and its complications. Table 5 Intra- and post-operative complications Complication type No. of patients Intra-op TDW* perforation 17/135 Wound conjunctival leak treated with bandage contact lens 2/135 Scleral flap leak repaired with tutoplast 1/135 Wound leak repaired with suturing 1/135 *Trabeculo-Descemet’s window Table 5 Intra- and post-operative complications References 1. Saunders LJ, Russell RA, Kirwan JF, McNaught AI, Crabb DP (2014) Examining visual field loss in patients in glaucoma clinics during their predicted remaining lifetime. Invest Ophthalmol Vis Sci 55:102–109 17. Kolker AE (1977) Visual prognosis in advanced glaucoma: a com- parison of medical and surgical therapy for retention of vision in 101 eyes with advanced glaucoma. Trans Am Ophthalmol Soc 75: 539–555 2. 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https://openalex.org/W4298164325	https://digitalcommons.usf.edu/cgi/viewcontent.cgi?article=1170&context=tlas_pub	Latin	null	Services and Resources to Engineers: A Case Study of Outreach and Marketing, Assessment, and Future Directions in a Research Library.	Issues in science and technology librarianship	2,015	cc-by	6,948	University of South Florida University of South Florida Digital Commons @ University of Digital Commons @ University of South Florida South Florida Academic Services Faculty and Staff Publications Tampa Library Winter 2015 Services and Resources to Engineers: A Case Study of Outreach Services and Resources to Engineers: A Case Study of Outreach and Marketing, Assessment, and Future Directions in a Research and Marketing, Assessment, and Future Directions in a Research Library Library Matt Torrence University of South Florida, torrence@usf.edu University of South Florida University of South Florida Digital Commons @ University of Digital Commons @ University of South Florida South Florida Academic Services Faculty and Staff Publications Tampa Library Winter 2015 Services and Resources to Engineers: A Case Study of Outreach Services and Resources to Engineers: A Case Study of Outreach and Marketing, Assessment, and Future Directions in a Research and Marketing, Assessment, and Future Directions in a Research Library Library Matt Torrence University of South Florida, torrence@usf.edu Follow this and additional works at: https://digitalcommons.usf.edu/tlas_pub Follow this and additional works at: https://digitalcommons.usf.edu/tlas_pub Part of the Engineering Commons, and the Library and Information Science Commons Part of the Engineering Commons, and the Library and Information Science Commo Scholar Commons Citation Scholar Commons Citation Torrence, Matt, "Services and Resources to Engineers: A Case Study of Outreach and Marketing, Assessment, and Future Directions in a Research Library" (2015). Academic Services Faculty and Staff Publications. 170. https://digitalcommons.usf.edu/tlas_pub/170 This Article is brought to you for free and open access by the Tampa Library at Digital Commons @ University of South Florida. It has been accepted for inclusion in Academic Services Faculty and Staff Publications by an authorized administrator of Digital Commons @ University of South Florida. For more information, please contact digitalcommons@usf.edu. Winter 2015 Issues in Science and Technology Librarianship DOI:10.5062/F43776Q0 Introduction The literature and research on the acquisition of knowledge by science and engineering students and faculty have demonstrated a number of clear and effective methods of outreach and library partnerships. Even the limited resources of a single subject specialist can provide advanced individual and group research services in concert with the requisite other professional librarian duties, such as collection development and library instruction. The focus of this article is the examination of the current promotion and provision of library research services to the science and engineering clientele, as well as a brief discussion on possible future improvements and applications. Services and Resources to Engineers: A Case Study of Outreach and Marketing, Assessment, and Future Directions in a Research Library Matt Torrence Academic Services Librarian for Science, Technology, Engineering, & Mathematics (STEM) Tampa Library University of South Florida Tampa, Florida torrence@usf.edu Abstract The literature, activities, and resource needs of engineering students and faculty provide insight into a demographic that is often among the earlyadopters of new technologies, tools, and methods of sharing information. Despite the often nonbibliographic nature of their research efforts, there are numerous elements of the traditional service model that remain relevant and integral to the engineering research patron base. New methods of in person and virtual instruction, the improved promotion of research consultation, and data management are prime targets for efficient and effective service updates. This article outlines the current engineering research environment, using a large research institution as a model and case study for evaluation and future planning for outreach and service marketing. Literature Review Studies of information seeking behavior of engineers show us their current preferences and research practices. Hemminger, et al., at UNC Chapel Hill (2007) found that faculty in the sciences have largely transitioned to free and subscription electronic search engines for their initial approach to searching, especially Google Scholar and other metasearch tools. This endeavor was followed by a national and multiinstitution distribution of the survey by Niu, et al., (2010) that found evidence that science researchers are heavily in favor of electronic means for searching and material delivery, but largescale use of unified institutional repositories and other digital means of scholarly communication are progressing at a much slower rate. Tucci (2011) investigated similar variables in the measure of research preferences and practices and noted the additional, perhaps unintended, importance of the study instrument as an opportunity to market library services and to collect data. This observation by Tucci emphasizes the importance of never missing an opportunity to assess and integrate the library's role, as well as that of the librarian, in the activities of science researchers and scholars. In much the same way, Brush (2010) used clickers to 1 evaluate the information needs of firstyear engineering students following instruction, while also making library services and resources increasingly visible and highlighting the importance of early and repeated library contact with science undergraduates. Brush also assessed audience response methods with engineering students and more traditional library orientation efforts. The science instruction offered at the USF Tampa Library will be discussed as it compares to these published examples. evaluate the information needs of firstyear engineering students following instruction, while also making library services and resources increasingly visible and highlighting the importance of early and repeated library contact with science undergraduates. Brush also assessed audience response methods with engineering students and more traditional library orientation efforts. The science instruction offered at the USF Tampa Library will be discussed as it compares to these published examples. Nesdill, Love, and Hunt (2010) offered an excellent review of the fiscal and staffing challenges facing providers of reference, as well as their own story of success in serving the sciences through a formalized team approach within the library. They emphasize the need to coordinate public services to compensate for the reduced number of subject specialists. Literature Review As with many libraries, staffing situations reported in their research led to a combination servicepoint approach; they described the move to a tiered system, allowing staff to respond to simple questions and referring up the chain to librarians and subject specialists for more advanced reference inquiries. They outlined the importance of unhinging from past service practices that have become impediments to progress. Lubker et al. (2010) discussed the concept of providing reference and research services in the halls and offices of the researchers, rather than waiting for them to visit specific libraries or librarian offices. Hunter, et al. (2010), focused on the combination of service provision by the Information Technology (IT) department and Reference to support "the full research lifecycle" in serving their engineering and science faculty clientele. Oxnam (2010) emphasized other types of these collaborative service points among specialized science and engineering libraries, as well as the federal governments' efforts to harvest and efficiently disseminate Government Printing Office (GPO) and other technical information integral to university and academic research. These reductions in resources are commonly found in the literature, but the ability to improvise and create partnerships in information sharing emerged as another central theme. Other inspiration comes from Nelson's 2007 article on engineering reference and information practices. This work provides an excellent survey of library activities related to outreach services, as well as documentation of projects initiated to establish satellite locations. Even in cases of unsuccessful efforts and innovations, much was learned and this, too, will be further discussed by reviewing similar efforts at the USF Tampa. The Environment at the University of South Florida This cohesive and organized effort allows for a common theme, but offers flexibility of emphasis for various programs and subject areas. The provision of electronic journals, databases, and other resources is integral to all fields of research, especially for the engineering faculty and students. The USF Libraries have access to most major science and engineering databases, including a comprehensive journal and book package from the publisher Springer and the complete ISI Web of Knowledge database (which includes the Journal Citation Reports, BIOSIS Previews, and the Web of Science resources). Regularly scheduled collection of this statistical information using WorldCat Collection Analysis Tool, often for accreditation purposes, illustrates the relative strength of USF's electronic subscriptions compare well to those of the peer and aspirant institutions cited by USF and the College of Engineering. The changes to the overall format and acquisition of collections have also been quite pronounced. A review of USF Library acquisition trends shows that in 20092010, 438,564 ebooks were available to authenticated affiliated patrons, which represents a rise of 196% from the 20042005 total of 147,943 e books in the USF Libraries collections. The numbers for ejournal collections are comparatively similar; ejournals from 20042005 totaled 23,986 titles and $1,560,804 in subscription costs and for 20092010, these figures rise to 77,884 titles at $2,881,439.53 (USF Libraries Academic Resources Annual Statistical Summary 2013). This is an increase of 225% in total number of titles and 85% in expenditures. As with many other large academic institutions, the USF Library has participated in several administrations of the LibQual+ survey, including the collection of data in February of 2011. The survey information provided by LibQual+ is valuable across subjects and patron types, but it must be carefully used. In many cases, there was no simple way to isolate the results for Engineering respondents, but it was possible to make inferences on the needs of this specific population. Many of the patterns and measures that will be discussed in the following paragraphs hold true across disciplines, and may be cautiously applied to engineering faculty and their activities. As evidenced by Hemminger, et al. (2007), and Niu, et al. (2010), there is a strong preference among engineering academics for selfdiscovery and for materials in electronic format. A particularly illustrative and brief example from LibQual+ deals with the specific question "A library Web site enabling me to locate information on my own". The Environment at the University of South Florida The USF College of Engineering is relatively large, consisting of six departments, 2,992 undergraduates, 789 graduate students, and 176 faculty members (University of South Florida InfoCenter 2013). The goal of effective communication with this population necessitates the standardization of research service offerings, while simultaneously suggesting new and innovative resources to a population of researchers heavily focused on the immediacy of new data and publications. There are over 700 electronic databases provided and managed by the USF Libraries and the USF Tampa Library. These resources are most commonly accessed by title, by subject using the MetaLIB system, or by the LibGuides designed specifically for engineering subject and course requirements. In the Engineering category, there are 83 online resources available, with further breakdown by type and/or subject content. Table 1 outlines current examples of the most popular USF resources in these areas and their levels of use. Database name Sessions Searches Fulltext Downloads ACS Publications – Vendor: American Chemical Society 26,431 11,218 49,875 Compendex (1884 ) (Engineering Village) – Vendor: Elsevier 2,129 7,887 NA IEEE Xplore – Vendor: IEEE NA 28,632 20,247 ScienceDirect – Vendor: Elsevier 186,696 66,531 460,313 Wiley Interscience – Vendor: Wiley 8,918 NA 32,251 ACM Computing Reviews – Vendor: ACM 19,826 3,633 NA Web of Knowledge – Vendor: Thomson Corp. 12,016 36,863 NA SpringerLink – Vendor: Springer NA NA 47,722 IoP Electronic Journals – Vendor: Institute of Physics 3,118 N/A 2,556 Table 1 2 Use of all of these and other major electronic engineering resources is extremely high, which helps to justify the spending for researchlevel resources in a number of areas within computer and engineering research. The USF Libraries pays over $6 million annually for resources, including almost $2 million for the following list of databases (USF Libraries Academic Resources Annual Statistical Summary 2013). In addition to prudent proactive individual purchases, the USF Libraries leverage consortia buying with effective negotiation and the packaging of resources to ensure maximum product per research dollar. This information is also packaged and regularly disseminated to engineering faculty using emails, blogs, and other forms of communication. The librarians and staff are constantly refining and honing these messages, including the method(s) of delivery, in both individual and group ways. The staff includes an expert in marketing and development and this person is available for consultation and uniformity of message in the delivery of electronic messages and newsletters. The Environment at the University of South Florida This represents one of the highest of the desired mean questions in the Information Control (IC) component in the 2011 results for USF and for a combination of ARL respondents. When comparing graduate student populations, for example, these individuals at USF report a minimum expectation of 7.43 (on a standard scale of 19), they perceive a score of 6.95, and they desire an 8.56. These numbers result in an adequacy figure of 0.48 (Association of Research Libraries & Texas A&M University 2011a). The ARL population of graduate students is slightly more forgiving, but only by a small margin. They report a minimum expectation of a 7.09, a perceived score of 7.21, and a desired level of 8.35 (Association of Research Libraries & Texas A&M University 2011b). This results in an adequacy margin of +0.12, but the most important facet of this example is the similarity of these numbers across surveys and populations. Libraries & Texas A&M University 2011a). The ARL population of graduate students is slightly more forgiving, but only by a small margin. They report a minimum expectation of a 7.09, a perceived score of 7.21, and a desired level of 8.35 (Association of Research Libraries & Texas A&M University 2011b). This results in an adequacy margin of +0.12, but the most important facet of this example is the similarity of these numbers across surveys and populations. When looking at more of the IC elements in both the USF and ARL results, the patterns of strong demand for easy and independent research are similar and pervasive. This data points even more directly to a growing need for effective and intuitive individual access to materials, primarily in electronic format. These results provided an extra incentive for the renovation of the USF Libraries web site, as well as the coordination and evaluation of other forms of marketing to the Engineering and other research populations. Early usability testing and feedback have encouraged the library's web team to continue the efforts to simplify, improve, and update the site. A final element of the current environment is a good look at the landscape. In 2009, all subject specialists in the USF Libraries prepared detailed environmental scans of the research efforts, publications, and grants of the faculty and other scholars. Table 2 The College of Engineering used the data to focus on new electronic resources and possible targets for instruction and the data directed the library to specific faculty for further consultation on collection and service needs. The use of additional collection analysis and development tools, such as WorldCat Collection Analysis Tool and Goldrush, allowed the identification and purchase of resources related to these and other specific areas of research centralization. This led to an expansion of the collection in the fields of Engineering Education, which appears on several department lists, as well as a variety of other topic and call number areas, such as Image Processing, Pattern Recognition, Artificial Intelligence, and Computer Vision. These areas also emerged as the topic research clusters and areas of emphasis for grants and faculty effort. After identifying peer and aspirant institutions, both inside and outside our current consortia units, comparisons of book and journal holdings allowed for the easy identification of core collection additions, as well as those more unique items necessary for distinct research areas. With fewer funds available for proactive collection development, making sure that new titles are relevant to the key research topics and personnel is of the utmost importance. The Environment at the University of South Florida This project was initiated as a quantitative and qualitative effort to identify priorities in teaching and external funding to more efficiently direct library A final element of the current environment is a good look at the landscape. In 2009, all subject specialists in the USF Libraries prepared detailed environmental scans of the research efforts, publications, and grants of the faculty and other scholars. This project was initiated as a quantitative and qualitative effort to identify priorities in teaching and external funding to more efficiently direct library 3 efforts and resource dollars. Tables were created for each department to correlate LC call number classes to specific subjects, classes (both undergraduate and graduate), and areas of research and grant focus that were gleaned from student, faculty, and USF sites. Here is an example (Table 2) for the department of Chemical & Biomedical Engineering: efforts and resource dollars. Tables were created for each department to correlate LC call number classes to specific subjects, classes (both undergraduate and graduate), and areas of research and grant focus that were gleaned from student, faculty, and USF sites. Here is an example (Table 2) for the department of Chemical & Biomedical Engineering: Class Subject Area OR Topic UG Courses Grad Courses Grad Topics Faculty Topics Gran Area QH573 671 Electrofusion/electrogenetheraphy (cytology) 2 QP301 336 Biomechanics 1 2 QP431 495 Human sensory processes/perception 1 1 QP519.7 519.9 Electrophoresis 1 R850 854 Biomedical systems/instrumentation 2 2 R856857 Biomaterials 6 3 R856857 and TA166167 Biomedical engineering 1 7 1 RC270.8 271 and RM260263 Chemotherapy/Electrochemotherapy 1 RD597 598.7 and WG (medical heading) Cardiovascular mechanics/artificial heart 3 2 2 = 70 (30%) Phone Existing Outreach & Services at the USF Tampa Library As with most libraries, especially a large and general academic library covering a wide variety of subject areas, there are many standard methods employed to promote new and existing research services and collections. In the USF environment, changes in general reference scheduling and resources have resulted in reduced general hours of oncall reference services at the physical library locations. The positive impact of these changes was the opportunity to apply these additional available hours of effort to new and innovative virtual reference services, all types of instruction and consultation services, and new modes of outreach and communication with the clientele. A number of individual forces were also affecting these changes, including staffing levels, statistical evaluation of service points and resource use, and LibQual+ and other environmental scanning endeavors. Another interesting trend was the rise in individual research consultation requests for engineers. The two sets of data in Table 3 represent various methods of contact and categories of research need for the month of September 2010 (our busiest month in the Fall semester) and September 2011. The 2010 data was collected just prior to the implementation of a new and updated library web site, which improved the visibility and availability of USF Library research services through more prominent links, blogs, and other methods of online communication and marketing to faculty and students. The second set of numbers represents the comparable data for September of 2011, following the launch of the new site and the concurrent promotion of the more numerous and accessible library services. Existing Outreach & Services at the USF Tampa Library September 2010 Contacts/Consultations: 233 Top methods of contact: 1 = 103 (44%) Face to Face 2 = 70 (30%) Phone 2 = 70 (30%) Phone 4 3 = 60 (26%) Email Top categories for purpose of contact: 1 = 33 (37%) Basic Reference Assistance 2 = 16 (18%) Advanced Reference Assistance/Research Consultation 3 = 12 (13%) Instructional Support 4 = 11 (12%) Vendor Communications September 2011 Contacts/Consultations: 174 Top methods of contact: 1 = 86 (49%) Email 2 = 62 (36%) Face to Face 3 = 26 (15%) Phone Top categories for purpose of contact: 1 = 39 (39%) Basic Reference Assistance 2 = 33 (33%) Advanced Reference Assistance/Research Consultation 3 = 10 (10%) Vendor Communications 4 = 6 (6%) Instructional Support Table 3 3 = 60 (26%) Email The combination of dramatic improvements to the library web site, better design and testing, and additional prominence of the virtual and personalized services on this upgraded platform suggested a positive influence on the ability of clients to be selfsufficient and find their library subject expert. The total number of interactions between USF affiliates and the engineering librarian was reduced by 25%, but this may have been partly due to a reduction in the overall number of hours spent working at standard reference and service desks. The number of research consultations during this same period rose by almost 100%. Enhancements to the organization of the site, the continued standardization of subject and course pages, and other planning and usability testing appear to have positively affected the availability and value of individual research assistance services, as well as the use by the science research community. The preference for mode of contact over this time period also demonstrated a dramatic change. In terms of staffing, individually assigned librarian desk hours in 2011 were reduced by an average of 1520%, which does account for some of this statistical change. It is likely that this is evidence of the desire for virtual assistance and, as hinted in LibQual+ results, further development of the increasingly self sufficient faculty researcher in these subject areas. With more and more of the advanced research clients using the increased email, chat, and other virtual reference services, or contacting their subject specialist directly, there are more available resources to better serve the needs of comprehensive and subject specific populations with other virtual reference services. Existing Outreach & Services at the USF Tampa Library The USF Libraries offered extensive and responsive email, chat, and other virtual modes of communication. The addition of links to the branded "AskALibrarian" electronic reference services, along with the additional hours of service gained by participating in an academic chat collaborative, warrant intensive future planning and assessment. Instruction, LibGuides, and Other Measures There were over 26 instructional sessions performed for courses in the College of Engineering in 2012 2013, including undergraduate and graduate classes, and this is generally indicative of the annual demand for library instruction from the various engineering departments. In addition to these individual class sessions, the library partnered with other USF groups to provide science and citation management workshops and other helpful information and research sessions. Demand tends to be heaviest in particular core courses, a specific example of which is detailed below, as well as in certain fields of engineering. The class titled ENC 3246: Communication for Engineers provided an opportunity to survey the students about the current and future professional value of information and information literacy. With multiple sections of this required class taught every semester, this represented an excellent opportunity to assess the needs of a current audience of students, some of whom will make up a future audience of faculty and industry clients. While these surveys are rough and informal in nature, the aggregate value (140 survey participants over the last two years) has provided ample discussion and assessment data for the core instructors teaching this class. A goal for the near future is to create a more formal program that extends the library and librarian relationship beyond this single class. The numbers in the following paragraphs will outline many of the successes of the instructional and virtual approach to a large portion 5 of the undergraduate engineering student population. Making sure they return to the library for additional information service and access remains a challenge to execute and to measure. The content of the survey was a simple sevenquestion instrument asking participants to estimate their presession library skills, assess the level and content of the session, and comment on areas of the workshop in need of improvement or clarification. Over the first two years of consistent survey administration, there have been very few openended comments, but extraordinarily high levels of response. The likely reasons for this are numerous, but the obvious cause of response success is the encouragement (and just short of requirement) of the instructors in getting their students to complete the instrument. The short nature of the survey, combined with positive reinforcement for completion, aid dramatically in the collection of the data. Instruction, LibGuides, and Other Measures A small change was made in the second year of survey administration and this allowed for more time to complete the survey. The additional time and explanation have assisted in the collection of more useful commentary by an increased number of survey participants. After discussion with the various instructors, the administration of the survey is now performed closer to the end of the semester and directly following submission of their final written product. There's more data to be collected and evaluated, but the early returns are positive. This will remain difficult to objectively quantify, but the acknowledgement of library value by this group of engineering undergraduates is a positive sign for this type of teaching and marketing effort. Each issuance of the survey has resulted in extremely positive impressions in terms of session value. The collected responses for all semesters demonstrate over 90% of the participants indicated their research skills had improved as a result of the session. These figures further support the value of collaborative instruction in one or more central and/or required undergraduate and graduate class sessions. The ability and occasion to contact second and thirdyear engineering students at a relevant point of need is critical. A majority of instructors for this course follow a standard curriculum and the program coordinator has afforded the library the chance to select and integrate resources into a LibGuide specific to this course. A link to this page was added to the BlackBoard portal for almost all sections. The ability to affect the syllabus, the types of tools and instruction afforded to the students, and the chance to assist engineers in a traditional (i.e., secondary and bibliographic) manner helped plant the seed for graduate and professional use of library resources and advanced services. LibGuides were also a great help in the simple and efficient estimation, at least in part, of the impact of both group and individual library instruction. The LibGuide created specifically for ENC 3246, the most likely course for an engineer to receive library instruction, has been used, assessed via survey, and updated every semester following its launch in the fall of 2009. This online course guide demonstrates high overall use and has tested well with several cohorts of students and instructors. Instruction, LibGuides, and Other Measures With 1,008 hits in 2010, 1,137 in 2011, and 1,520 in 2012, it's among the most consistently used course guides on the USF site and the numbers are on the rise. It's likely that those attending class sessions are producing the majority of these numbers, but student contacts and faculty feedback have provided evidence that instructors teaching multiple sections of ENC 3246 are heavily promoting the use of this guide to their students. There have been many documented cases of direct communication from participants these courses, as well as contact from course participants at other USF campus locations that are not receiving direct library instruction. Despite emails to faculty, blog announcements, and other marketing efforts, the general engineering subject guide has garnered 1,252 hits in 2012; a bit fewer than anticipated based on the population that should ideally be interested in using this page as a research starting point. As a point of comparison, the College of Education counted a total of 3,279 students (compared to 3,784 in Engineering according to USF's InfoCenter) and the main LibGuide for this college received 12,489 hits. There was far more embedded librarianship and instruction going on with the College of Education, but there remains a strong desire to get the demonstrated use of this Engineering gateway a bit higher. And while the current numbers are not exactly great news, it showed increased use of library resources with the direct connection to classroom assignments at the undergraduate level. With the complete list of engineering databases also available via our master list on the MetaLIB platform, it's possible that some students and researchers were missing the value in these subject and course portals. Where holes and failures were made visible, it simply means marketing opportunities await. The use of LibGuides and the redesign of the web site to enhance the visibility of new marketing initiatives have proved an invaluable tool in the battle to maintain a high ratio of value to effort. The addition of a new and experienced individual for the development and coordination of the library site brought not only an increase in available technical skills, but also observations from the outside/corporate world. This input further enhanced the organization, design, and visibility of the subject and course LibGuides in the new 2011 version of the USF Libraries web site. Piloting & Assessing New Methods Throughout the academic years 2006 and 2008, the USF Tampa Library and several of the librarians, embarked on a pilot project to provide ondemand reference services to students and faculty outside the library walls. The statistics, even if slightly dated, still tell an interesting part of the tale, but it remains hard to qualify these efforts as a comprehensive success, or failure. Despite initial benefits in the form of serendipitous faculty and student contacts, asking librarians to provide two to four hours of weekly onsite reference services was received with mixed results. While Lubker, et al., (2010) were able to employ this model and enjoy success in the specialized medical library environment, efforts to reach out from a large multidisciplinary library to locations in the buildings of the College of Art, the Natural & Environmental Sciences, and Engineering were not as productive. The USF pilot of this service was launched in early 2006 and continued through to the end of 2008. The Spring 2007 semester was the busiest for the service point at the College of Engineering, but still yielded only nine complex and 12 simple inquiries over a total of 28 service hours for the term. At less than one question per hour, the project was eliminated and these efforts turned to other points of focus, such as virtual reference and oncall consultation services. Despite low use, there may have been benefits to this attempt, even if only to help break the librarian stereotype. In the case of providing satellite reference stations, the gains were small, but educational. This program was not particularly successful in creating demand, at least at this institution and the locations attempted, but there was some serendipity associated with geography. especially in terms of faculty interaction which lead to new contacts for instruction, research guidance, and collaboration. The satellite service was halted in 2008 allowing the USF Libraries to move forward with other more effective marketing and promotional efforts. Blogs are another common tool employed by subject specialists at the USF Libraries in a variety of news and scholarly applications. From 2008 to 2011, this author contributed heavily to the content and dissemination of the STM (for Science, Technology, and Mathematics) blog created by colleagues to serve the combined populations of the sciences. It was, in the early days, updated regularly with content useful to all areas science and engineering faculty and students. Instruction, LibGuides, and Other Measures As evidenced by increased overall traffic to the LibGuides, the new design has allowed for better discovery of the 6 engineering and other subject portals. The previous site exhibited far less overall standardization and developmental usability testing, as well as less formal organization and guidelines for the creation and development of subject and course pages using the LibGuides platform. engineering and other subject portals. The previous site exhibited far less overall standardization and developmental usability testing, as well as less formal organization and guidelines for the creation and development of subject and course pages using the LibGuides platform. Other important areas of instruction and outreach include graduate and new faculty orientations, which were often easy opportunities to promote new databases and service initiatives. It's also common to use the role of collection liaison to gain contact with the faculty and insight into their research. Identifying areas of crossover with engineering, or new clusters of research formed with interdisciplinary faculty, is another approach to consider when courting new populations for research services. At USF, these areas are identified by new classes, degrees, and research centers in areas that overlap with science and technology and include the programs of entrepreneurship, medicine, public health, and more. Many of these clients are traditional and existing library users, but these new and hybrid programs represent another great chance to sell the library and its professional information services. These simple acts of additional communication, as well as the ability of these faculty groups to directly express research and collection needs, may be used to spread the word about available library expertise. Conclusions and Future Planning The relative successes and moderate failures described above have all been beneficial to the current and future marketing and dissemination of new engineering resources and research services. The literature and LibQual+ survey results have demonstrated a strong preference for the electronic searching and delivery of research materials, which is serendipitously encouraged by the changing structure of collection development. With reduced discretionary funding available for proactive purchase and acquisition, making sure what is purchased gets used and what is needed gets purchased is of the utmost importance. Continued development of new modes for the electronic marketing of resources and services is a given, but on the horizon are additional opportunities for automation and collaboration. At the USF Tampa Library, plans are in place for additional automation in collection building and the delivery of reference services. The staff in the Academic Resources department has implemented a purchaseondemand system for identifying and acquiring new monographic materials. By partnering with the vendors, plans have been implemented that act behind the scenes to bring patrons the books and other items they need. This is a strong benefit to engineering patrons, as they tend to be among the academic populations most interested in selfsufficiency and electronic access (Niu, et al. 2010). The USF LibQual+ results for 2011 demonstrate that faculty and graduate students, the two largest research populations, place a large emphasis on "A library Web site enabling me to locate information on my own". The library is still showing a deficiency in this area for both populations, at 0.53 for faculty and 0.48 for graduate students (LibQual+ Survey: University of South Florida 2011). With the further evolution and development of SFX, as well as the future consideration of a discovery tool and improved federated searching, these numbers should improve by the next rounds of library assessment. Rather than attempt to predict the direction of research collections using approval plans and predictive purchases, the relevant populations in science and engineering will be able to guide the collections with their research needs using these and other traditional services, such as interlibrary loan and direct request of new materials. In conjunction with Google Scholar, the only drawback to these increasingly seamless finding aids is that the users often don't seem to know that the library is involved in the wonderful list of resources that are quickly and magically accessible. As previously documented by Hemminger, et al. Piloting & Assessing New Methods While well designed and heavily publicized via emails, word of mouth, signage, and various other means, the usage statistics for this virtual publication never reached that of its counterpart efforts in the College of Education (the EdLib Report). As the hits trailed off, so did the frequency of update and, consequently, the value to the engineering scholar. This author's own efforts to create a regular audience through email blasts, signage in the college, and direct communication to faculty were ineffective, but the passage of time has inspired another try. The availability of new and more effective blog and electronic dissemination platforms, enhanced technical assistance, and increased promotional expertise through more organized communication efforts prompted the USF Tampa Library to take another swing at blogging in the sciences and other subject areas. A new engineering missive, titled The Library Engineer's Report, was launched in October of 2011. The use of the WordPress platform, better web design, and content management and message coordination by a new library web master should prove to be beneficial to patrons. As with the work of Niu, et al. (2010), this researcher found this form of informal scholarly communication to be of only minor impact, at least based on the early returns. The total number of unique page views for October 2011 to July 2013 was 278; there is potential for more investigation into the efficacy of blogs as a means of effective information exchange with science faculty. 7 Joint vendorlibrary workshops are also underway at the USF Libraries and in October 2011, the USF Tampa Library featured several IEEE Xplore workshops cohosted by both corporate representatives and librarians. These sessions are interesting not just due to their collaborative nature, but due to the fact that the sessions are being marketed to engineers, artists, and other multidisciplinary areas of the university. Many of the databases and other resources listed under the heading of engineering have much to offer other researchers and there is a great deal the engineering scholar can glean from using databases outside their subject designation. Crossover use and marketing of these new and increasingly subjectblended materials will also be of utmost importance. Conclusions and Future Planning (2007), and Niu, et al. (2010), this type of expanded metasearch is directly in line with the preferences of researchers in the fields of science and engineering. On the service end, there are also plans in place for new and innovative research assistance and the marketing of new resources available using the aforementioned blogs, library workshops, and web site development. A new set of basic, advanced, and subject/resource specific library workshops are currently being piloted under the name "Research Rescue". These sessions are offered in both physical and virtual (Elluminate) settings and cover a variety of subject and resource areas, including Engineering, citation management, and vendor/library partnered workshops. While in the early stages of development, initial offerings have been well received by engineering students and faculty. Marketing of the first set of sessions has been cautiously effective, with over 42 students, faculty, and other researchers attending the voluntary advanced library sessions covering offered specifically to the College of Engineering. As stated above, improved means of communication and message delivery stand ready to further improve these statistics and the library's rate of return on marketing efforts. Other items on the horizon include plans to automate the inclusion of subject and course LibGuides into the BlackBoard/CANVAS course mechanisms. Work is currently underway to partner with IT to ensure the effective and efficient management of mapping these relationships. This will benefit the engineers in multiple ways, especially with the ability to centralize access to library resources in a virtual spot where authentication has already been established. Further review and enhancement of the site is also a major part of any future planning efforts and will additionally benefit the marketing of new services and resources. The establishment of focus groups is currently underway to better collect information and serve the engineering scholar. The final points of focus for the improvement of outreach and marketing to science and engineering patrons involve stressing the importance of library partnerships and collaborative efforts. As mentioned 8 above with IT and course shell efforts, the library resources and staff must continue to prove their relevance and value. Colleagues at the USF Libraries are at work creating and marketing a new institutional repository and mode of data management to help cement a universitywide niche for the library in this arena of scholarly communication. Conclusions and Future Planning By cultivating partnerships with other educational units, such as Tutoring & Learning and the USF Writing Center, the USF Libraries will continue to increase demand for valueadded information services for engineering and other research clients. References Association of Research Libraries & Texas A&M University. 2011a. LibQual+ 2011 survey: University of South Florida University of South Florida, Tampa Library. Washington D.C.: Association of Research Libraries. Association of Research Libraries & Texas A&M University. 2011b. LibQual+ 2011 survey: Association of Research Libraries. Washington D.C.: Association of Research Libraries. Brush, D.A. 2010. Evaluation of an audience response system in library orientations for engineering students. Issues in Science & Technology Librarianship [Internet]. [Cited 2011 Aug 24]; 60 (Winter). Available from: http://www.istl.org/10winter/article1.html Hemminger, B.M., Lu, D., Vaughan, K.T.L., & Adams, S.J. 2007. Information seeking behavior of academic scientists. Journal of the American Society for Information Science and Technology 58(14):220525. Hunter, C., Lake, S., Lee, C., & Sallans, A. 2010. A case study in the evolution of digital services for science and engineering libraries. Journal of Library Administration 50(4):33547. Lubker, I.M., Henderson, M.E., Canevari, C.S., & Wright, B.A. 2010. Refocusing reference services outside the library building: One library's experience. Medical Reference Services Quarterly 29(3):218. Nelson, M.S. 2007. Initiating engineering outreach reference services: Background and practice. Reference Services Review 35(2):26584. Nesdill D., Love A., & Hunt M. 2010. From subject selectors to college and interdisciplinary teams. Science & Technology Libraries 29(4):30714. Niu, X., Hemminger, B.M., Lown, C., Adams, S., Brown, C., Level, A., McLure, M., Powers, A., Tennant, M.R., & Cataldo, T. 2010. National study of information seeking behavior of academic researchers in the United States. Journal of the American Society for Information Science and Technology 61(5):86990. Oxnam, M. 2010. Collaboration and the power of partnership in science and engineering libraries. Journal of Library Administration 50(3):25363. Tucci, V.K. 2011. Assessing informationseeking behavior of computer science and engineering faculty. Issues in Science & Technology Librarianship [Internet]. [Cited 2011 Aug 26]; 64 (Winter). Available from: http://www.istl.org/11winter/refereed5.html USF InfoCenter [Internet]. [Cited 2011 Aug 15]. Tampa (FL): University of South Florida. Available from: http://usfweb3.usf.edu/infocenter/ USF Libraries Academic Resources Annual Statistical Summary. [Internet]. [Cited 2011 Sept 9]. Tampa (FL): University of South Florida Libraries. Available from: http://guides.lib.usf.edu/academicresources This work is licensed under a Creative Commons Attribution 4.0 International License. 9
https://openalex.org/W2141406826	https://www.atmos-chem-phys.net/16/715/2016/acp-16-715-2016.pdf	English	null	Aircraft-measured indirect cloud effects from biomass burning smoke in the Arctic and subarctic	Atmospheric chemistry and physics	2,016	cc-by	20,212	Aircraft-measured indirect cloud effects from biomass burning smoke in the Arctic and subarctic 8Foundation for Research and Technology – Hellas, Patras, Greece 11Institute for Ion Physics and Applied Physics, University of Innsbruck, Innsbruck, Austria 12Paciﬁc Northwest National Laboratory, Richland, WA, USA y pp y y 12Paciﬁc Northwest National Laboratory, Richland, WA, USA Correspondence to: L. M. Zamora (lauren.m.zamora@nasa.gov) Received: 7 August 2015 – Published in Atmos. Chem. Phys. Discuss.: 26 August 2015 Revised: 16 November 2015 – Accepted: 22 December 2015 – Published: 21 January 2016 Received: 7 August 2015 – Published in Atmos. Chem. Phys. Discuss.: 26 August 2015 Revised: 16 November 2015 – Accepted: 22 December 2015 – Published: 21 January 2016 Abstract. The incidence of wildﬁres in the Arctic and sub- arctic is increasing; in boreal North America, for exam- ple, the burned area is expected to increase by 200–300 % over the next 50–100 years, which previous studies suggest could have a large effect on cloud microphysics, lifetime, albedo, and precipitation. However, the interactions between smoke particles and clouds remain poorly quantiﬁed due to confounding meteorological inﬂuences and remote sensing limitations. Here, we use data from several aircraft cam- paigns in the Arctic and subarctic to explore cloud micro- physics in liquid-phase clouds inﬂuenced by biomass burn- ing. Median cloud droplet radii in smoky clouds were ∼40– 60 % smaller than in background clouds. Based on the re- lationship between cloud droplet number (Nliq) and vari- ous biomass burning tracers (BBt) across the multi-campaign data set, we calculated the magnitude of subarctic and Arc- tic smoke aerosol–cloud interactions (ACIs, where ACI = (1/3) × dln(Nliq)/dln(BBt)) to be ∼0.16 out of a maxi- mum possible value of 0.33 that would be obtained if all aerosols were to nucleate cloud droplets. Interestingly, in a separate subarctic case study with low liquid water content (∼0.02 g m−3) and very high aerosol concentrations (2000– 3000 cm−3) in the most polluted clouds, the estimated ACI value was only 0.05. In this case, competition for water va- por by the high concentration of cloud condensation nuclei (CCN) strongly limited the formation of droplets and reduced the cloud albedo effect, which highlights the importance of cloud feedbacks across scales. Aircraft-measured indirect cloud effects from biomass burning smoke in the Arctic and subarctic Using our calculated ACI val- ues, we estimate that the smoke-driven cloud albedo effect may decrease local summertime short-wave radiative ﬂux by between 2 and 4 W m−2 or more under some low and homo- geneous cloud cover conditions in the subarctic, although the changes should be smaller in high surface albedo regions of the Arctic. We lastly explore evidence suggesting that numer- ous northern-latitude background Aitken particles can inter- act with combustion particles, perhaps impacting their prop- erties as cloud condensation and ice nuclei. 1 Introduction They cite a study by Costantino and Breón (2010), where it was demonstrated that not co- locating aerosol–cloud layers in the vertical column dramat- ically lowered ACI estimates from 0.24 to 0.04 over marine stratocumulus clouds inﬂuenced by African biomass burn- ing. This bias seems to be apparent in many ACI estimates globally; from a literature search, McComiskey and Fein- gold (2012) revealed that remote-sensing-derived ACI values worldwide are lower than those derived from in situ, model- ing, and/or ground-based studies. They also showed that in addition to errors in the co-location of clouds and aerosols, the comparatively low spatial resolution of remote sensing observations can further enhance the low bias in ACI esti- mates. However, the interactions between smoke particles and Arctic clouds are poorly quantiﬁed, in part due to the con- founding effects of meteorology and surface conditions (e.g., Earle et al., 2011; Jackson et al., 2012; Jouan et al., 2012), and in part due to satellite sampling constraints over the Arc- tic, such as caused by the presence of many low contrast re- gions, multi-layer clouds (Intrieri et al., 2002), and reduced sunlight. One common way in which aerosol–cloud interac- tions (ACIs) are quantiﬁed is by assessing how a cloud prop- erty changes relative to some aerosol tracer or, in this case, biomass burning aerosol tracer (BBt). Following Eq. (1), ACI estimates for a given location can be derived from aircraft measurements of cloud droplet number, Nliq; they can also be derived from ground-based or remote sensing retrievals of changes in cloud properties such as droplet effective radius (re) or cloud optical depth (τ) at constant liquid water path (LWP) (Feingold et al., 2001; McComiskey et al., 2009): In the Arctic, these biases can be substantial. In a study in northern Finland, ACI estimates derived over the same gen- eral time period and location from both ground-based and remote sensing methods were ∼0.25 and 0.09 ± 0.04, re- spectively (Lihavainen et al., 2010), a more than twofold dif- ference. For reference, the range of Arctic remote-sensing- derived ACI estimates for all aerosol sources is −0.01 to 0.09 (Lihavainen et al., 2010; Tietze et al., 2011); in situ, ground- based, and model estimates range between 0.05 and 0.3 (Gar- rett et al., 2004; Lihavainen et al., 2010; Zhao et al., 2012). 1 Introduction respond with the last two terms, which are derived at con- stant LWP from the following theoretical relationships: reα LWP/τ (Stephens, 1978) and ταN1/3 liq (Twomey, 1977). Note that although each term in Eq. (1) should equal each other’s term, in practice, measurement-derived biases can cause ap- parent differences between the terms. This issue will be dis- cussed further in later sections. The incidence of wildﬁres in the Arctic and subarctic is in- creasing dramatically (Flannigan et al., 2009; Moritz et al., 2012; Stocks et al., 1998), and in some areas, such as boreal North America, it is expected to grow by 200–300 % over the next 50–100 years (Balshi et al., 2009). Already, periods of intense wildﬁres can increase regional aerosol concentrations in the Arctic twofold (Warneke et al., 2010), and the impact of smoke is increasingly being recognized as a strong con- tributor to Arctic haze (Hegg et al., 2009, 2010; McConnell et al., 2007; Shaw, 1995; Stohl et al., 2006, 2007). Increases in biomass burning aerosols could have a large effect on cloud dynamics (Earle et al., 2011; Jouan et al., 2012; Lance et al., 2011; Lindsey and Fromm, 2008; Rosenfeld et al., 2007; Ti- etze et al., 2011); in turn, smoke-derived changes to cloud microphysics may result in changes to precipitation and re- gional heating that are strong enough to affect dwindling re- gional sea ice (Kay et al., 2008; Kay and Gettelman, 2009; Lubin and Vogelmann, 2006; Vavrus et al., 2010). One study convincingly demonstrated that smoke reduces cloud droplet effective radius and enhances cloud albedo in Arctic liquid clouds (Tietze et al., 2011). In that study, mod- eled BBt concentrations were combined with remote sensing of cloud properties, enabling the authors to reduce meteoro- logical bias by basing their conclusions on tens of thousands of clouds sampled over a variety of meteorological condi- tions throughout the Arctic. Smoke ACI values derived from relative changes in cloud re were estimated to be between 0.04 and 0.11 out of a maximum 0.33. (Note however that in that study, clouds were binned by temperature and pressure, rather than by LWP as in Eq. 1 above.) However, despite being able to conclusively demonstrate a smoke cloud albedo effect, Tietze et al. (2011) noted that they might have underestimated the magnitude of satellite-derived ACI values because of difﬁculties constraining aerosol con- centrations and locations. Published by Copernicus Publications on behalf of the European Geosciences Union. 716 1 Introduction The degree of bias at other global sites has led McComiskey and Feingold (2012) to assert that the albedo effect can only be assessed accurately from aircraft or ground-based in situ data. ACI = 1 3 dlnNliq dlnBBt = −∂lnre ∂lnBBt LWP = ∂lnτ ∂lnBBt LWP . (1) (1) The ACI term as deﬁned by Eq. (1) was originally de- scribed as the “indirect effect” (IE) (Feingold et al., 2001, 2003). Here, similarly to McComiskey et al. (2009), we use “ACI” instead of “IE” to differentiate the fact that the met- ric in Eq. (1) is more directly associated with aerosol-driven changes to cloud microphysical responses than with radiative forcing. The maximum value of ACIs as derived from Eq. (1) is 0.33. An ACI value of 0.33 corresponds with the 1.0 max- imum possible change in lnNliq relative to lnBBt, which would occur if every aerosol were to nucleate a cloud droplet. The ﬁrst term of Eq. (1) is divided by 3 in order to cor- To better understand the impacts that expected increases in smoke will have on the Arctic, it is important to bet- ter constrain remote sensing and model estimates of smoke- www.atmos-chem-phys.net/16/715/2016/ www.atmos-chem-phys.net/16/715/2016/ Atmos. Chem. Phys., 16, 715–738, 2016 L. M. Zamora et al.: Aircraft-measured indirect cloud effects L. M. Zamora et al.: Aircraft-measured indirect cloud effects 717 Figure 1. Sampling locations for the following campaigns: ARC- TAS (light orange), NRC FIRE.ACE (dark orange), UW FIRE.ACE (dark blue), and ISDAC (light blue). The locations of clouds sam- pled are shown in Fig. 4. have never been compared directly to each other. Here we note only brieﬂy a few relevant points about the data sets and how they are inter-compared. y p First, during the ISDAC and FIRE.ACE ﬂights, multi- ple passes inside clouds were often obtained, and aerosols were intentionally sampled above- and below-cloud. In con- trast, during ARCTAS there was very limited resampling of a given region and generally only one pass through a cloud was obtained. This difference in sampling impacts our re- sults only in that there are not as many vertical proﬁles through the ARCTAS clouds as in the other data sets. Sec- ond, the UW FIRE.ACE data set contains some gaps in po- sitional data (latitude, longitude, and altitude), which range most frequently between 1 and 10 s, with rare instances of gaps > 1 min. 2.2.1 ARCTAS In ARCTAS, cloud liquid water content (LWC) was deter- mined from droplet size spectra gathered with the CAPS- CAS instrument (Baumgardner et al., 2001) based on inte- grated volume droplet size distributions between 0.75 and 50 µm. Throughout this size range, precision was estimated to be 20 % within each size bin based on pre-calibrations with sized glass and polystyrene latex spheres. We expect accuracy to also be ∼20 %, since pre-campaign calibrations were performed with spheres of known size, and since post- campaign tests with latex spheres were consistent with the expected sizes. Unfortunately, we could not validate in situ accuracy because simultaneously collected hot-wire probe LWC data were unobtainable due to high noise in out-of- cloud samples. For this reason, in-cloud hot-wire LWC data are not reported here other than to note that they showed qualitatively consistent trends with the CAPS-CAS LWC data. Liquid-phase cloud presence was deﬁned by LWC val- ues ≥0.01 g m−3 (Matsui et al., 2011), a value that corre- sponds well with cloud presence veriﬁed from the on-ﬂight video. Because neither ice water content (IWC) nor cloud particle images were directly measured during ARCTAS, we are unable to accurately verify cloud phase at temperatures < 0 ◦C in the ARCTAS data set. Therefore, we limited our focus within the ARCTAS data set to clouds present at tem- peratures > −0.5 ◦C (i.e., those clouds highly likely to be in the liquid phase). We also excluded clouds that the video 1 Introduction If the data were out-of-cloud and if the gap in positional data is < 1 min, we linearly interpolate the lati- tude, longitude, and/or altitude. Otherwise, occasional gaps > 1 min and data without positional information were ex- cluded. Thirdly and most importantly, we have made our best effort to use data that are as comparable as possible between campaigns. However, when high-quality measurements are not available from the same instrument in all campaigns, we use the most similar measurement available and we discuss the uncertainties this raises in the text. Figure 1. Sampling locations for the following campaigns: ARC- TAS (light orange), NRC FIRE.ACE (dark orange), UW FIRE.ACE (dark blue), and ISDAC (light blue). The locations of clouds sam- pled are shown in Fig. 4. speciﬁc ACIs in the Arctic using in situ aircraft data. The biggest challenge in obtaining representative aircraft-based ACI values is the fact that they are more prone to uncertain- ties caused by the inﬂuences of poorly constrained meteoro- logical factors (Shao and Liu, 2006) than other methods due to logistical limitations in sample size. We confront this is- sue in two ways. First, we focus on a case study day from the Arctic Research of the Composition of the Troposphere from Aircraft and Satellites (ARCTAS) campaign (Fuelberg et al., 2010; Jacob et al., 2010) in which several clouds were sampled under very similar conditions. We derive ACI esti- mates for all clouds that were either veriﬁably clean or are clearly inﬂuenced by biomass burning aerosols, and contrast the observed cloud properties. Second, to increase sample size, we consolidated data from four separate aircraft cam- paigns in the Arctic. In addition to ARCTAS, these data sets include: the First ISCCP (International Satellite Cloud Cli- matology Project) Regional Experiment Arctic Clouds Ex- periment (FIRE.ACE), which included portions ﬂown by the University of Washington Convair-580 (UW FIRE.ACE) and the Canadian National Research Council Convair-580 (NRC FIRE.ACE) (Curry et al., 2000), and the Indirect and Semi-Direct Aerosol Campaign (ISDAC) (McFarquhar et al., 2011). We then compare these ﬁndings with those from the ARCTAS case study. 2.1 Data set description The dates and ﬂight locations of data used in this study are shown in Fig. 1, and the data used are listed in Tables 1– 4. The ARCTAS, FIRE.ACE, and ISDAC data sets have each been extensively described previously (e.g., Curry et al., 2000; Fuelberg et al., 2010; Jacob et al., 2010; Korolev et al., 2003; McFarquhar et al., 2011; Rangno and Hobbs, 2001; Soja et al., 2008). However, to our knowledge, they www.atmos-chem-phys.net/16/715/2016/ Atmos. Chem. Phys., 16, 715–738, 2016 L. M. Zamora et al.: Aircraft-measured indirect cloud effects 718 718 L. M. Zamora et al.: Aircraft measured indirect cloud effects Table 1. Instrumentation used in this study from the ARCTAS data set. Data were collected at 1 s resolution, unless noted otherwise. ARCTAS-A 1–19 April; -CARB 29 June; -B 1–13 July 2008 Instrument Range Uncertainty Nliq, re, and LWC Cloud, Aerosol and Precipitation Spectrometer – Cloud and Aerosol Spectrometer (CAPS-CAS) 0.5–50 µm 20 %a Phase none (see text) liquid only NA CN TSI Condensation Particle Counter (CPC) 3010 > 0.01 µm precision 5 % TSI CPC 3025 > 0.003 µm precision 10 % TSI Aerodynamic Particle Sizer (APS) 3321 0.583–7.75 µm NA DMT Ultra-High Sensitivity Aerosol Spectrometer (UHSAS) 0.0609–0.986 µm ∼5 %, but increases in air with > 3000 particles cm−3 (Cai et al., 2008) Temperature Rosemount 102 E4AL −65 to +35 ◦C ±1 ◦C Relative humidity Aircraft-Integrated Meteorological Measurement System (AIMMS-20) – 2 % CCN DMT continuous-ﬂow, stream-wise thermal-gradient CCN counter – 7–16 % (Moore et al., 2011) CO Tunable Diode Laser Absorption Spectrometer (TDLAS) – ±2 % (Sachse et al., 1987) Submicron sulfateb Time-of-Flight Aerosol Mass Spectrometer – ±35 % (DeCarlo et al., 2008) Submicron OAb Time-of-Flight Aerosol Mass Spectrometer – 38 % (Huffman et al., 2005) BC massb Single-Particle Soot Photometer (SP2) – ±10 % (Moteki and Kondo, 2008) CH3CN Proton Transfer Reaction – Mass Spectrometer (PTR-MS) – ±10 % (Wisthaler et al., 2002) CH2Clc 2 Electron Capture Detection and Mass Spectrometer – ±10 % or ±2 pptv (Colman et al., 2001) Total backscatter (550 nm)b TSI 3563 Integrating Nephelometer > 0.1 Mm−1 0.5 Mm−1 Submicron scatter (550 nm)b Radiance Research Model M903 Nephelometer 1 Mm−1 0.5 Mm−1 a Based on pre- and post-campaign comparisons with sized glass and latex spheres. b Data were collected at 10 s resolution. c Data were collected at ∼40 s resolution. Table 2. Instrumentation used in this study from the ISDAC data set. Data were collected at 1 s resolution. www.atmos-chem-phys.net/16/715/2016/ ISDAC, 1–29 April 2008 Instrument Range Uncertainty Nliq, re, LWC DMT Cloud Droplet Probe (CDP) 2–50 µm ∼20 % (Earle et al., 2011) Nliq, LWC, re Forward Scattering Spectrometer Probe (FSSP) model 100 0.3–47 µm ∼17 % (Nliq), ∼34 % (LWC, re) (Baumgardner, 1983) Phase Cloud Particle Imager (CPI) 40 µm–2 mm NA CN PMS airborne Passive Cavity Aerosol Spec- trometer Probe (PCASP)-100X ∼0.12–3 µm 7 % (Earle et al., 2011) TSI CPC 3775 > 0.004 µm (Shantz et al., 2014) ± 10 % (Shantz et al., 2014) Temperature Rosemont 102 probe −65 to +35 ◦C ±1 ◦C CCN DMT continuous-ﬂow, stream-wise thermal- gradient CCN counter (reported between 14 and 37 % supersaturation) – 7–16 % (Moore et al., 2011) Total and submicron dry backscatter (550 nm) TSI 3563 Integrating Nephelometer > 0.1 M m−1 1–2 ± 0.5 M m−1 For days when high-quality CDP data were unavailable, following Earle et al. (2011). Table 2. Instrumentation used in this study from the ISDAC data set. Data were collected at 1 s resolution. Atmos. Chem. Phys., 16, 715–738, 2016 www.atmos-chem-phys.net/16/715/2016/ L. M. Zamora et al.: Aircraft-measured indirect cloud effects 719 L. M. Zamora et al.: Aircraft-measured indirect cloud effects Table 3. Instrumentation used in this study from the NRC FIRE.ACE data set. Data were collected at 1 s resolution. NRC FIRE.ACE, 1–29 April 1998 Instrument Range Uncertainty Nliq FSSP-100 0.3–47 µm ∼17 % (Baumgardner, 1983) LWC, re FSSP-100 0.3–47 up to 25 % (Peng et al., 2002) LWC King probe 0.05–3 g m−3 ±10 % or larger (Peng et al., 2002) Nevzorov probe ∼0.006–1 g m−3 ±15 % (Korolev et al., 1998) Phase CPI 40 µm–2 mm not available Temperature Rosemont probe −65 to +35 ◦C ±1 ◦C in-cloud, ±2–3 ◦C out-of-cloud CN PCASP 100X 0.12–3 µm 7 % (Earle et al., 2011) CCN Cloud condensation nucleus counter (reported at 57–72 % supersaturation) NA ±10 % Table 3. Instrumentation used in this study from the NRC FIRE.ACE data set. Data were collected at 1 s resolution. umentation used in this study from the NRC FIRE.ACE data set. Data were collected at 1 s resolution. Table 4. Instrumentation used in this study from the UW FIRE.ACE data set. Data were collected at 1 s resolution. 4. Instrumentation used in this study from the UW FIRE.ACE data set. Data were collected at 1 s resolution. Table 4. www.atmos-chem-phys.net/16/715/2016/ Instrumentation used in this study from the UW FIRE.ACE data set. Data were collected at 1 s resolution. UW FIRE.ACE, 19 May–24 June 1998 Instrument Range Uncertainty Nliq FSSP-100 0.3–47 µm ∼17 % (Baumgardner, 1983) LWC, re FSSP-100 0.3–47 µm see Table 5 LWC Gerber Scientiﬁc PVM-100X 0.01–0.75 g m−3 12 % (Garrett and Hobbs, 1999) Phase CPI 40 µm–2 mm NA CN PCASP 100X 0.12–3 µm 7 % (Earle et al., 2011) Total dry backscatter (550 nm) MS Electron Integrating Nephelometer > 0.1 M m−1 NA indicated were affected by drizzle or ice precipitation from cloud layers above. evant sampling periods used in this study. We believe this dis- crepancy to be due to a deﬁciency in the FSSP-96 data during this time period, because the FSSP-96 underestimated King and Nevzorov probe LWCs by ∼23 and 26 %, respectively, whereas the FSSP-124 data estimated King and Nevzorov probe data to within 8 %, on average (Table 5). Therefore, the FSSP size distribution data reported here for the NRC FIRE.ACE campaign are based on FSSP-124 data between 5 and 47 µm. 2.2.2 FIRE.ACE and ISDAC During the UW and NRC FIRE.ACE campaigns, LWC was determined from droplet size spectra gathered from the For- ward Scattering Spectrometer Probe (FSSP-100) measure- ments for particles with diameters between 0.5 and 47 and 5 and 47 µm, respectively. These measurements are func- tionally very similar to the CAPS CAS measurements from ARCTAS. During the sampling periods where air mass clas- siﬁcation matched the criteria described in Sect. 2.4, the FSSP data had a close relationship to hot-wire probe mea- surements of LWC for both campaigns (Table 5). For the NRC FIRE.ACE campaign, two FSSP probes were available (serial numbers 96 and 124, denoted hereafter as FSSP-96 and FSSP-124). The FSSP-96 is normally recommended for use by the data originators because the FSSP-124 had an intermittent hardware problem during the NRC FIRE.ACE campaign, and because it may have undersized particles > 30 µm diameter. In this analysis, the hardware problem did not occur during our time periods of interest, and the FSSP- 124 droplet distribution for droplets with diameters within 30–47 µm closely matched those of the FSSP-96. However, the FSSP-124 had higher droplet numbers in particles with diameters < 30 µm compared to the FSSP-96 during the rel- µ During ISDAC, LWC was determined from cloud droplet probe (CDP) data. These data agreed within 15 % of the bulk probe values. Following Earle et al. (2011), FSSP data were used on days when high-quality CDP data were unavailable; the FSSP data are estimated to agree with CDP data to within 20 %. Note that similarly to ice particles (e.g., Korolev et al., 2011), very large droplets may shatter on any of the cloud droplet probe tips. This may introduce some potential ar- tifacts when droplet sizes are very large (e.g., for some of the reference measurements available in FIRE.ACE and IS- DAC). For comparability with ARCTAS clouds, the presence of liquid clouds in the FIRE.ACE and ISDAC data sets was determined by simultaneous measurements of LWC > 0.01 g m−3. Also, for inter-campaign comparisons we focused on clouds sampled for ≥20 s in order to both increase representativeness of the average measured proper- ties of the clouds and to enhance meteorological similarity Atmos. Chem. Phys., 16, 715–738, 2016 www.atmos-chem-phys.net/16/715/2016/ (2002) as τ = 3 2 LWCHc reρw , (3) τ = 3 2 LWCHc reρw , (3) where Hc is cloud thickness (again only available in verti- cal cloud transects) and ρw is the density of water. In addi- tion to vertical transects, we also used horizontal transects within clouds to obtain information on horizontal variabil- ity of within-cloud properties and to obtain increased sample numbers for re. Here, we deﬁne liquid cloud phase by the lack of any ice particles in the CPI data throughout the entire cloud tran- sect, based on a roundness criterion (Lawson et al., 2001). When possible (i.e., in the NRC FIRE.ACE and ISDAC data sets), we veriﬁed that there was no detectable ice water along the cloud transects. This relatively stringent deﬁnition of liq- uid phase clouds is used to describe as best as possible the liquid-phase end-member cloud characteristics. Because air- craft cloud transects can only sample a portion of a cloud, we must assume that the portion of the cloud sampled is repre- sentative of the rest of the cloud. This may introduce uncer- tainties, particularly in persistent large-scale stratus clouds. Nonetheless, as discussed in Sect. 3.1, we believe that errors from this assumption are not likely to have a large impact on our results. In some instances in the multiple-campaign analysis, the same cloud or very similar clouds were sampled more than once, often intentionally, either through an entire vertical cloud transect or through a portion of a cloud. In order to reduce the potential for pseudo-replication in the analysis, transects that were deemed to be from the same cloud or from very similar clouds were averaged to provide one aggre- gated proﬁle or re and Nliq value for those instances. Clouds were determined as being related in part by a combination of time and location sampled. Here, the range of distance and time between clouds deemed as related or the same ranged from 0.4 to 76 km and several seconds to 2.5 h apart, depend- ing on the conditions and cloud type (the 2.5 h time frame included eight separate transects through a stratus cloud). In addition, in all clouds we assessed cloud pressure, loca- tion, temperature, and on-ﬂight video (when available). www.atmos-chem-phys.net/16/715/2016/ Atmos. Chem. Phys., 16, 715–738, 2016 L. M. Zamora et al.: Aircraft-measured indirect cloud effects L. M. Zamora et al.: Aircraft-measured indirect cloud effects 720 720 L. M. Zamora et al.: Aircraft-measured indirect cloud effects Table 5. Comparison of LWC measurements (g m−3) from various instruments. Campaign LWC determination method Slope y intercept R2 value UW FIRE.ACE FSSP vs. Gerber Scientiﬁc PVM-100Xa (Gerber et al., 1994) 0.92 −0.018 0.88 NRC FIRE.ACEb FSSP-124 vs. King probe (King et al., 1978) 1.08 −0.006 0.96 FSSP-124 vs. Nevzorov probe (Korolev et al., 1998) 1.01 0.045 0.82 Nevzorov vs. King 0.87 0.001 0.82 a For Gerber LWC < 0.5 g m−3. Above that, the FSSP missed known rain/drizzle events with larger droplets, and that began to impact the linear relationship. bSamples with LWC < the detection limit were not included. ble 5. Comparison of LWC measurements (g m−3) from various instruments. of clouds. Sometimes entrainment from outside air caused pockets of low to no LWC (i.e., LWC < 0.001 g m−3) within a cloud body; these pockets of air were not included when determining the average cloud droplet effective radius. re = R r3 n(r) dr R r2 n(r) dr , (2) (2) There is no consistent deﬁnition for cloud phase in the lit- erature. In remote sensing studies for example, cloud phase is usually determined by cloud radiative properties – thus, clouds with some mixed particles can be included in liquid- or ice-phase classiﬁcations if they are mostly liquid or mostly ice (e.g., Baum et al., 2012; Platnick et al., 2003). Due to in- strumentation limitations, aircraft studies sometimes also de- ﬁne a cloud with small fractions of ice particles as being a “liquid” cloud (e.g., Korolev et al., 2003). Alternatively, dis- tinct portions of a cloud may be classiﬁed as different phases if a primarily liquid portion of a cloud is far away (∼1–2 km) from a mixed portion of a cloud mass (McFarquhar et al., 2007; Zuidema et al., 2005). where r is the radius and n(r) is the cloud particle size distri- bution. LWP is deﬁned as the vertical integral of LWC from the base to the top of the cloud. LWP values were only de- termined when vertical proﬁles through the cloud were avail- able, thus providing the cloud base and top heights. We de- ﬁne τ following Peng et al. www.atmos-chem-phys.net/16/715/2016/ In biomass burning cases we also assessed nearby aerosol con- ditions (as determined in ISDAC by SPLAT II particle com- position and in ARCTAS by CH3CN, black carbon (BC), submicron SO2− 4 and submicron organic aerosol, or OA, con- centrations). Within the multi-campaign analysis, two of the eight biomass burning clouds contained aggregated transects, as did four of the 16 background clouds. One background 2.4 Air mass classiﬁcation For this work, distinguishing smoke-inﬂuenced conditions from background cloud conditions is critical. During ARC- TAS, background conditions were selected by a combina- tion of in-cloud gas concentrations (average CO < 123 ppbv and average acetonitrile (CH3CN) < 0.14 ppbv) and near- cloud SO2− 4 and BC concentrations (< 0.3 µg m−3 and < 0.12 µg C m−3, respectively). In ideal cases, near-cloud air masses were deﬁned as half the width of the cloud if it was a vertical proﬁle, and within 10 s before and after the cloud if it was a horizontal transect. However, sometimes the presence of a neighboring cloud or the vertical changes in the aircraft track forced us to use slightly smaller samples. Because within-cloud gas concentrations were not avail- able, we used average near-cloud (as deﬁned above) aerosol concentrations to deﬁne background conditions in the FIRE.ACE data. To reduce the risk of any potential humidi- ﬁcation effects, we excluded near-cloud air masses that had any observations of cloud particles in the CPI or that had LWC values ≥0.001 g m−3. g To classify background air masses, we used the Passive Cavity Aerosol Spectrometer Probe (PCASP) aerosol con- centrations (CNPCASP) directly adjacent to the cloud. The PCASP measures dehumidiﬁed particles with diameters be- tween 0.12 and 3 µm. Previous authors have noted the pres- ence of large numbers of small nucleation- to Aitken-mode particles (between ∼15 and 85 nm) in the spring and sum- mertime Arctic that appear to have natural sources (Garrett et al., 2004; Howell et al., 2014; Leaitch et al., 2013; Leck and Bigg, 1999; O’Dowd et al., 2010; Ström et al., 2009; Tunved et al., 2013; Zhao and Garrett, 2015). However, the relatively large minimum size cutoff of the PCASP (∼120 nm) ex- cludes these particles, while including low altitude particles from pollution and biomass burning sources, which tend to be in the accumulation mode (Earle et al., 2011; Lathem et al., 2013; Warneke et al., 2010). Thus, CNPCASP tends to be a fairly good indicator of non-background conditions. The 123 ppbv CO cutoff value represents the upper quar- tile range of time periods with concurrently low CO, CH3CN, and BC (all separate indicators of combustion), and the CH3CN cutoff is the median for these values. For compar- ison, Lathem et al. (2013) and Moore et al. L. M. Zamora et al.: Aircraft-measured indirect cloud effects ditions across the wide temporal and spatial region covered during ARCTAS. cloud in the case study included aggregated transects. To as- sess the impact of cloud transect aggregation on our analysis, we calculated differences in ACI values using the maximum and minimum values of Nd within the aggregated samples. Calculated differences in ACI values were 1 %, indicating that uncertainties caused by aggregation had only minor im- pacts on our results. ARCTAS biomass-burning-inﬂuenced air masses were classiﬁed following the procedure of Lathem et al. (2013), where BB-inﬂuenced air masses have concentrations of > 175 ppbv and 0.2 ppbv CO and CH3CN, respectively. A manual scan indicated that aerosol pollutant tracers BC and submicron SO2− 4 were always elevated with respect to back- ground concentrations under these conditions in this data set. For comparison, Lance et al. (2011) used a concentration of > 200 ppbv CO for “polluted” (mostly biomass burning) cases. LWC among aggregated clouds was generally similar (within 30 % of each other). However, in some cases it was more variable; in one biomass burning aggregation, the set of eight related cloud transects had LWCs ranging from 0.12 to 0.54 g m−3. The relationship of LWC with re suggests that entrainment could have inﬂuenced LWC variability within this particular cloud. Although we cannot constrain the inﬂu- ence of entrainment to a high degree of certainty within an individual cloud aggregate, as discussed in Sect. 3.1, the ACI values derived across all clouds did not deviate from adia- batic values calculated from cloud parcel theory. During the two FIRE.ACE campaigns, the combination of relevant high-quality and/or high-resolution aircraft chemical data for completely characterizing air mass sources was not collected, and remote sensing products useful for air mass classiﬁcation were also unavailable. As a result, biomass- burning-derived haze events were indistinguishable from an- thropogenic pollution events in the FIRE.ACE data sets. Therefore, we only use FIRE.ACE clouds sampled under unpolluted background conditions for inter-comparison with the other data sets. 2.3 Cloud microphysical properties We used aircraft vertical proﬁles to assess cloud droplet ef- fective radius (re), cloud liquid water path (LWP) and cloud optical depth (τ), and to gather information on aerosol prop- erties above and below cloud. The re was derived by Eq. (2), following Hansen and Travis (1974): Atmos. Chem. Phys., 16, 715–738, 2016 www.atmos-chem-phys.net/16/715/2016/ 721 2.4 Air mass classiﬁcation (2011) deﬁned background air masses as having CO and CH3CN values at < 170 and 0.1 ppbv, respectively, and Lance et al. (2011) used a criterion of ∼160 ppbv CO. Such high background CO values are observed periodically over springtime Alaska due to higher emissions from Asia during spring and re- duced photochemical loss during winter months (Brock et al., 2011). In 2008 speciﬁcally (during a similar time pe- riod as ARCTAS-A), background CO was elevated further due to unusually early and frequent Asian wildﬁres that year (Moore et al., 2011). However, background Arctic CO lev- els can frequently be lower than these values. For exam- ple, during a separate summer campaign in 2011 over east- ern Canada, Sakamoto et al. (2015) observed and used a lower background CO threshold of 120 ppbv. Our chosen CO threshold of 123 ppbv was chosen in part because it enabled the use of a consistent value to characterize background con- To be classiﬁed as background, air masses had to have CNPCASP concentrations of ≤127 particles cm−3 (Shantz et al., 2012). This CNPCASP cutoff is a more stringent crite- rion for determining clean conditions than those adopted by Jackson et al. (2012), Earle et al. (2011), and Peng et al. (2002), where respective values of < 200, 250, and 300 particles cm−3 were used, but the criterion applied here appears to exclude biomass burning and pollution aerosols fairly effectively (Table 6). However, the upper 95 % CH3CN www.atmos-chem-phys.net/16/715/2016/ L. M. Zamora et al.: Aircraft-measured indirect cloud effects In cases when the data are not normally distributed, this method is more appropriate than a linear regression be- cause it reduces the impact of outliers. concentrations are higher than typical background condi- tions, indicating that our chosen cutoff value is generally, but not completely, effective at removing air masses inﬂu- enced by smoke. Therefore, the FIRE.ACE samples have a more uncertain background classiﬁcation than the ARCTAS and ISDAC data sets, where actual chemical tracers verify the presence of pollution and biomass burning aerosols. For ISDAC samples, background conditions were determined by out-of-cloud CNPCASP concentrations, in order to be con- sistent with the FIRE.ACE campaigns. However, the TSI aerosol concentrations (CNTSI) and backscatter values were not used to assign a background classiﬁcation (see Sect. 3.2 for further details). As previously mentioned, ARCTAS was the only cam- paign where biomass burning gaseous tracers were directly quantiﬁable in-cloud (here we use BBt = CH3CN (de Gouw et al., 2003) and BBt = CO (Tietze et al., 2011)), measured in ppbv. Both CO (Bian et al., 2013) and CH3CN have ap- preciable background concentrations in the Arctic (as can be seen in Fig. 3a). Therefore, approximate background CO and CH3CN concentrations of 99.2 and 0.088 ppbv, respec- tively, were subtracted prior to deriving ACI values from Eq. (1) in the case study. These background values were de- rived from the mean of the Kendall robust line-ﬁt method analyses of ARCTAS CCN (cloud condensation nuclei) and CNPCASP equivalent concentrations vs. CO (or CH3CN) con- centrations. In the multi-campaign analysis, background val- ues of 0.018 ppbv CH3CN were subtracted, due to lower background concentrations in the cleanest samples. Although for simplicity we deﬁne a single background Arctic CH3CN level here, background CH3CN can range from ∼0.050 ppbv in the Arctic marine boundary layer to ∼0.14 ppbv at alti- tudes of ∼8 km (Kupiszewski et al., 2013; Warneke et al., 2009; A. Wisthaler, personal communication, 2015). A maxi- mum error of 0.038 ppbv in background CH3CN would equal at most 18 % of the CH3CN signal in biomass burning sam- ples. For that reason, and because CH3CN was only one of six tracers used to derive ACI values, the range of possi- ble background CH3CN concentrations is expected to have only minor impacts on the analysis. L. M. Zamora et al.: Aircraft-measured indirect cloud effects Table 6. A comparison of background concentrations of biomass burning and pollution tracers as previously reported to those in the ARCTAS-B data set in air masses that would be deﬁned as background using only the CNPCASP equivalenta cutoff of ≤127 particles cm−3. Data are out-of-cloud and from altitudes < 2.1 km due to instrument limitations above this level. Table 6. A comparison of background concentrations of biomass burning and pollution tracers as previously reported to those in the ARCTAS-B data set in air masses that would be deﬁned as background using only the CNPCASP equivalenta cutoff of ≤127 particles cm−3. Data are out-of-cloud and from altitudes < 2.1 km due to instrument limitations above this level. Tracer Median 95th Previously reported (units) (interquartile range) percentile backgroundb concentrations CO (ppbv) 96 (96–109) 135 120–170f–i CH3CN (ppbv) 0.08 (0.06–0.10) 0.19 0.1h,j BC (µg C m−3) 0.001 (0.001–0.004) 0.016 0.029f Submicron SO2− 4 (µg m−3)c 0.010 (0.005–0.070) 0.33 0.1–0.9f,j–l a CNPCASP values were not available in ARCTAS, and were thus approximated from the CN concentrations from the APS and UHSAS for the same size range as would be measured in the PCASP. b Submicron SO2− 4 concentrations are reﬂective of average, not background, conditions. c Following Fisher et al. (2011), we assume ARCTAS submicron sea-salt SO2− 4 is negligible, and that total submicron SO2− 4 is approximately equal to submicron non-sea-salt SO2− 4 . d Stohl et al. (2007), e,f Warneke et al. (2009, 2010), g Brock et al. (2011), h Moore et al. (2011), i Shinozuka et al. (2015), j Lathem et al. (2013), k,l Quinn et al. (2000, 2002). a CNPCASP values were not available in ARCTAS, and were thus approximated from the CN concentrations from the APS and UHSAS for the same size range as would be measured in the PCASP. b Submicron SO2− 4 concentrations are reﬂective of average, not background, conditions. c Following Fisher et al. (2011), we assume ARCTAS submicron sea-salt SO2− 4 is negligible, and that total submicron SO2− 4 is approximately equal to submicron non-sea-salt SO2− 4 . d Stohl et al. (2007), e,f Warneke et al. (2009, 2010), g Brock et al. (2011), h Moore et al. (2011), i Shinozuka et al. (2015), j Lathem et al. (2013), k,l Quinn et al. (2000, 2002). tributed. L. M. Zamora et al.: Aircraft-measured indirect cloud effects Arctic background CO is more consistent than CH3CN, and in that case, the dif- ferences in background CO as computed from CNPCASP vs. CCN line-ﬁt analyses (93.0 and 105.4 ppbv, respectively) led to only a 2.6 % change in the derived ACI values. A biomass burning classiﬁcation was assigned in ISDAC data when a cloud had contact with discernable amounts of biomass burning aerosols, as determined by single particle mass spectrometer, SPLAT II (Zelenyuk et al. 2009, 2015), based on the mass spectral analysis of individual aerosol par- ticles (Fig. 2). This method has been similarly employed to determine biomass burning inﬂuence in the ISDAC data set previously (Earle et al., 2011; McFarquhar et al., 2011; Shantz et al., 2014). www.atmos-chem-phys.net/16/715/2016/ Atmos. Chem. Phys., 16, 715–738, 2016 722 L. M. Zamora et al.: Aircraft-measured indirect cloud effects L. M. Zamora et al.: Aircraft-measured indirect cloud effects Cloud& &&& Cloud&&&&&& Al(tude&(m)& Nliq&(cm23)& CN&(cm23)& Frac(on&(%)& Ice&crystals&(m23)& Time&UTC&(hh:mm:ss)& Time&UTC&(hh:mm:ss)& (a) (c) (d) (e) (g) (h) F25& F26& (b) (f) Figure 2. ISDAC 2008 aerosol and ﬂight characteristics near and in selected clouds inﬂuenced by biomass burning from 19 April (left) and 20 April (right). Flight characteristics shown include: (a) altitude, (b) LWC (blue) and IWC (pink), (c) aerosol concentration from the PCASP (black), SPLAT (red), and UHSAS (green) instruments, and (d) bulk aerosol SPLAT chemical composition. Tan shading indicates SPLAT sampling through the in-cloud CVI inlet. Cloud& &&& Cloud&&&&&& Al(tude&(m)& Nliq&(cm23)& CN&(cm23)& Frac(on&(%)& Ice&crystals&(m23)& Time&UTC&(hh:mm:ss)& Time&UTC&(hh:mm:ss)& (a) (c) (d) (e) (g) (h) F25& F26& (b) (f) Figure 2. ISDAC 2008 aerosol and ﬂight characteristics near and in selected clouds inﬂuenced by biomass burning from 19 April (lef and 20 April (right) Flight characteristics shown include: (a) altitude (b) LWC (blue) and IWC (pink) (c) aerosol concentration from th Figure 2. ISDAC 2008 aerosol and ﬂight characteristics near and in selected clouds inﬂuenced by biomass burning from 19 April (left) and 20 April (right). Flight characteristics shown include: (a) altitude, (b) LWC (blue) and IWC (pink), (c) aerosol concentration from the PCASP (black), SPLAT (red), and UHSAS (green) instruments, and (d) bulk aerosol SPLAT chemical composition. Tan shading indicates SPLAT sampling through the in-cloud CVI inlet. (b) CH2Cl2"(pptv)" (a) CO"(ppbv)" CH3CN"(ppbv)" (a) (b) CO"(ppbv)" CH3CN"(ppbv)" CH2Cl2"(pptv)" Figure 3. Carbon monoxide (ppbv) during the 1 July 2008 ARCTAS-B ﬂight as a function of (a) the biomass burning tracer CH3CN (ppbv) and (b) the fossil fuel combustion tracer CH2Cl2 (pptv). ters > 3 and 10 nm were measured during ARCTAS from TSI models 3025 and 3010, respectively. Because CNPCASP val- ues were not measured during ARCTAS, we combined APS- and UHSAS-sized aerosol data collected during that cam- paign into a similar size distribution as the CNPCASP mea- surements (0.124–3.278 µm). UHSAS and APS measure- ments are not actively dried like PCASP samples are (Earle et al., 2011; Strapp et al., 1992), but sample humidity de- creases signiﬁcantly upon heating in the cabin and measure- ments are taken at dry relative humidity; in addition, particles are exposed to dried sheath air prior to detection. There are some limitations of the ACI approach. L. M. Zamora et al.: Aircraft-measured indirect cloud effects First, a systematic bias can be introduced when aerosol and cloud properties are averaged or co-located in low spatial or tem- poral resolution data sets (McComiskey and Feingold, 2012). This particular systematic bias is generally not a large con- cern for in-cloud aircraft studies such as this one where gas and/or aerosol measurements and Nliq measurements are ei- ther collected simultaneously or in very close proximity. Sec- ondly, the magnitudes of derived ACIs can vary depending on the BBt tracers used, and any one tracer may be biased by random error and a variety of other reasons that may cause Figure 3. Carbon monoxide (ppbv) during the 1 July 2008 ARCTAS-B ﬂight as a function of (a) the biomass burning tracer CH3CN (ppbv) and (b) the fossil fuel combustion tracer CH2Cl2 (pptv). used were CNPCASP concentrations, backscatter at 550 nm, BC concentrations, and when available, CCN (not available in the UW FIRE.ACE campaign). For comparison to the PCASP, aerosol concentrations with diameters > 4 nm were measured with a TSI 3775 in ISDAC. Aerosols with diame- Atmos. Chem. Phys., 16, 715–738, 2016 2.5 Assessment of indirect effects from biomass burning As mentioned before, the impact of smoke aerosols on cloud droplet activation was assessed by looking at aerosol–cloud interactions of biomass burning aerosols on cloud droplet number. The ACI values were derived from changes in cloud droplet number relative to measured biomass burning tracers, BBt, following Eq. (1) and using a nonparametric Kendall ro- bust line-ﬁt method. The Kendall robust line-ﬁt model (also commonly known as the Theil–Sen method) (Sen, 1968; Theil, 1950) derives a linear model of a data set from the median of the slopes between each two points in the data set. While this method is not as commonly used as linear re- gressions, it performs similarly when data are normally dis- Because the in-cloud CO and CH3CN values were not available in the ISDAC or FIRE.ACE campaigns, we also compared aerosol tracers of smoke/polluted particles adja- cent to the cloud as a BBt quantity. The aerosol tracers www.atmos-chem-phys.net/16/715/2016/ Atmos. Chem. Phys., 16, 715–738, 2016 723 Atmos. Chem. Phys., 16, 715–738, 2016 www.atmos-chem-phys.net/16/715/2016/ L. M. Zamora et al.: Aircraft-measured indirect cloud effects 724 L. M. Zamora et al.: Aircraft-measured indirect cloud effects M. Zamora et al.: Aircraft-measured indirect cloud effects Figure 4. Map of cloud sample locations from all campaigns. Red points indicate biomass burning samples, blue cases indicate back- ground samples, and gray points indicate intermediate samples. the tracer to imperfectly approximate actual cloud droplet nu- clei. To reduce the biases inherent to any one tracer, we use a combination of up to six BBt tracers to derive ACIs, as avail- able. A third potential problem is the risk that a snapshot of a cloud in time is not representative of the net cloud properties over its lifetime (Duong et al., 2011). Currently, only models can fully characterize cloud lifetime properties, but interpret- ing the model output can be challenging for other reasons. Within aircraft in situ data, this source of sampling error is best minimized in aircraft in situ data by resampling through- out the cloud’s life cycle. Resampling was sometimes, but not always, carried out for individual cloud cases presented here, and was not speciﬁcally carried out throughout the lifetime of the cloud. However, based on the results presented in Duong et al. (2011), the magnitude of this type of error is unlikely to have a large impact on our results, although we cannot with full conﬁdence assess how cloud life stage might have impacted the way aerosols were interacting with the clouds. Figure 4. Map of cloud sample locations from all campaigns. Red points indicate biomass burning samples, blue cases indicate back- ground samples, and gray points indicate intermediate samples. The fourth limitation of the ACI method is that Nliq has a sublinear relationship with CCN (e.g., Morales et al., 2011; Morales and Nenes, 2010), with particularly noticeable devi- ations from linear behavior expected when a cloud contains high CCN concentrations (e.g., Moore et al., 2013). This be- havior is driven by increased competition for water vapor, which in turn decreases cloud supersaturation and reduces the tendency to form additional drops. Because ACI values are typically derived from linear-type regressions, apparent ACI values can be reduced if clouds with high CCN are in- cluded in the analysis. We discuss the potential for this type of interaction where applicable in the text. www.atmos-chem-phys.net/16/715/2016/ Finally, the most difﬁcult problem to address is the potential bias introduced if one does not account for meteorological conditions (Shao and Liu, 2006). We discuss the relationship of derived ACIs with meteorology in Sects. 2.6 and 3. present at 1–3 km altitude. With one exception (an ARCTAS- B background case from 8 July 2008), the stratiform clouds were not present below a strong temperature or moisture in- version. In our data set, none of the biomass burning cases were present below an inversion either; such inversions oc- curred only in four of the clean background cases, indicating generally unimpeded aerosol mixing from above and below for biomass burning clouds in these data. The cumuliform clouds were also found between 1 and 3 km, and although they were less optically thick than the stratiform clouds, opti- cally thin (τ < 15) and multi-layer clouds dominated all sam- ples. Across all clouds sampled during the four campaigns, there was substantial variation between cloud properties (Ta- ble 7) and the physical locations of the clouds (Fig. 4). For example, background clouds were primarily sampled over the open ocean and at higher latitudes, whereas the smoky clouds were primarily sampled at lower latitudes over land. For this reason, in addition to comparing median character- istics of all background and clean cases, we also focus on a case study where multiple clean and smoky clouds were observed under very similar meteorological and surface con- ditions (Sect. 3.1). 3.1 Indirect effects of smoke in Arctic liquid-phase clouds On 1 July 2008 during the ARCTAS-B campaign, a variety of small cumuliform clouds were sampled during ﬂight 18 over inland Saskatchewan, Canada. The physical character- istics of the clouds were very similar (Table 8), being small (∼0.7 km high, and ∼0.2–7 km wide) non-precipitating clouds present between 1680 and 2650 m altitude, and far from any major temperature or water vapor inversions. All clouds were liquid phase, with low median LWC values of 0.02 g m−3 (the implications of which are discussed further down). All clouds had temperatures ranging from −0.1 to 3.1 ◦C. All were sampled within 97 km2 and 5.2 h of each other, during which time each cloud experienced a similar northeasterly wind direction. ACI estimates can also sometimes be inﬂuenced or even overwhelmed by systematic differences in local meteorolog- ical conditions associated with cleaner versus more polluted clouds (Hegg et al., 2007; Shao and Liu, 2006). For the case study, that possibility is unlikely because of the relatively small area and time frame considered and the similar me- teorological conditions in which the clouds were sampled. However, because case study smoky clouds had a combi- nation of very low LWC, very high aerosol concentrations from a fresh ﬁre, and consequently, very small droplet sizes (Fig. 6), it is likely that smoky case study clouds were less sensitive to further additions of smoke aerosols than clouds with lower aerosol concentrations. Such nonlinear behavior is predicted when high CCN levels cause increased com- petition for water vapor, which in turn decreases cloud su- persaturation and reduces the tendency to form additional drops (e.g., Moore et al., 2013; Morales et al., 2011; Morales and Nenes, 2010). Additionally, possible enhanced entrain- ment of outside air in smoky clouds compared to background clouds (Ackerman et al., 2004; Bretherton et al., 2007; Chen et al., 2012; Lebsock et al., 2008) could enhance droplet evaporation and further reduce ACI values from the expected adiabatic ACI maximum value at a given aerosol level. Despite being exposed to similar meteorological and sur- face conditions, aerosol inputs to these clouds ranged signif- icantly, with average CH3CN and PCASP equivalent particle numbers ranging between 0.092 and 0.55 ppbv and 107 and 3001 cm−3, respectively. The large range in chemical prop- erties was due to the aircraft track, which repeatedly cov- ered areas up- and downwind of local fresh smoke plumes from the Lake McKay ﬁre. L. M. Zamora et al.: Aircraft-measured indirect cloud effects 3 Results a given tracer approximates the sub-population of aerosols that are participating in cloud droplet activation (Lihavainen et al., 2010). As plumes age, there may also be increas- ing uncertainty in biomass burning aerosol co-location with gaseous tracers such as CO and CH3CN, as these are subject to different depositional processes (Hecobian et al., 2011). However, in this case the ﬁres were relatively fresh so this issue is unlikely to be an important source of uncertainty. 3.1 Indirect effects of smoke in Arctic liquid-phase clouds Other researchers have previously noted differences in calculated ACIs when these interactions are computed from different tracers (e.g., McComiskey et al., 2009; Lihavainen et al., 2010; Zhao et al., 2012), and these differences probably reﬂect a combination of measurement error and how well As shown in Fig. 5, smoke is clearly correlated with re- duced cloud droplet radius in the seven clouds studied (with an average 59 % reduction relative to background clouds, Ta- ble 8). As expected, there was a concurrent increase in cloud droplet number (Fig. 5). Based on this increase, we compute a combined median ACI of 0.05 (bootstrapped 95 % conﬁ- dence interval 0.04–0.06) across all tracers shown in Fig. 5. Although linear regressions were not used to derive ACIs, we plot them for each tracer in Fig. 5 to show the degree of variation between individual tracer ACI values. Other researchers have previously noted differences in calculated ACIs when these interactions are computed from different tracers (e.g., McComiskey et al., 2009; Lihavainen et al., 2010; Zhao et al., 2012), and these differences probably reﬂect a combination of measurement error and how well For these reasons, although the 1 July 2008 case is in some ways ideal, in that the clouds were sampled in very simi- lar environmental conditions, it is not necessarily represen- tative of typical cloud conditions in the Arctic. The clouds were present relatively far south in the subarctic (52–56◦N) and were cumuliform compared to the more dominant Arc- tic stratus-type clouds. Moreover, the case study clouds were L. M. Zamora et al.: Aircraft-measured indirect cloud effects 3 Results 3.1 Indirect effects of smoke in Arctic liquid-phase clouds 725 Atmos. Chem. Phys., 16, 715–738, 2016 3.1 Indirect effects of smoke in Arctic liquid-phase clouds This ﬁre is comprehensively de- scribed in the combination of Cubison et al. (2011), Alvarado et al. (2010), and Raatikainen et al. (2012). In Fig. 3, we show that CO < 500 ppbv is strongly re- lated to the smoke tracer CH3CN and that it shows no cor- relation to the fossil fuel combustion tracer dichloromethane (CH2Cl2) (see Kondo et al., 2011 for further discussion on use of this tracer during ARCTAS). Given that CO has both pollution and biomass burning sources, this ﬁnding indicates smoke was the dominant aerosol contributor on that day, not pollution. Back trajectories also support this conclusion (Al- varado et al., 2010). Of the clouds sampled during this ﬂight, two clouds met the classiﬁcation criteria for being biomass- burning-inﬂuenced, three were classiﬁed as intermediate, and two met the ARCTAS background criteria. g Because in situ ACI derivations assume linearity in the re- sponse of Nliq to BBt, and such as assumption does not hold well at high CCN levels, we would expect to derive lower in situ ACI estimates if clouds with very high CCN levels are included in the analysis (Rosenfeld et al., 2014). That ACI values would increase to 0.08 (95 % conﬁdence interval 0.05–0.12) if the two biomass burning clouds were excluded suggests that nonlinear processes could have affected the re- duced ACI values in the case study. For reference, at case study smoky CNPCASP equivalent concentrations of ∼2000– 3000 cm−3, modeled adiabatic ACI values were ∼0.06–0.16 (Moore et al., 2013). The range in modeled ACI values de- pended on factors such as cloud vertical velocity and CCN hygroscopicity (the CCN spectrum). Given these model un- certainties and our estimated case study ACI value, any po- tential effects of entrainment were not clearly noticeable in our data. As shown in Fig. 5, smoke is clearly correlated with re- duced cloud droplet radius in the seven clouds studied (with an average 59 % reduction relative to background clouds, Ta- ble 8). As expected, there was a concurrent increase in cloud droplet number (Fig. 5). Based on this increase, we compute a combined median ACI of 0.05 (bootstrapped 95 % conﬁ- dence interval 0.04–0.06) across all tracers shown in Fig. 5. Although linear regressions were not used to derive ACIs, we plot them for each tracer in Fig. 5 to show the degree of variation between individual tracer ACI values. 2.6 Overview of surface and meteorological conditions Ambient conditions such as cloud type and presence of driz- zle from an overlying cloud deck were determined from available video, photos, ﬂight notes, and AVHRR images. Although in situ chemical and physical measurements were primarily used to determine end-member situations (i.e., where only smoke or only background air were the dom- inant sources of aerosols interacting with clouds), in some cases we discuss out-of-cloud aerosols with potentially more mixed sources. In these cases we supplemented chemical and physical data with 5-day HYSPLIT back trajectories (Draxler, R. R. and Rolph, G. D. HYSPLIT (HYbrid Single- Particle Lagrangian Integrated Trajectory) model, accessed via the NOAA ARL READY website (http://www.arl.noaa. gov/HYSPLIT.php), NOAA Air Resources Laboratory, Col- lege Park, MD) to determine recent air mass history. Using video, photos, and ﬂight notes, clouds were also classiﬁed as either stratiform or cumuliform. Stratiform clouds were www.atmos-chem-phys.net/16/715/2016/ Atmos. Chem. Phys., 16, 715–738, 2016 L. M. Zamora et al.: Aircraft-measured indirect cloud effects 726 ies and ranges for all background and biomass burning cloud cases in the multi-campaign assessment. Table 7. Median properties and ranges for all background and biomass burning cloud cases in the multi-campaign assessment. Property Background (n = 19) Biomass burning (n = 8) Aerosol number concentration (CN∗ PCASP), cm−3 42 (1–97) 584 (58–2001) CCN, cm−3 31 (6–332) 437 (68–6670) Backscatter at 550 nm, Mm−1 0.7 (−0.19 to 1.13) 8.8 (0.3–44.1) Temperature, ◦C −5 (−20 to 7) 2 (−9 to 10) Pressure, mbar 848 (505–995) 776 (687–909) Liquid water content (LWC), g m−3 0.07 (0.01–0.25) 0.03 (0.01–0.27) Cloud droplet effective radius (re), µm 8.7 (5.7–12.6) 5.0 (1.9–7.8) Droplet number concentration (Nliq), cm−3 41 (12–525) 338 (188–782) ∗CNPCASP equivalent data. Table 8. Mean properties and ranges for the 1 July 2008 ARCTAS case study, including background, intermediate, and biomass burnin loud cases. Property Background (n = 2) Intermediate (n = 3) Biomass burning (n = 2) Aerosol number concentration (CN∗ PCASP), cm−3 249 (107–390) 294 (147–427) 2604 (2207–3001) CCN, cm−3 545 (205–592) 722 (462–908) 10 879 (10 348–11 411) Backscatter at 550 nm, Mm−1 1.7 (0.9–2.5) 3.3 (1.6–4.7) 35.7 (31.2–40.2) Temperature, ◦C 0.8 (0.2–0.9) 0.1 (−0.1 to 3.1) 2.8 (2.4–3.1) Pressure, mbar 766 (762–770) 786 (763–826) 808 Liquid water content (LWC), g m−3 0.07 (0.03–0.12) 0.02 (0.01–0.04) 0.01 (0.01–0.02) Cloud droplet effective radius (re), µm 4.8 (3.7–5.8) 2.6 (2.1–3.3) 1.9 (1.9–2.0) Droplet number concentration (Nliq), cm−3 454 (384–525) 749 (621–907) 936 (824–1048) ∗Or CNPCASP equivalent for ARCTAS data. ranges for the 1 July 2008 ARCTAS case study, including background, intermediate, and biomass burning Table 8. Mean properties and ranges for the 1 July 2008 ARCTAS case study, including background, intermediate, and biomass burning cloud cases. Table 8. Mean properties and ranges for the 1 July 2008 ARCTAS case study, including background, intermediate, and biomass burning cloud cases. ∗Or CNPCASP equivalent for ARCTAS data. 6.4 µm (n = 13 and 14), respectively. However, the combined median ACI estimate from all tracers shown in Fig. 7 is 0.16 (95 % conﬁdence interval 0.14–0.17). This value is three times that of the case study, which is further evidence to sug- gest that cloud sensitivity to aerosols in the case study was lowered by aerosol-driven adiabatic reductions in cloud su- persaturation (and possibly enhanced entrainment). subjected to fresh concentrated smoke rather than aged di- luted smoke, as one would expect at higher latitudes. www.atmos-chem-phys.net/16/715/2016/ www.atmos-chem-phys.net/16/715/2016/ Atmos. Chem. Phys., 16, 715–738, 2016 L. M. Zamora et al.: Aircraft-measured indirect cloud effects 727 Figure 5. Based on seven samples from the ARCTAS-B 1 July 2008 case study, here we show the relationships between ln(re) (top row) and ln(Nliq) (bottom row) and ln(BBt) derived from six indicators (where BBt = CO (ppbv) (× indicates background values of 99.2 ppbv have been subtracted), CH3CN (ppbv) (× indicates background values of 0.088 ppbv have been subtracted), CCN (cm−3), backscatter at 550 nm (Mm−1), BC (µg C m−3), and CNPCASP equivalent values (cm−3), as calculated from UHSAS and APS measurements. Biomass burning samples are noted in red, and background samples are noted in blue. To show variation between tracers, linear regressions and associated ACI estimates are shown in light gray (but note that ﬁnal ACI values are not derived from individual regressions, but rather a combination of all six tracers). Figure 5. Based on seven samples from the ARCTAS-B 1 July 2008 case study, here we show the relationships between ln(re) (top row) and ln(Nliq) (bottom row) and ln(BBt) derived from six indicators (where BBt = CO (ppbv) (× indicates background values of 99.2 ppbv have been subtracted), CH3CN (ppbv) (× indicates background values of 0.088 ppbv have been subtracted), CCN (cm−3), backscatter at 550 nm (Mm−1), BC (µg C m−3), and CNPCASP equivalent values (cm−3), as calculated from UHSAS and APS measurements. Biomass burning samples are noted in red, and background samples are noted in blue. To show variation between tracers, linear regressions and associated ACI estimates are shown in light gray (but note that ﬁnal ACI values are not derived from individual regressions, but rather a combination of all six tracers). 0.5 2.0 10.0 50.0 200.0 1e-01 1e+00 1e+01 1e+02 1e+03 1 1 Smoky Clean 0.5$ 2$ 20$ 50$100$ 500$ 10'1$ 100$ 101$ 102$ 103$ 28$μm$ Droplet$diameter$(μm)$ dN/dlogDp$(cm'3)$ 1$ 10$ 5$ Figure 6. Mean cloud droplet size distributions (µm) for individ- ual case study biomass burning clouds (thin orange lines) and clean background clouds (thick blue lines). The 28 µm line is marked in gray. 0.5 2.0 10.0 50.0 200.0 1e-01 1e+00 1e+01 1e+02 1e+03 1 1 Smoky Clean 0.5$ 2$ 20$ 50$100$ 500$ 10'1$ 100$ 101$ 102$ 103$ 28$μm$ Droplet$diameter$(μm)$ dN/dlogDp$(cm'3)$ 1$ 10$ 5$ ties in far-off, non-sampled portions. Uncertainties are also higher in clouds that were only transected horizontally, be- cause mixed-phase clouds in the Arctic frequently have ver- tical layers of ice and liquid particles (Morrison et al., 2012). L. M. Zamora et al.: Aircraft-measured indirect cloud effects We cannot fully rule out that non-sampled portions of the clouds in the multi-campaign analysis contained ice parti- cles, or that different vertical layers had different re val- ues. However, if the six ISDAC and FIRE.ACE background clouds that were either stratiform or that contained only horizontal transects are excluded, the results of the multi- campaign analysis are nearly the same (ACI = 0.15 and me- dian background cloud re = 7.0 vs. 7.6 µm). Thus we do not believe that uncertainties in cloud phase had a major impact on our results. Figure 6. Mean cloud droplet size distributions (µm) for individ- ual case study biomass burning clouds (thin orange lines) and clean background clouds (thick blue lines). The 28 µm line is marked in gray. 3.2 Implications for radiation and precipitation Based on model output by McComiskey and Feingold (2008) (their Fig. 2a), we estimate that given the case study median ACI value of 0.05, the smoke-derived cloud albedo effect on summertime local short-wave radiative forcing could be be- tween −2 and −4 W m−2 for regions with surface albedo of ∼0.15. Typical short-wave spectrum broadband (0.3– 5.0 µm) albedos over subarctic Canada range from ∼0.09 to 0.17, compared to ∼0.23 to 0.71 in the winter (Davidson and Wang, 2005); thus, any local forcing in winter from smoke ACI effects would likely be reduced, compared to the sum- mer. The McComiskey and Feingold (2008) output was also based on the assumption of homogeneous, unbroken clouds with CCN concentrations of 600 cm−3, an LWP of 50 g m−2, and a cloud base height of 500 m. Such surface albedo and cloud/aerosol conditions are similar to some of the summer terrestrial conditions sampled over Canada during ARCTAS- deﬁning clean conditions was higher than ours, and they did not include any samples that met our background criteria (which were only present during the 4 April 2008 ISDAC ﬂight). Also note that the biomass-burning-inﬂuenced cloud cases assessed by Earle et al. (2011) did not overlap with the clouds assessed in this study. As noted previously, because the aircraft could only sam- ple transects of clouds, we had to assume that the observed cloud phase was representative of the whole cloud. In the case study, all clouds were sampled at temperatures > 0 ◦C, and this assumption holds well. Where we expect this as- sumption to be most uncertain is in stratiform clouds in the multi-campaign analysis, which might have different proper- L. M. Zamora et al.: Aircraft-measured indirect cloud effects There- fore, as explained above, we expect case study clouds already affected by high smoke concentrations to have reduced sen- sitivity to additional smoke, particularly given the low LWC of the case study clouds. To assess the impact of smoke on liquid clouds more gen- erally, we compared background and biomass burning cloud properties sampled over the larger region shown in Fig. 4. This more expansive set of clouds includes a broader range of high-latitude meteorological conditions, making it more rep- resentative of overall conditions in the Arctic region. How- ever, the greater heterogeneity also makes trends in the data more difﬁcult to interpret, as we cannot describe in full detail the degree to which meteorological inﬂuences affected each cloud given the limitations of the data sets. Observed smoke-driven reductions in liquid cloud droplet size and increases in cloud droplet number in both the case study and the multi-campaign analysis are in line with several other studies in the Arctic. Peng et al. (2002) found a simi- lar difference in re of 4.8 µm to the multi-campaign analysis in two combined data sets in the Arctic (one of which was the NRC FIRE.ACE data set), in conditions where PCASP values were > and < 300 particles cm−3, although they did not speciﬁcally focus on biomass-burning-related samples. Tietze et al. (2011) also found signiﬁcant changes in LWP, τ, and re using remote sensing cloud observations combined with a modeled biomass burning tracer. In contrast, Earle et al. (2011) did not see a reduction in re in biomass-burning- inﬂuenced clouds based on selected ISDAC samples. They attributed this ﬁnding to a combination of meteorological and microphysical factors. It is possible that some of the differ- ences with our study are also caused by reduced contrast be- tween selected clean and polluted cases, as their cutoff for Despite the uncertain meteorological inﬂuence, we see qualitatively similar trends to those in the 1 July 2008 ARC- TAS case study (Fig. 7). We ﬁnd a 3.7 µm (42 %) me- dian reduction in re between the smoky and background cases (Table 7). Concurrently, median Nliq increased from 41 droplets cm−3 in background clouds to 338 droplets cm−3 in smoky clouds. Within stratiform-only and cumuliform- only liquid clouds, groupings that are somewhat more com- parable meteorologically, the mean re differences are 2.5 and www.atmos-chem-phys.net/16/715/2016/ Atmos. Chem. Phys., 16, 715–738, 2016 L. M. L. M. Zamora et al.: Aircraft-measured indirect cloud effects Zamora et al.: Aircraft-measured indirect cloud effects L. M. Zamora et al.: Aircraft-measured indirect cloud effects Periodic broken cloud conditions, cloud heterogeneity (McComiskey and Feingold, 2008), and the patchiness of smoke will all reduce the net cloud albedo ra- diative forcing over wider spaces and times. Therefore, the −2 to −4 W m−2 range is only applicable in the subarctic in some summertime conditions. Nonetheless, this estimate at least provides a rough indication of how important these local effects might be during the most relevant time periods (i.e., when burning is most likely to occur). B. The summer subarctic biomass burning clouds we de- scribe from ARCTAS-B CCN and LWP levels bracket the model’s assumptions, ranging between 1 and 94 g m−2 and 68 and 6670 cm−3, respectively. However, cloud base heights were typically higher than the model-assumed 500 m, and al- though unbroken clouds are frequently observed in the Arc- tic and subarctic, the ACI value we use was determined from samples that included some clouds within broken cloud sys- tems, which may possibly have different microphysical re- sponses to aerosols. Periodic broken cloud conditions, cloud heterogeneity (McComiskey and Feingold, 2008), and the patchiness of smoke will all reduce the net cloud albedo ra- diative forcing over wider spaces and times. Therefore, the −2 to −4 W m−2 range is only applicable in the subarctic in some summertime conditions. Nonetheless, this estimate at least provides a rough indication of how important these local effects might be during the most relevant time periods (i.e., when burning is most likely to occur). 1 Droplet%diameter%(μm)% 1 Figure 8. Mean cloud particle size distributions (µm) for all non- case study biomass burning clouds (yellow dots) and clean back- ground clouds (light blue dots). The 28 and 50 µm lines are marked in gray. Thick red and darker blue lines indicate median values for binned size classes for smoky and clean clouds, respectively, in- cluding zero values not shown on the log–log plot. Due to the high number of zero values above > 50 µm diameter, the mean values above this level are also shown (dashed lines) for comparison. In contrast to the subarctic, in the Arctic, high surface albedo will lessen the expected impact of the cloud albedo effect. L. M. Zamora et al.: Aircraft-measured indirect cloud effects 728 Figure 7. Same as in Fig. 5, but for data from the multi-campaign analysis. As in Fig. 5, CO* indicates that background values of 99.2 ppbv have been subtracted. For CH3CN, the * indicates background values of 0.018 ppbv have been subtracted (due to low background CH3CN levels in some of the samples). Figure 7. Same as in Fig. 5, but for data from the multi-campaign analysis. As in Fig. 5, CO* indicates that background values of 99.2 ppbv have been subtracted. For CH3CN, the * indicates background values of 0.018 ppbv have been subtracted (due to low background CH3CN levels in some of the samples). 1 2 5 10 20 50 100 200 500 1e-05 1e-03 1e-01 1e+01 1e+03 1 1 10#5% 10#3% 10#1% 101% 103% 28%μm% Droplet%diameter%(μm)% dN/dlogDp%(cm#3)% 1 2 5 10 20 50 1e-05 1e-03 1e-01 1e+01 1e+03 1 1 Smoke Background Median smoke Median background Mean smoke Mean background Figure 8. Mean cloud particle size distributions (µm) for all non- case study biomass burning clouds (yellow dots) and clean back- ground clouds (light blue dots). The 28 and 50 µm lines are marked in gray. Thick red and darker blue lines indicate median values for binned size classes for smoky and clean clouds, respectively, in- cluding zero values not shown on the log–log plot. Due to the high number of zero values above > 50 µm diameter, the mean values above this level are also shown (dashed lines) for comparison. 1 2 5 10 20 50 100 200 500 1e-05 1e-03 1e-01 1e+01 1e+03 1 1 10#5% 10#3% 10#1% 101% 103% 28%μm% Droplet%diameter%(μm)% dN/dlogDp%(cm#3)% 1 2 5 10 20 50 1e-05 1e-03 1e-01 1e+01 1e+03 1 Smoke Background Median smoke Median background Mean smoke Mean background B. The summer subarctic biomass burning clouds we de- scribe from ARCTAS-B CCN and LWP levels bracket the model’s assumptions, ranging between 1 and 94 g m−2 and 68 and 6670 cm−3, respectively. However, cloud base heights were typically higher than the model-assumed 500 m, and al- though unbroken clouds are frequently observed in the Arc- tic and subarctic, the ACI value we use was determined from samples that included some clouds within broken cloud sys- tems, which may possibly have different microphysical re- sponses to aerosols. www.atmos-chem-phys.net/16/715/2016/ Atmos. Chem. Phys., 16, 715–738, 2016 L. M. Zamora et al.: Aircraft-measured indirect cloud effects Atmos. Chem. Phys., 16, 715–738, 2016 3.3 Interactions of background aerosols with dilute biomass burning particles: a potential uncertainty in ACI values g g However, the nucleation- and Aitken-mode background particles are not ubiquitous throughout the year. They tend to accumulate mainly in the spring and summer, which is thought to be due to a combination of three factors: (1) there is more sunlight available for the photochemical reactions key to new particle formation (Engvall et al., 2008; Tunved et al., 2013), (2) reduced sea ice and enhanced primary pro- duction likely lead to greater emissions of marine precur- sor gases and nanogels (Leaitch et al., 2013; O’Dowd et al., 2010; Tunved et al., 2013), and (3) during Arctic summer there tend to be fewer larger particles such as smoke for these small particles to coagulate and condense upon. However, Arctic summertime smoke events do occur (e.g., Fuelberg et al., 2010; Iziomon et al., 2006) and may be increasing (Moritz et al., 2012). In the subarctic, wildﬁres peak in the summer (Giglio et al., 2006). Thus, although the inﬂuence of the small background particles on subarctic and Arctic smoke ACI values is probably minor, deviations from the lin- ear ACI expectations derived here might occur during dilute summertime Arctic smoke events and in subarctic locations, for example when smoke is diluted over or near marine envi- ronments. As mentioned previously, large numbers of nucleation- and Aitken-mode particles are frequently observed in the spring and summer Arctic and subarctic (Engvall et al., 2008; Leck and Bigg, 1999; Ström et al., 2009; Zhao and Garrett, 2015). These particles are thought to have a marine origin via some combination of new particle formation from marine gases (Allan et al., 2015; Leaitch et al., 2013; O’Dowd et al., 2010; Tunved et al., 2013) and direct oceanic nanogel emissions (Heintzenberg et al., 2006; Karl et al., 2012, 2013; Leck and Bigg, 1999; Orellana et al., 2011). Chemical data from the ARCTAS data set also show the presence of numerous small particles with a natural background source (Fig. 9). Previous studies also suggest that the small particles can condense upon larger particles (e.g., smoke) when such par- ticles are present (Engvall et al., 2008; Leaitch et al., 2013; Tunved et al., 2013). This coagulation process may explain why Arctic smoke aerosols have been shown to sometimes contain organic components likely derived from smaller, non-biomass-burning particles mixed with sulfates and ma- rine particles (Earle et al., 2011; Zelenyuk et al., 2010). L. M. Zamora et al.: Aircraft-measured indirect cloud effects study suffer large uncertainties related to their collection over heterogeneous conditions, their droplet distributions support the hypothesis of smoke-induced reductions in drizzle. upon condensation, the small background particles might act as surfactants or otherwise modify smoke CCN character- istics, causing deviations from the ACI value as derived in Sect. 3.1 at low smoke concentrations. This hypothesis is dif- ﬁcult to test because, excepting three intermediate instances in the case study, the data presented in Sect. 3.2 only included background and high smoke conditions. study suffer large uncertainties related to their collection over heterogeneous conditions, their droplet distributions support the hypothesis of smoke-induced reductions in drizzle. 3.3 Interactions of background aerosols with dilute biomass burning particles: a potential uncertainty in ACI values To get some idea of how important the background particles may be, we estimated the maximum mean aerosol volume change that would occur if high concentrations of small background aerosols were to mix with and condense upon diluted smoke particles. Concentrations of background particles were esti- mated at 5000 cm−3 (based on high-end values observed in Fig. 9 and at another Arctic site; Ström et al., 2009). Diluted smoke concentrations were estimated at 450 particles cm−3 (low-end values from Fig. 9). Volumes were calculated from the size ranges observed in ARCTAS background and smoky aerosols (see Appendix A for details). In this hypothetical scenario, we estimate that background aerosols could in- crease dilute smoke aerosol volume by up to 2–15 %, al- though volume increases are likely substantially less in most air masses. L. M. Zamora et al.: Aircraft-measured indirect cloud effects Although future sea ice losses and associated reduc- tions in surface albedo may affect the relative importance of the cloud albedo effect on Arctic clouds, others (e.g., Garret et al., 2004) have suggested that in the Arctic, a more im- portant impact of reduced cloud droplet size may be greater long-wave opacity, which can lead to enhanced snowmelt. Relatedly, smaller droplets may affect cloud lifetime either by extending it via reduced precipitation (the “second indi- rect effect”; Ackerman et al., 2000; Albrecht, 1989) or by reducing it via enhanced water vapor competition and evap- oration, as may have occurred in the case study. droplet radius. This narrowing is likely to lessen the eventual probability of precipitation (Tao et al., 2012), as it moves median droplet size further away from the 28 µm effective diameter threshold at which collision/coagulation processes are thought to become efﬁcient enough to induce precipita- tion (Rosenfeld et al., 2012). Cloud droplet spectra from the multi-campaign clouds are shown for comparison in Fig. 8. There is not as obvious a narrowing of spectra as for the case study, but median droplet concentrations in smoky clouds never reached above 28+µm diameter, whereas median droplet diameter in background clouds did reach above this point (Fig. 8). Also, small droplet concentrations (those most susceptible to evaporation) in- creased in smoky conditions, and rainfall was only noted in clean conditions, as shown in Fig. 8 by elevated (> 0.1 cm−3) cloud droplet concentrations with diameters > 50 µm (King et al., 2013). Therefore, although clouds outside the case Cloud droplet spectra from the 1 July 2008 ARCTAS case study clouds are shown in Fig. 6. Although sample size is small, the presence of smoke appears to narrow the droplet spectra from a dispersion of 0.84 in background clouds to 0.55 in smoky clouds, as calculated by the ratio between the standard deviation of the size distribution and the mean Atmos. Chem. Phys., 16, 715–738, 2016 www.atmos-chem-phys.net/16/715/2016/ 729 L. M. Zamora et al.: Aircraft-measured indirect cloud effects 4 Discussion and conclusions The challenge of separating the inﬂuence of meteorology and aerosol indirect effects on clouds introduces relatively large uncertainty in our understanding of how smoke impacts clouds. Using in situ aircraft data, we quantiﬁed these im- pacts in both a subarctic cumulus cloud case study and in a multi-campaign data assessment of clouds north of 50◦N. The multi-campaign assessment suggests an ACI value of 0.16 (95 % conﬁdence interval 0.10–0.13), which is on the high end of previous satellite-based assessments (0.04–0.11) (Tietze et al., 2011). Given a known low bias in remote- sensing-derived estimates of ACIs (e.g., McComiskey and Feingold, 2012), our ﬁndings suggest that smoke-derived in- creases in cloud albedo may be higher than previously de- rived in the region. We reduced confounding meteorological effects by including data from as wide a geographic region as possible, applying very stringent conditions to identify clean and smoky clouds, and reducing the impact of outliers on ACI derivations by using the Kendall robust line-ﬁt method instead of normal linear regressions. However it is important to note that meteorological effects are still imperfectly con- strained in this assessment due to inherent limitations in the in situ data set size and content. Interestingly, the small Arctic marine particles appear to be fairly hygroscopic (Lathem et al., 2013; Lawler et al., 2014; Zhou et al., 2001), and they can be surface-active (Lohmann and Leck, 2005). One study using ARCTAS data showed that background aerosol values of the hygroscopic- ity parameter, κ, were on average nearly 2 times higher than average smoke κ values (0.32 ± 0.21 vs. 0.18 ± 0.13, respec- tively), although there was a high degree of variability and overlap in the κ values (Lathem et al., 2013). Previous studies also suggest that volume increases alone might affect Arctic particle hygroscopicity, independent of chemistry (Moore et al., 2011). Given this information, we cannot rule out that Atmos. Chem. Phys., 16, 715–738, 2016 L. M. Zamora et al.: Aircraft-measured indirect cloud effects All quality- ﬂagged data were excluded, as well as suspicious ISDAC values within 17 km and < 1 km altitude of Fairbanks airport, Alaska. Very small background aerosols appear to dominate the high aerosol number concentration/low scatter particles seen in (a)–(c), as shown by their disappearance when a diameter cutoff of 140 nm is used (d). For comparison to the multi-campaign analysis, we also analyzed the 1 July 2008 ARCTAS case in the subarctic, where multiple clean and smoky clouds were found under similar meteorological conditions. The case study smoke cases had a combination of low cloud LWC, high in-plume aerosol concentrations, and very small cloud droplets. From these samples, we derived an ACI estimate of 0.05 (95 % conﬁdence interval 0.04–0.06), which is smaller than that of the multi-campaign analysis. Based on theory (e.g., Moore et al., 2013), as the number of smoke CCN increases (through some combination of enhanced aerosol number and/or in- creased hygroscopicity for existing particles), there is greater water vapor competition. This competition makes supersat- uration development and cloud droplet activation increas- ingly difﬁcult, which would reduce ACI values. Therefore, we speculate that the 0.05 ACI case study value falls at the low-end of typical smoke ACI values for the larger subarc- tic/Arctic region. Reductions in droplet activation and po- tential enhanced evaporation would also limit the maximum magnitude of smoke cloud albedo effects. its inhibition of precipitation and cloud lifetime effect, as ev- idenced by the observed reductions in cloud droplet radius of ∼50 % in both the case study and the multi-campaign as- sessment. Smaller cloud droplets can have various consequences. Smoke-driven reductions or delays in precipitation may af- fect the distribution of aerosol and moisture deposition. Longer cloud lifetime could impact not only Arctic albedo but also long-wave radiation (Stone, 1997), and previous studies suggest that even small changes in the above parame- ters may affect sensitive Arctic sea ice (Kay et al., 2008; Kay and Gettelman, 2009; Lubin and Vogelmann, 2006; Vavrus et al., 2010). Additionally, changes in cloud cover might also have indirect effects on ocean photosynthesis and biogeo- chemistry (Bélanger et al., 2013). It is our hope that the im- proved quantiﬁcation of smoke-derived ACI values will help quantify these impacts in future model studies. One obvious limitation of our study is that we do not address the impacts of smoke on existing mixed- and ice- phase clouds. L. M. Zamora et al.: Aircraft-measured indirect cloud effects 10#2% 10#1% 100%101% 102% 103% 10#2% 10#1% 100%101% 102% 103% 10#2% 10#1% 100%101% 102% 103% 10#2% 10#1% 100%101% 102% 103% 102% 103% 104% 105% 102% 103% 104% 105% (d) ISDAC ####>#140#nm#diam.# (c) ISDAC ####>#4#nm#diam.# (a)  ARCTAS-B## #######>#3#nm#diam.# (b) ARCTAS-B ####>#3#nm#diam.# CO#(ppbv)# CH3CN#(ppbv)# Submicron#sca6er#at#550#nm#(M#m<1)# Submicron#sca6er#at#550#nm#(M#m<1)# Aerosol#no# concentraAon# (cm<1)# .# Figure 9. Log relationships between ARCTAS-B and ISDAC aerosol number concentration and submicron scatter. In panels (a) and (b), the combustion tracer, CO, and the biomass burning tracer, CH3CN, are also shown in out-of-cloud air masses. The black squares in panels (a) and (b) indicate where background aerosol concentrations of 5000 cm−3 and dilute smoke concentrations of 450 cm−3 would be relative to other points. Measurements are from the following instruments: (a) and (b) TSI 3025, (c) TSI 3775, and (d) PCASP. ARCTAS-B summertime samples were taken at altitudes < 5.2 km; ISDAC samples were taken at < 3.65 km due to TSI3025 instrument limitations. All quality- ﬂagged data were excluded, as well as suspicious ISDAC values within 17 km and < 1 km altitude of Fairbanks airport, Alaska. Very small background aerosols appear to dominate the high aerosol number concentration/low scatter particles seen in (a)–(c), as shown by their disappearance when a diameter cutoff of 140 nm is used (d). 10#2% 10#1% 100%101% 102% 103% 10#2% 10#1% 100%101% 102% 103% 102% 103% 104% 105% (a)  ARCTAS-B## #######>#3#nm#diam.# (b) ARCTAS-B ####>#3#nm#diam.# CO#(ppbv)# CH3CN#(ppb Submicron#sca6er#at#550#nm#(M#m<1)# Aerosol#no# concentraAon# (cm<1)# .# 10#2% 10#1% 100%101% 102% 103% 10#2% 10#1% 100%101% 102% 103% 10#2% 10#1% 100%101% 102% 103% 10#2% 10#1% 100%101% 102% 103% 102% 103% 104% 105% 102% 103% 104% 105% (d) ISDAC ####>#140#nm#diam.# (c) ISDAC ####>#4#nm#diam.# (a)  ARCTAS-B## #######>#3#nm#diam.# (b) ARCTAS-B ####>#3#nm#diam.# CO#(ppbv)# CH3CN#(ppbv)# Submicron#sca6er#at#550#nm#(M#m<1)# Submicron#sca6er#at#550#nm#(M#m<1)# Aerosol#no# concentraAon# (cm<1)# .# Submicron#sca6er#at#550#nm#(M#m<1)# Submicron#sca6er#at#550#nm#(M#m<1)# Figure 9. Log relationships between ARCTAS-B and ISDAC aerosol number concentration and submicron scatter. In panels (a) and (b), the combustion tracer, CO, and the biomass burning tracer, CH3CN, are also shown in out-of-cloud air masses. The black squares in panels (a) and (b) indicate where background aerosol concentrations of 5000 cm−3 and dilute smoke concentrations of 450 cm−3 would be relative to other points. Measurements are from the following instruments: (a) and (b) TSI 3025, (c) TSI 3775, and (d) PCASP. ARCTAS-B summertime samples were taken at altitudes < 5.2 km; ISDAC samples were taken at < 3.65 km due to TSI3025 instrument limitations. www.atmos-chem-phys.net/16/715/2016/ Atmos. Chem. Phys., 16, 715–738, 2016 730 L. M. Zamora et al.: Aircraft-measured indirect cloud effects Additionally, we cannot account for the ways in which smoke might have affected the sample phase. For example, ice nuclei presence might facilitate the conversion of an otherwise liquid-phase cloud into a mixed-phase cloud that was excluded in this assessment. Alternatively, we could have included liquid clouds in our assessment that might oth- erwise have been present as mixed- or ice-phase clouds if not for the inhibition of freezing by soluble smoke compounds via the Raoult effect (discussed in Tao et al., 2012). Based on a previous model study by McComiskey and Feingold (2008), the ACI value of 0.05 from the case study suggests that smoke may reduce local summertime radiative ﬂux via the cloud albedo effect by between 2 and 4 W m−2 or more under low and homogeneous cloud cover conditions in the subarctic. At higher latitudes where surface albedo is al- ready high, the impact on radiative ﬂux is likely to be smaller. In those regions, a more important effect of smoke might be www.atmos-chem-phys.net/16/715/2016/ www.atmos-chem-phys.net/16/715/2016/ Atmos. Chem. Phys., 16, 715–738, 2016 Atmos. Chem. Phys., 16, 715–738, 2016 L. M. Zamora et al.: Aircraft-measured indirect cloud effects 731 Finally, we have presented evidence to suggest that coagu- lation of the numerous nucleation- and Aitken-mode back- ground particles frequently present in clean summertime Arctic air masses might increase the volume of diluted smoke aerosols by up to 2–15 %. Previous studies suggest that such interactions with background particles may increase smoke aerosol hygroscopicity, which in turn could cause deviations from the ACI value derived here. Future remote sensing or ground-based analyses may be able to more completely ad- dress the different impacts of dilute vs. concentrated smoke aerosols in Arctic clouds. Atmos. Chem. Phys., 16, 715–738, 2016 www.atmos-chem-phys.net/16/715/2016/ Appendix A: Calculations for maximum potential contribution of background aerosol to diluted smoke aerosol volume We ﬁrst estimate the volume of smoke particles at di- lute concentrations of 450 particles cm−3. Arctic/subarctic smoke aerosol size distributions were taken from Kondo et al. (2011) and Sakamoto et al. (2015), where log-normal aerosol size distributions were characterized by geometric mean diameters of 224 ± 14 and 230 nm and geometric stan- dard deviations of 1.33 ± 0.05 and 1.5, respectively. From the corresponding size distributions, we estimate smoke aerosol volumes of ∼2.9–6.0 µm3 per cm−3 of air at smoke concen- trations of 450 cm−3. The degree to which aerosol properties can be affected by the collection of Arctic nucleation- and Aitken-mode back- ground particles onto larger smoke and pollution particles also depends in part on the size ranges and concentrations of the background particles. These can be quite variable (En- gvall et al., 2008) (also see Fig. A1). To estimate average background concentrations, we use the observed geometric mean ratio range in 6-year Svalbard summertime data (En- gvall et al., 2008), which indicated that Aitken-mode par- ticle concentrations were ∼1.5–3 times greater than those of accumulation-mode particles. Given this range in ratios, we would expect background particle concentrations to be ∼ 675–1350 cm−3 at smoke concentrations of 450 cm−3. We then combine the expected small background aerosol con- centrations with ARCTAS background aerosol spectra from events from 12 April, 10 July, and 13 July 2008 (Fig. A1) for particles < 80 nm in diameter. Based on these values, the small background aerosol volume is estimated at 0.012– 0.114 µm3 cm−3. A comparison of this volume with the pre- viously estimated smoke aerosol volume suggests that back- ground aerosols could contribute only ∼0.2–4 % of total di- luted smoke aerosol volume in average summertime condi- tions. This estimate does not account for the fact that all else being equal, small particles are usually more likely to co- agulate onto the largest sized particles (Seinfeld and Pandis, 1998), which would reduce the contribution to average parti- cle volume even further. Figure A1. Mean out-of-cloud aerosol particle size distributions for several ARCTAS background aerosol events. Some days had multi- ple background aerosol events; these are distinguished by color and the letters (a)–(c). The light gray line shows the 80 nm cutoff used here to distinguish Aitken-mode particles from accumulation-mode particles. Alternatively, we can estimate what the background aerosol volume might be if particle concentrations were as high as 5000 cm−3. www.atmos-chem-phys.net/16/715/2016/ www.atmos-chem-phys.net/16/715/2016/ M. Zamora et al.: Aircraft-measured indirect cloud effects Figure A1. Mean out-of-cloud aerosol particle size distributions for several ARCTAS background aerosol events. Some days had multi- ple background aerosol events; these are distinguished by color and the letters (a)–(c). The light gray line shows the 80 nm cutoff used here to distinguish Aitken-mode particles from accumulation-mode particles. L. M. Zamora et al.: Aircraft-measured indirect cloud effects M. Zamora et al.: Aircraft-measured indirect cloud effects 732 Appendix A: Calculations for maximum potential contribution of background aerosol to diluted smoke aerosol volume Although such events are not common in the Arctic and subarctic, similar high-end concentrations of background particles are observed in Fig. 9 and have been observed elsewhere in the Arctic as well (Ström et al., 2009). Again assuming the same range of particle size distributions observed in Fig. A1, the small background aerosol volume at 5000 particles cm−3 is estimated to be between 0.092 and 0.422 µm3 per cm−3 of air. Thus, in this case, background aerosols could add at most 2–15 % of total aerosol volume in diluted smoke with concentrations of 450 particles cm−3. Atmos. Chem. Phys., 16, 715–738, 2016 L. M. Zamora et al.: Aircraft-measured indirect cloud effects Acknowledgements. The authors would like to thank A. Aknan, B. Anderson, E. Apel, G. Chen, M. Couture, T. Garrett, K. B. Huebert, A. Khain, A. Korolev, T. Lathem, P. Lawson, R. Leaitch, J. Limbacher, J. Nelson, M. Pinsky, W. Ridgeway, A. Rangno, S. Williams, S. Woods, and Y. Yang for data and/or advice or help with various aspects of this project, and all others who were involved in collecting and funding the collection of the data sets we have used. We acknowledge the Atmospheric Radiation Measurement (ARM) Program sponsored by the U.S. Department of Energy, Ofﬁce of Science, Ofﬁce of Biological and Environmental Research, Climate and Environmental Sciences Division for providing the ISDAC data set. The authors also gratefully acknowledge the NOAA Air Resources Laboratory (ARL) for the provision of the HYSPLIT transport and dispersion model and/or READY website (http://www.ready.noaa.gov) used in this publication. Plots were made with Ocean Data View (Schlitzer, R., Ocean Data View, http://odv.awi.de, 2015) and R (R Core Team, 2013). 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https://openalex.org/W3208344936	https://escholarship.org/content/qt4gs1g3n9/qt4gs1g3n9.pdf?t=rii5rx	English	null	Utilization of Telehealth Solutions for Patients with Opioid Use Disorder Using Buprenorphine: A Scoping Review	Telemedicine and e-health	2,021	cc-by	773	UC Irvine Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health Title Utilization of Telehealth Solutions for Patients with Opioid Use Disorder Using Buprenorphine: A Scoping Review Permalink https://escholarship.org/uc/item/4gs1g3n9 Journal Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health, 23(5.1) ISSN 1936-900X Authors Guillen, Aileen Chakravarthy, Bharath Reddy, Minal et al. Publication Date 2022 DOI 10.5811/westjem.58895 Copyright Information Copyright 2022 by the author(s).This work is made available under the terms of a Creativ Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ UC Irvine Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health Title Utilization of Telehealth Solutions for Patients with Opioid Use Disorder Using Buprenorphine: A Scoping Review Permalink https://escholarship.org/uc/item/4gs1g3n9 Journal Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health, 23(5.1) ISSN 1936-900X Authors Guillen, Aileen Chakravarthy, Bharath Reddy, Minal et al. Publication Date 2022 DOI 10.5811/westjem.58895 Copyright Information Copyright 2022 by the author(s).This work is made available under the terms of a Creativ Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ UC Irvine Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health Copyright Information Copyright 2022 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ eScholarship.org Powered by the California Digital Library University of California eScholarship.org Powered by the California Digital Library University of California Powered by the California Digital Library University of California MEMC Abstracts 2022 COVID-19 on mental health. Figure 1. Prisma Flow Diagram. 5 Utilization of Telehealth Solutions for Patients with Opioid Use Disorder Using Buprenorphine: A Scoping Review Aileen Guillen; Bharath Chakravarthy; Minal Reddy; Soheil Saadat Objectives: A scoping review was conducted to examine the breadth of evidence related to telehealth innovations being utilized in the treatment of Opioid Use Disorder (OUD) with buprenorphine and its effect on patient outcomes and healthcare delivery. Background: With the opioid epidemic worsening from year to year, there is a critical need to connect with this growing population and get them access to life-saving interventions. Buprenorphine is shown to be associated with lower overdose rates and a decrease in opioid-related acute care, but has historically been underutilized in treatment for OUD. Previous studies have determined that geographical barriers and lack of access to DEA-waivered providers are common obstacles towards starting MAT. Telehealth presents itself as a solution to this discrepancy and is becoming more feasible to integrate into clinical practice. Figure 1. Prisma Flow Diagram. Figure 1. Prisma Flow Diagram. Western Journal of Emergency Medicine 6 Variation in Trauma Team Response Fees in United States Trauma Centers 6 Arianna Neeki; David Wong; Fanglong Dong; Jan Serrano; Louis P. Tran; Mason Chan; Michael M. Neeki; Pamela R. A. Lux Methods: The authors systematically searched seven databases and websites for peer-reviewed and gray literature related to telehealth solutions for buprenorphine treatment published between 2008 and March 18, 2021. There were 69 articles which met inclusion criteria. Objectives: Investigate the variation of the trauma team response fee (TTRF) among all levels of Trauma Centers (TC) Level I-IV, in different geographic regions in the U.S. (Midwest, West, South, Northeast U.S.). Results: According to the reviewed literature, incorporation of telehealth technology with Medication Assisted Treatment (MAT) for OUD is associated with higher patient satisfaction, comparable rates of retention, and an overall reduction in health care costs. Background: Investigate Hospital Medical Directors (HMD) and Trauma Medical Directors (TMD) knowledge of TTRF dollar amount in their institution. Conclusion: Utilization of synchronous videoconferencing has reportedly been effective in increasing access to and usage of buprenorphine by overcoming both geographical and logistical barriers. This has been made possible through the expansion of telehealth technologies and a substantial push towards relaxed federal guidelines, both of which were quickly escalated in response to the COVID-19 pandemic. Future research is needed to fully quantify the effect of these factors; however, the results appear promising thus far and should urge policymakers to consider making these temporary policy changes permanent. Methods:Setting 525 American College of Surgeons verified trauma centers (TC) in the U.S. Level I-IV TCs. TC’s in the continental U.S including Alaska and Hawaii. Data Collection Cross-sectional convenience sample. Online survey development cloud-based software, Survey Monkey. Responses from October 8, 2019 through March 11, 2020. Results: True costs of TTRF’s in the U.S remains elusive due to inadequate data. TTRF’s were higher in level II TC’s in the West compared to Level I’s. No statistically significant difference in TTRF’s despite geographical and cost of living differences. 41.3% of HMD are aware of dollar amount of TTRF’s. 56.5% of TMD are aware of dollar amount of TTRF’s. Volume 23, Issue 4 Supplement: September 2022 Western Journal of Emergency Medicine S3
https://openalex.org/W4383956447	https://f1000research.com/articles/12-811/pdf	English	null	Investigating the link between frontal sinus morphology and craniofacial characteristics with sex: A 3D CBCT study on the South Indian population	F1000Research	2,023	cc-by	7,413	Ceena Denny 1, Mohana Bhoraskar1, Sabiha Abdul Aziz Shaikh 1, Bastian T S2, Nanditha Sujir 1, Srikant Natarajan 3 Ceena Denny 1, Mohana Bhoraskar1, Sabiha Abdul Aziz Shaikh 1, Bastian T S2, Nanditha Sujir 1, Srikant Natarajan 3 1Oral Medicine and Radiology, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka 575001 Mangalore India g 2Oral and Maxillofacial Pathology, MAHE Institute of Dental Sciences, Chalakara, Puducherry, 673010, India 3Oral and Maxillofacial Pathology, Manipal College of Dental Sciences, Mangalore Manipal Academy of Higher Education, Manipal, Karnataka.India-575001, Mangalore, India Open Peer Review Approval Status 1 2 version 2 (revision) 11 Sep 2023 view version 1 11 Jul 2023 view view First published: 11 Jul 2023, 12:811 https://doi.org/10.12688/f1000research.137008.1 Latest published: 11 Sep 2023, 12:811 https://doi.org/10.12688/f1000research.137008.2 v2 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 RESEARCH ARTICLE Investigating the link between frontal sinus morphology and craniofacial characteristics with sex: A 3D CBCT study on the South Indian population [version 2; peer review: 2 approved] Ceena Denny 1, Mohana Bhoraskar1, Sabiha Abdul Aziz Shaikh 1, Bastian T Nanditha Sujir 1, Srikant Natarajan 3 1Oral Medicine and Radiology, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipa Karnataka-575001, Mangalore, India 2Oral and Maxillofacial Pathology, MAHE Institute of Dental Sciences, Chalakara, Puducherry, 673010, India 3Oral and Maxillofacial Pathology, Manipal College of Dental Sciences, Mangalore Manipal Academy of Higher Education, Man Karnataka.India-575001, Mangalore, India First published: 11 Jul 2023, 12:811 https://doi.org/10.12688/f1000research.137008.1 Latest published: 11 Sep 2023, 12:811 https://doi.org/10.12688/f1000research.137008.2 v2 Open Peer Review Approval Status RESEARCH ARTICLE Investigating the link between frontal sinus morphology and craniofacial characteristics with sex: A 3D CBCT study on the South Indian population [version 2; peer review: 2 approved] Abstract Background: Measurement of craniofacial parameters plays an important role in sex determination in forensic science. The present study was done using cone beam computed tomography (CBCT) scans to evaluate the morphologic structure of the frontal sinuses and compare it with the width of nasal, cranial, maxillary and mandibular width which might help us in sex determination. Background: Measurement of craniofacial parameters plays an important role in sex determination in forensic science. The present study was done using cone beam computed tomography (CBCT) scans to evaluate the morphologic structure of the frontal sinuses and compare it with the width of nasal, cranial, maxillary and mandibular width which might help us in sex determination. Methods: A cross-sectional retrospective study was conducted using 142 full field of view (FOV) scans of patients archived from the department. The width of the nose, cranium, maxilla, and mandibular width was measured and compared with the frontal sinus between the two sexes. view view Methods: A cross-sectional retrospective study was conducted using 142 full field of view (FOV) scans of patients archived from the department. The width of the nose, cranium, maxilla, and mandibular width was measured and compared with the frontal sinus between the two sexes. Sudeendra Prabhu, Yenepoya (Deemed to be University), Mangalore, India 1. Arvind Babu Rajendra Santosh , The University of The West Indies, Kingston, Jamaica 2. Results: A paired t-test was done to compare the linear measurements for both sexes' right and left frontal sinuses. The measurements were higher in males when compared to females. There was a statistically significant asymmetry (larger dimension on the left side) of the anterioposterior (p-value of 0.012) and superior- inferior dimensions in males (p-value of 0.135). Spearman's correlation showed that frontal sinus correlated with other craniofacial parameters like nasal, cranial, maxillary and mandibular width among both sexes. The frontal sinus, nasal, cranial, maxillary and mandibular widths were higher in males when compared to females (independent t-test). Discriminant function scores showed 66- 68% accuracy to discriminate sex, using the anteroposterior dimension and mandibular width. Page 1 of 14 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 Conclusions: The measurement of craniofacial parameters using CBCT can aid in determining the sex of unidentified and decomposed bodies. Corresponding author: Srikant Natarajan (srikant.n@manipal.edu) Author roles: Denny C: Conceptualization, Data Curation, Formal Analysis, Methodology, Project Administration, Writing – Original Draft Preparation; Bhoraskar M: Data Curation; Abdul Aziz Shaikh S: Data Curation; T S B: Writing – Review & Editing; Sujir N: Data Curation, Investigation; Natarajan S: Data Curation, Formal Analysis, Project Administration, Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2023 Denny C et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Denny C, Bhoraskar M, Abdul Aziz Shaikh S et al. Investigating the link between frontal sinus morphology and craniofacial characteristics with sex: A 3D CBCT study on the South Indian population [version 2; peer review: 2 approved] F1000Research 2023, 12:811 https://doi.org/10.12688/f1000research.137008.2 First published: 11 Jul 2023, 12:811 https://doi.org/10.12688/f1000research.137008.1 Keywords y Frontal sinus, Craniofacial parameters, Forensic science, Cone Beam Computed Tomography, Sex determination This article is included in the Manipal Academy This article is included in the Manipal Academy This article is included in the Manipal Academy of Higher Education gateway. of Higher Education gateway. Introduction Determining the identity of human remains through visual assessment of craniofacial parameters using linear measure- ments is a common practice in forensic science. Unfortunately, accidents, natural disasters, and fires can cause severe decomposition or damage to the body, making identification difficult. In these cases, anthropological knowledge is crucial in identifying the remains. The skull, being the most complex and stable part of the human body, can withstand force and assist in forensic investigations.1 The frontal sinus is a crucial component of the human skull, located in the pneumatized cavity of the frontal bone. While typically paired, these sinuses are seldom symmetrical and can vary in size. Radiographically visible by the 2nd or 3rd year of life, the frontal sinus completes its growth by the age of 20. Interestingly, these sinuses are unique to each individual, even when compared in monozygotic twins.2 By analyzing the shape and size of the frontal sinus, we can gain valuable insights into an individual's craniofacial parameters. This information can be used to diagnose and treat various medical conditions, including sinusitis and facial trauma. It is important to note that the morphology of the frontal sinus can vary significantly between individuals, making it a valuable tool in personalized medicine. Radiographs are crucial in identifying anthropological structures when DNA samples and soft tissues are not accessible. Radiology plays a significant role in determining these structures, particularly when considering morphological parameters.3 Cone beam computed tomography (CBCT) is a cutting-edge diagnostic imaging technique that provides a three-dimensional view of a specific region in the head and neck. This technology allows for the visualization of anatomical structures from different angles, without any superimposition, distortion, or magnification. The accuracy of measurements obtained through CBCT makes it a superior imaging modality compared to traditional two-dimensional imaging.4 One of the most significant advantages of CBCT is its cost-effectiveness, as well as its ability to expose individuals to less radiation. This makes it a safer and more accessible option for patients. While CBCT has been widely used in the field of anthropology, it has recently gained more attention in the field of forensic identification.5 Although the pelvis, skull, and femur were initially used for identification purposes, these bones are rarely found intact during accidents. In contrast, the frontal sinus is highly resistant to damage due to its protected location. Corresponding author: Srikant Natarajan (srikant.n@manipal.edu) Page 2 of 14 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 Ethics and consent Ethical approval was obtained from the Manipal College of Dental Sciences' Institutional Ethics Committee (protocol ref no: 21056, dated 20/9/21). As part of the dental treatment, participants were asked to provide written consent for the use of their anonymized data in future research and publications. Introduction Extensive research has been conducted in the past using radiographs to determine the significance of the frontal sinus in identifying an individual's characteristics. However, the evaluation of frontal sinuses through Cone Beam Computed Tomography (CBCT) has emerged as a crucial method, especially in challenging scenarios like mass disasters, accidents, or when antemortem reports are unavailable.5 However, very few studies have been conducted using CBCT in the South Indian population. Our study aims to fill this gap by focusing on the South Indian population, which has distinct craniofacial features compared to other populations. Through this research, we hope to enhance the understanding of the relationship between frontal sinus morphology and craniofacial parameters in this specific population. The purpose of this study is to evaluate the morphological structure of the frontal sinuses and compare it with the nasal, maxillary, cranial and the mandible widths. This information can be useful in identifying and determining human characteristics. REVISED Amendments from Version 1 REVISED Amendments from Version 1 I have added a few more points in both discussion and conclusion. We have highlighted the important parameter having good correlation quoted studies using CBCT for sex dimorphism added justification for studying the morphometric characterization of the frontal sinus with consideration of gender. An f rther responses from the re ie ers can be fo nd at the end of the article I have added a few more points in both discussion and conclusion. We have highlighted the important parameter having good correlation quoted studies using CBCT for sex dimorphism added justification for studying the morphometric characterization of the frontal sinus with consideration of gender. Any further responses from the reviewers can be found at the end of the article Methods Ethics and consent Participants The frontal sinuses were evaluated in patients who were above 20 years with no history of trauma, cranial anomalies, associated pathologies in the sinus, skull/orthognathic surgery, and endocrine disorders. 66 scans were of the male sex and 77 scans were of the female sex in the age range of 20-54 years old. Design and sample size Design and sample size A cross-sectional retrospective study was conducted after obtaining ethical clearance. 142 full field of view (FOV) scans of patients were retrieved from the department of Oral Medicine and Radiology. The images selected were those of South-Indian population which included a subset of residents of the Dakshina Kannada district catering to the West coast of India. Page 3 of 14 Page 3 of 14 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 Based on the article “Association between frontal sinus morphology and craniofacial parameters: A forensic view published in the Journal of Forensic Legal Medicine by authors S.K. Buyuk et al. in the year 2017,6 the correlation coefficient derived/reported was 0.231. With an alpha error of 5% and a power of 80%, the Z values of the given alpha and beta values are 1.959 and 0.842. With the correlation coefficient and using the above formula the required sample size was 142. The sampling was done in such a way to match the age for each individual of the opposite sex. Data collection The cone beam computed tomographic (CBCT) scans were acquired using the Promax 3DMid (Planmeca Oy., Helsinki, Finland) CBCT unit. The large FOV, low dose images that allowed complete visualization above the frontal sinuses were taken with the exposure parameters being 90 kVp, 5.6 mA, and exposure time of 18 seconds (DAP-925 mGycm2, CTDI-3.7 mGy). A slice thickness of 0.400 mm was used to assess the sections. The exposure parameters according to the standard default values were based on the FOV. The images were reconstructed using the Romexis software version 4.6.2.R (ITK-SNAP 4.0 I can be used as an alternative). Statistical analysis The various dimensions of the frontal sinus and cranial parameters were compared between the left and right sides to check for asymmetry using paired t test. The various parameters of the frontal sinus and the cranial parameters were correlated with each other using Spearmans rank correlation. To identify the variation between the males and females independent t test was performed. All comparisons were considered significant at a level of 95% (p<0.05). Analysis was performed using IBM SPSS 20.0 (IBM incorp. Chicago). Measurements Measurements The linear measurements were done on the reconstructed image in the following way (Figure 1): Measurements The linear measurements were done on the reconstructed image in the following way (Figure 1): • Frontal sinus - For assessment of maximum supero-inferior measurements, the coronal section was utilized and measurement was taken between the highest and lowest point of both the right and left frontal sinus, while the axial section was usedtomeasure the maximum antero-posterior and mesiodistal widthofthe left and rightfrontal sinus. • Cranium - For the measurement of the maximum width of the cranium, the axial section was adjusted to the level of the superior border of the orbit (while looking at the coronal view), and at this level, measurement was taken from the inner cortical plate on one side to the inner cortical plate on the contralateral side. • Cranium - For the measurement of the maximum width of the cranium, the axial section was adjusted to the level of the superior border of the orbit (while looking at the coronal view), and at this level, measurement was taken from the inner cortical plate on one side to the inner cortical plate on the contralateral side. Figure 1. Cone-beam computed tomography image showing the measurements of A) mesio-distal dimension of frontal sinus; B) antero-posterior measurement of frontal sinus; C) supero-inferior measurement of frontal sinus; D) cranial width; E) nasal width; F) maxillary width; G) mandibular width. Pag Figure 1. Cone-beam computed tomography image showing the measurements of A) mesio-distal dimension of frontal sinus; B) antero-posterior measurement of frontal sinus; C) supero-inferior measurement of frontal sinus; D) cranial width; E) nasal width; F) maxillary width; G) mandibular width. Page 4 of 14 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 • Nasal - The maximal nasal width was taken in the axial section after adjusting it at the level of the zygomatic arch. • Nasal - The maximal nasal width was taken in the axial section after adjusting it at the level of the zygomatic arch. • Maxilla - Axial section was skimmed till the maximal width of the maxilla was observed and in this section, measurement was taken. • Maxilla - Axial section was skimmed till the maximal width of the maxilla was observed and in this section, measurement was taken. Measurements • Mandibular width at antegonial notch - The maximum width of the mandible was taken at the level of the antegonial notch. For this purpose, first, the axial view was angulated so that in the sagittal section, the gonial and antegonial notch were observed. The axial view was then adjusted to the level of the antegonial notch and in this section, the maximal mandibular width was taken. • Mandibular width at antegonial notch - The maximum width of the mandible was taken at the level of the antegonial notch. For this purpose, first, the axial view was angulated so that in the sagittal section, the gonial and antegonial notch were observed. The axial view was then adjusted to the level of the antegonial notch and in this section, the maximal mandibular width was taken. L: left, R: right, N: number, SD: standard deviation. Red and underline signifies that P value <0.05 are statistically significant. L: left, R: right, N: number, SD: standard deviation. , g , , Red and underline signifies that P value <0.05 are statistically significant. L: left, R: right, N: number, SD: standard deviation. Red and underline signifies that P value <0 05 are statistically significant Results There was asymmetry detected of the frontal sinus in all the patients in terms of the mesiodistal, anteroposterior and superoinferior dimensions with the majority showing the left side larger than the right side.25 However, only the anteroposterior dimension was significantly more on the left side in males (p-value 0.012, paired-test) (Table 1). Mandibular parameter correlates moderately with anteroposterior dimension on the left and right side in males but not in females. The anteroposterior dimension of the left side and the mesiodistal dimension of the left side correlate significantly (r=0.357, p=0.003) in males but not in females. On the other hand, the anteroposterior dimension of the right side correlates with the mesiodistal dimension of the right side in both females and males. The cranial parameter shows a moderate negative correlation with the anteroposterior dimension of the left side (r value of-0.235, p-value of 0.039) and nasal dimension (r value of -0.248, p-value of 0.03) in females but not in males. Maxillary dimension showed a significant positive correlation with mandible (r value of 0.326 and p value of 0.004) in females and not in males. Nasal dimensions correlated with superoinferior dimensions on the right side in females (0.233 and p-value of 0.041) but not in males. Superoinferior dimensions correlated positively with mesiodistal as well as anteroposterior dimensions in males and females (p<0.05, r ranges from 0.248 to 0.461) (Table 2). Table 1. Paired t-test to compare the frontal sinus on either side in both sexes. N Mean SD Mean difference SD t P value Male Pair 1 Mesiodistal L 65 30.967.7 0.589.86 0.48 0.635 Mesiodistal R 65 30.377.6 Pair 2 Anterioposterior L 65 12.014.64 1.263.92 2.59 0.012 Anterioposterior R 65 10.752.68 Pair 3 Superioinferior L 65 29.496.14 0.94.78 1.51 0.135 Superioinferior R 65 28.595.14 Female Pair 1 Mesiodistal L 77 29.369 0.4710.91 0.38 0.707 Mesiodistal R 77 28.898.57 Pair 2 Anterioposterior L 77 9.162.82 -0.222.8 -0.68 0.499 Anterioposterior R 77 9.373.5 Pair 3 Superioinferior L 77 26.995.83 0.614.66 1.16 0.252 Superioinferior R 77 26.376.03 Table 1. Paired t-test to compare the frontal sinus on either side in both sexes. F1000Research 2023, 12:811 Last updated: 10 OCT 2024 Table 2. Spearman s correlation between either sexes. Results Mesio-Distal L Mesio-Distal R Anterio- Posterior L Anterio- Posterior R Superio- Inferior L Superio- Inferior R Nasal Mandible Cranial Male Mesio-distal R 0.192 (0.125) Anterio-posterior L .357* (0.003) 0.223 (0.074) Anterio-posterior R 0.134 (0.287) .282* (0.023) .644 (<0.001) Superio-inferior L .381 (0.002) 0.231 (0.064) .312* (0.012) 0.178 (0.155) Superio-inferior R .431** (<0.001) .285* (0.021) .336** (0.006) .248* (0.046) .629** (<0.001) Nasal 0.08 (0.527) 0.214 (0.087) -0.076 (0.548) 0.209 (0.094) 0.047 (0.707) 0.218 (0.081) Mandible 0.206 (0.099) 0.102 (0.419) .262* (0.035) .271* (0.029) -0.066 (0.603) 0.069 (0.585) 0.167 (0.183) Cranial 0.032 (0.803) -0.159 (0.206) 0.109 (0.389) 0.119 (0.344) -0.029 (0.818) 0.013 (0.916) -0.147 (0.243) -0.125 (0.322) Maxilla 0.026 (0.839) 0.14 (0.265) -0.051 (0.684) 0.02 (0.877) 0.117 (0.352) 0.087 (0.489) .419** (0.001) 0.193 (0.123) -0.141 (0.262) Female Mesio-distal R .248* (0.03) Anterio-posterior L 0.1 (0.388) 0.164 (0.153) Anterio-posterior R 0.122 (0.291) .341 (0.002) .454 (<0.001) Superio-inferior L .348 (0.002) .439 (<0.001) .249* (0.029) .334 (0.003) Superio-inferior R 0.221 (0.054) .468 (<0.001) .313 (0.006) .461 (<0.001) .684** (<0.001) Nasal -0.002 (0.988) 0.101 (0.383) 0.098 (0.399) 0.203 (0.076) 0.159 (0.168) .233* (0.041) Mandible 0.049 (0.671) -0.021 (0.858) -0.014 (0.905) 0.073 (0.526) -0.042 (0.716) -0.02 (0.86) -0.021 (0.857) Cranial -0.16 (0.164) -0.062 (0.591) -.235* (0.039) -0.138 (0.232) -0.143 (0.213) 0.021 (0.855) -.248* (0.03) 0.06 (0.602) Maxilla -0.073 (0.529) 0.059 (0.612) 0.209 (0.069) .286* (0.012) 0.029 (0.801) 0.064 (0.579) 0.025 (0.831) .326** (0.004) 0.038 (0.743) *Significant with p value <0.05. F1000Research 2023, 12:811 Last updated: 10 OCT 2024 Table 3. Independent t-test to compare values between males and females. Male (n=65) Female (n=77) t P value Mean sd Mean sd Mesiodistal L 30.967.7 29.369 1.124 0.263 Mesiodistal R 30.377.6 28.898.57 1.08 0.282 Anterioposterior L 12.014.64 9.162.82 4.324 <0.001 Anterio posterior R 10.752.68 9.373.5 2.587 0.011 Superioinferior L 29.496.14 26.995.83 2.485 0.014 Superioinferior R 28.595.14 26.376.03 2.334 0.021 Nasal 24.652.73 24.232.66 0.933 0.353 Mandible 80.986.86 76.25.21 4.604 <0.001 Cranial 129.9816.05 127.266.59 1.362 0.175 Maxilla 60.44.41 58.274.11 2.976 0.003 Red and underline signifies that P value <0.05 are statistically significant. Table 3. Independent t-test to compare values between males and females. An independent t-test was done to compare the measurements between the two sexes and it was found that there was a statistically significant difference between them. Results The frontal sinus, nasal, cranial, maxillary and mandibular widths were higher in males when compared to the females and this was statistically significant except for the nasal width, cranial and mesiodistal measurements of the frontal sinus (Table 3). Comparing the discriminant function scores we found that the male values are consistently higher than the female values in all the cases. The discriminant function equations and the demarcation points are given in Table 4. The highest accuracy was seen with the mandibular width with 68.3% accuracy, followed by anteroposterior left dimensions with 66.90%. Both the mandible and the left anterior-posterior dimensions were more accurate in females than in males. The other variables like the superio-inferior and mesio-distal measurements of frontal sinus and nasal dimensions were below 60% accuracy. The discriminant function equations can be used with sectioning points of roughly zero to demarcate the sex. Discussion Sex, age and stature are crucial factors in identifying unknown individuals through skeletal remains, especially when the body is decomposed or fragmented beyond recognition. Radiographs are often used to aid in this process, as the anatomic structures and their variations are unique to each individual.7 In forensic dentistry, craniometric parameters also play a significant role in both postmortem and antemortem evaluations of bodies. Cranial dimensions vary among different populations, and measuring the cranium can help determine racial differences.8 Zuckerkandl reported in 1895 that the morphology of the frontal sinus is unique to each individual and can be used for identification purposes.9 Our study was a retrospective cone beam computed tomographic study, utilizing archived images from the South-Indian population, specifically a subset of the Dakshina Kannada district on the West coast of India. This research aims to establish specific anthropometric patterns for different populations, highlighting the importance of considering regional variations in forensic investigations. The frontal sinus is a structure that becomes visible on radiographs at around 5-6 years of age and stops growing around 20 years.7 It is intriguing to note that no two individuals have the same frontal sinus.10 Research has shown that the frontal sinus is larger in males than in females,11–13 which is consistent with our own findings. Moreover, when comparing the size of the frontal sinus on each side, we observed that the left side was larger than the right side. This finding is in line with previous studies conducted by Rubira-Bullen IR et al. (2010), Kanat A et al. (2015), Soman BA et al. (2016), and Denny C et al. (2018),12–16 but not with the study by Camargo et al. 2007.11 The difference in size could be attributed to the independent development of the sinuses, resulting in varying sizes even among individuals of the same age.14 Kanat et al. (2015)15 suggested that handedness and footedness may play a role in predicting cerebral dominance, with right-handed individuals exhibiting left hemispheric dominance and vice versa for left-handed individuals. In their recent study, Shamlou and Tallman (2022) discovered a noteworthy disparity in the height and depth of sinuses across various sexes and populations. However, intriguingly, they did not observe any variation in sinus shape. Discussion This finding sheds light on the intricate anatomical differences within different groups, emphasizing the importance of considering these factors in future research and medical practices.17 Page 7 of 14 Page 7 of 14 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 Table 4. Discriminant function analysis for sex. Parameter Male mean values Male SD Female mean values Female SD Equation Percentage of females correctly classified Percentage of males correctly classified overall accuracy Male centroid Female centroid Sectioning point Demarcating point Mesio- distal L 30.9562 7.69745 29.3599 9.00332 Discriminant function (D)=-3.569 +(0.119) x (Mesiodistal L) 61.5 54.5 57.70% 0.103 -0.087 -2.8169E-05 29.9916 Mesio- distal R 30.3722 7.60354 28.8914 8.57115 Discriminant function (D)=-3.631 +(0.123) x (Mesiodistal R) 49.2 62.3 56.30% 0.099 -0.083 0.000309859 29.52033 Anterio- posterior L 12.0088 4.64454 9.1569 2.81874 Discriminant function (D)=-2.779 +(0.266) x (Anterioposterior L) 58.5 74 66.90% 0.411 -0.347 -2.8169E-05 10.44737 Anterio- posterior R 10.7475 2.6839 9.374 3.49745 Discriminant function (D)=-3.174 +(0.317) x (Anterioposterior R) 55.4 70.1 63.40% 0.236 -0.199 0.000119718 10.01262 Superio- inferior L 29.4866 6.13768 26.9856 5.83334 Discriminant function (D)=-4.708 +(0.167) x (Superioinferior L) 58.5 59.7 59.20% 0.227 -0.192 -0.000204225 28.19162 Superio- inferior R 28.5909 5.14431 26.3719 6.0325 Discriminant function (D)=-4.853 +(0.177) x (Superioinferior R) 55.4 58.4 57.00% 0.213 -0.18 -0.000105634 27.41808 Nasal 24.6549 2.72772 24.2325 2.65697 Discriminant function (D)=-9.082 +(0.372) x (Nasal) 52.3 53.2 52.80% 0.085 -0.072 -0.000133803 24.41398 Mandible 80.9786 6.86311 76.199 5.21295 Discriminant function (D) =-13.013+(0.166) x (Mandible) 67.7 68.8 68.30% 0.43 -0.363 -7.04225E-06 78.39157 Cranial 129.9837 16.04581 127.2568 6.58519 Discriminant function (D) =-10.813+(0.084) x (Cranial) 69.2 57.1 62.70% 0.124 -0.105 -0.000176056 128.7262 Maxilla 60.404 4.41019 58.2729 4.11417 Discriminant function (D) =-13.934+(0.235) x (Maxilla) 67.7 55.8 61.30% 0.272 -0.229 0.000330986 59.29362 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 In summary, the frontal sinus is a distinctive anatomical structure that exhibits variations in size and shape among individuals. Our study corroborates previous research indicating that males tend to have larger frontal sinuses than females, and that the left side is typically larger than the right. These differences may be attributed to independent development and handedness/footedness, which could potentially impact cerebral dominance. Discussion Our study found that the maximal cranial width measurement was comparable to the research conducted by Ulcay T and Kamaşak B (2021).18 Additionally, we discovered that cranial width was significantly greater in males than in females.19,20 This dimorphism in the male skull is attributed to pubertal changes that result in increased muscular attachments, while the female skull retains juvenile features.21 According to a study conducted by Buyuk SK (2017), it was discovered that males have a greater maxillary width than females.6 Dr. G Venkat Rao and G Kiran (2016), stated in their article that dental arch width is indicative of the size of the basal bone. As the basal bone of the jaws is larger in males, this explains why males have a larger maxillary arch.22 The width of the mandible was found to be greater in males, which is consistent with the findings of previous studies conducted by Sreelekha D et al. (2020)23 and Albalawi AS et al. (2019).3 The present study investigated nasal width in both males and females and determined that there were no significant differences between the two groups. This finding is consistent with previous research conducted by Buyuk SK (2017) and M. I. Marini et al. (2020).6,24 Spearman's correlation analysis reveals that there is a significant correlation between the right side anterolateral and mesiodistal/superior inferior dimensions in both males and females. Additionally, mandibular dimensions show a significant correlation with anteroposterior dimensions in males, while cranial dimensions exhibit a negative correlation with anteroposterior and nasal dimensions in females. Furthermore, maxillary dimensions display a significant corre- lation with anteroposterior and mandibular dimensions in females. These observations can provide valuable insights for future research by comparing findings across different ethnicities and nationalities. However, to ensure a more accurate representation of the population, it is necessary to conduct further studies with larger sample sizes. Conclusions Cone beam computed tomography has revolutionized the way we approach diagnostic imaging, providing a more comprehensive and accurate view of the head and neck region. Its benefits extend beyond the medical field, making it an essential tool for forensic investigations. With its increasing popularity, CBCT is sure to continue to play a vital role in the future of diagnostic imaging. Our study revealed that the morphological structure of the frontal sinuses can be useful in identifying and determining human characteristics. This study also has the potential to offer valuable insights to medical professionals and researchers in the field of craniofacial medicine. By leveraging advanced imaging technology and focusing on a specific population, we can gain a deeper understanding of the intricate relationship between the frontal sinus and craniofacial parameters. Data availability Underlying data y Underlying data figshare: Investigating the Link between Frontal Sinus Morphology and Craniofacial Characteristics with Sex: A 3D CBCT study on the South Indian Population. https://doi.org/10.6084/m9.figshare.23559849.v1.25 This project contains the underlying measurement data (please note code 1 represents male, and 2 represents female in the sex column). Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Page 9 of 14 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 References 1. Harshitha K, Mohamed S, et al.: Evaluation and Correlation of Tooth Morphometrics in the Maxillary Arch for Sex Identification. Oral Maxillofac. Patho. J. 2019; 10(2): 57–60. among Indian adults. J. Forensic Dent. Sci. 2014; 6(1): 25–30. PubMed Abstract\|Publisher Full Text\|Free Full Text 1. Harshitha K, Mohamed S, et al.: Evaluation and Correlation of Tooth Morphometrics in the Maxillary Arch for Sex Identification. Oral Maxillofac. Patho. J. 2019; 10(2): 57–60. 2. Suman JL, Jaisanghar N, et al.: Configuration of frontal sinuses: A forensic perspective. J. Pharm. Bioallied Sci. 2016; Oct; 8(Suppl 1): S90–S95. PubMed Abstract\|Publisher Full Text\|Free Full Text 3. Albalawi AS, Alam MK, Vundavalli S, et al.: Mandible: An Indicator for Sex Determination - A Three-dimensional Cone-Beam Computed Tomography Study. Contemp. Clin. Dent. 2019; 10(1): 69–73. PubMed Abstract\|Publisher Full Text\|Free Full Text 4. Botticelli S, Verna C, Cattaneo PM, et al.: Two- versus three- dimensional imaging in subjects with unerupted maxillary canines. Eur. J. Orthod. 2011; 33(4): 344–349. PubMed Abstract\|Publisher Full Text 5. Choi IGG, Duailibi-Neto EF, Beaini TL, et al.: The Frontal Sinus Cavity Exhibits Sexual Dimorphism in 3D Cone-beam CT Images and can be Used for Sex Determination. J. Forensic Sci. 2018; 63(3): 692–698. PubMed Abstract\|Publisher Full Text 6. Buyuk SK, Karaman A, Yasa Y: Association between frontal sinus morphology and craniofacial parameters: A forensic view. J. Forensic Legal Med. 2017; 49: 20–23. PubMed Abstract\|Publisher Full Text 7. Rao KA, Doppalapudi R, Al-Shammari NT, et al.: Evaluation of frontal sinus index in establishing sex dimorphism using three-dimensional cone beam computed tomography in Northern Saudi Arabian population. J. Forensic Sci. Med. 2022; 8: 1–5. Publisher Full Text 8. Viloria A, Pineda OB, Romero L: Predicting the anthropometric properties of cranial structures using big data. Procedia Comput. Sci. 2020; Jan 1; 170: 905–910. Publisher Full Text 9. da Silva RF, Pinto RN, Ferreira GM, et al.: Importance of frontal sinus radiographs for human identification. Braz. J. Otorhinolaryngol. 2008; 74(5): 798. PubMed Abstract\|Publisher Full Text\|Free Full Text 10. Buckland-Wright JC: A radiographic examination of frontal sinuses in early British populations. Man. 1970; 5: 512–517. Publisher Full Text 11. Camargo JR, Daruge E, Prado FB, et al.: The frontal sinus morphology in radiographs of Brazilian subjects: Its forensic importance. Braz. J. Morphol. Sci. 2007; 24: 239–243. 12. References Denny C, Jacob AS, Ahmed J, et al.: Frontal Sinus as an aid in Gender Identification in Forensic Dentistry: A Retrospective Study using Cone Beam Computed Tomography. World J. Dent. 2018; 9 (1): 34–37. Publisher Full Text 13. Belaldavar C, Kotrashetti VS, Hallikerimath SR, et al.: Assessment of frontal sinus dimensions to determine sexual dimorphism 14. 14. Rubira-Bullen IR, Rubira CM, Sarmento VA, et al.: Frontal sinus size on facial plain radiographs. J. Morphol. Sci. 2010; 27(2): 77–81. 2. Suman JL, Jaisanghar N, et al.: Configuration of frontal sinuses: A forensic perspective. J. Pharm. Bioallied Sci. 2016; Oct; 8(Suppl 1): S90–S95. PubMed Abstract\|Publisher Full Text\|Free Full Text 15. Kanat A, Yazar U, Ozdemir B, et al.: Frontal sinus asymmetry: Is it an effect of cranial asymmetry? X-ray analysis of 469 normal adult human frontal sinus. J. Neurosci. Rural Pract. 2015; Oct-Dec; 06(4): 511–514. PubMed Abstract\|Publisher Full Text\|Free Full Text 3. Albalawi AS, Alam MK, Vundavalli S, et al.: Mandible: An Indicator for Sex Determination - A Three-dimensional Cone-Beam Computed Tomography Study. Contemp. Clin. Dent. 2019; 10(1): 69–73. PubMed Abstract\|Publisher Full Text\|Free Full Text 25. Denny C: Investigating the Link between Frontal Sinus Morphology and Craniofacial Characteristics with Sex: A 3D CBCT study on the South Indian Population. [Dataset]. figshare. 2023. Publisher Full Text 06(4): 511 514. PubMed Abstract\|Publisher Full Text\|Free Full Text 16. Soman BA, Sujatha GP, Lingappa A: Morphometric evaluation of the frontal sinus in relation to age and gender in subjects residing in Davangere, Karnataka. J. Forensic Dent. Sci. 2016 Jan-Apr; 8(1): 57. PubMed Abstract\|Publisher Full Text\|Free Full Text 4. Botticelli S, Verna C, Cattaneo PM, et al.: Two- versus three- dimensional imaging in subjects with unerupted maxillary canines. Eur. J. Orthod. 2011; 33(4): 344–349. PubMed Abstract\|Publisher Full Text 5. Choi IGG, Duailibi-Neto EF, Beaini TL, et al.: The Frontal Sinus Cavity Exhibits Sexual Dimorphism in 3D Cone-beam CT Images and can be Used for Sex Determination. J. Forensic Sci. 2018; 63(3): 692–698. PubMed Abstract\|Publisher Full Text 17. Shamlou AA, Tallman SD: Frontal Sinus Morphological and Dimensional Variation as Seen on Computed Tomography Scans. Biology. 2022; 11(8): 1145. PubMed Abstract\|Publisher Full Text\|Free Full Text https://doi.org/10.5256/f1000research.154636.r205338 © 2023 Santosh A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. PubMed Abstract\|Publisher Full Text 18. Ulcay T, Kamaşak B: Evaluation of craniometric measurements in human skulls. J. Health Sci. Med. 2021; 4(1): 38–44. Publisher Full Text 6. Buyuk SK, Karaman A, Yasa Y: Association between frontal sinus morphology and craniofacial parameters: A forensic view. J. Forensic Legal Med. 2017; 49: 20–23. PubMed Abstract\|Publisher Full Text 19. Ilayperuma I: On the Prediction of Personal Stature from Cranial Dimensions. Int. J. Morphol. 2010; 28(4): 1135–1140. Publisher Full Text 7. Rao KA, Doppalapudi R, Al-Shammari NT, et al.: Evaluation of frontal sinus index in establishing sex dimorphism using three-dimensional cone beam computed tomography in Northern Saudi Arabian population. J. Forensic Sci. Med. 2022; 8: 1–5. Publisher Full Text 20. Khan MA, Chaudhry MN, Altaf FMN: Cranial measurements; estimation of stature from cranial measurements. Professional Med. J. 2015; 22(8): 1034–1038. Publisher Full Text 8. Viloria A, Pineda OB, Romero L: Predicting the anthropometric properties of cranial structures using big data. Procedia Comput. Sci. 2020; Jan 1; 170: 905–910. Publisher Full Text 21. Scheuer L: Application of osteology to forensic medicine. Clin. Anat. 2002; 15(4): 297–312. Publisher Full Text 22. Venkat DG, Rao GK: Sex Determination by means of Inter-Canine and Inter-Molar Width- A Study in Telangana population. APJHS. 2016 Dec 30 [cited 2023 Apr 4]; 3(4): 171–175. Publisher Full Text\|Reference Source 9. da Silva RF, Pinto RN, Ferreira GM, et al.: Importance of frontal sinus radiographs for human identification. Braz. J. Otorhinolaryngol. 2008; 74(5): 798. PubMed Abstract\|Publisher Full Text\|Free Full Text 23. Sreelekha D, Madhavi D, Jothi SS, et al.: Study on mandibular parameters of forensic significance. J. Anat. Soc. India. 2020; 68: 21–24. 24. Marini MI, Angrosidy H, Kurniawan A, et al.: The anthropological analysis of the nasal morphology of Dayak Kenyah population in Indonesia as a basic data for forensic identification. Transl. Res. Anat. 2020 Jun; 19: 100064. Publisher Full Text Page 10 of 14 Open Peer Review Current Peer Review Status: F1000Research 2023, 12:811 Last updated: 10 OCT 2024 Open Peer Review https://doi.org/10.5256/f1000research.150156.r186253 © 2023 Santosh A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Arvind Babu Rajendra Santosh Faculty of Medical Sciences, Mona Campus, The University of The West Indies, Kings I am writing to provide my assessment of the revisions made by the authors in response to the reviewer comments for the manuscript titled "Investigating the link between frontal sinus morphology and craniofacial characteristics with sex: A 3D CBCT study on the South Indian population." In continuation of a previous comment regarding the title that mentions "with sex," I recommend that the objective should explicitly state the investigation of gender-related variations in frontal sinus morphology. Therefore, I suggest that the purpose be revised as follows: "The purpose of this study is to evaluate the morphological structure of the frontal sinuses, compare them with the nasal, maxillary, cranial, and mandible widths, and investigate gender- related variations in these characteristics among the South Indian population." After carefully reviewing the revised version, I am pleased to report that the authors have satisfactorily addressed all the concerns raised during the peer review process. The aforementioned comment can be revisited, and if the authors agree with the suggestion, they can revise the purpose of the study accordingly. With that said, I support the indexing of the manuscript, either in its current form or with the revised purpose of the study. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Primary focus is on oral disease, and secondary attention is given towards forensic dentistry Page 11 of 14 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Arvind Babu Rajendra Santosh 1 Faculty of Medical Sciences, Mona Campus, The University of The West Indies, King 2 Faculty of Medical Sciences, Mona Campus, The University of The West Indies, Kingston, Jamaica 2 Faculty of Medical Sciences, Mona Campus, The University of The West Indies, King The researchers undertook an investigation into the essential areas of forensic dental sciences, particularly focusing on the morphometric characterization of the frontal sinus with respect to gender. The title of the study clearly highlights this specific area of interest. However, in the last two paragraphs of the introduction, it would be advisable for the researchers to provide a more comprehensive justification for studying the morphometric characterization of the frontal sinus with consideration of gender. 1. The title of the study clearly highlights this specific area of interest. However, in the last two paragraphs of the introduction, it would be advisable for the researchers to provide a more comprehensive justification for studying the morphometric characterization of the frontal sinus with consideration of gender. 1. The study's methodology was well-structured and comprehensive, facilitating reliable data collection. However, for the data analysis section, I recommend seeking input from a statistician. Did the study use age-gender matching in their sampling strategy? 2. The most highlighting and promising results of the study should be emphasized in the initial section of discussion. 3. Add a statement that directly reflects striking findings of your study in the conclusion that answer your hypothesis. 4. Overall, this study is a valuable contribution to the literature, and we commend the authors for their excellent work. I endorse the manuscript for indexing with minor revisions. https://doi.org/10.5256/f1000research.150156.r186254 © 2023 Prabhu S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Reviewer Report 19 July 2023 Is the work clearly and accurately presented and does it cite the current literature? Yes Yes Page 12 of 14 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 Is the study design appropriate and is the work technically sound? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Primary focus is on oral disease, and secondary attention is given towards forensic dentistry I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Reviewer Report 19 July 2023 Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed Sudeendra Prabhu Sudeendra Prabhu 1 Department of Oral Pathology, Forensic Odontology, Yenepoya Dental College, Yenepoya (Deemed to be University), Mangalore, Karnataka, India 2 Department of Oral Pathology, Forensic Odontology, Yenepoya Dental College, Yenepoya (Deemed to be University), Mangalore, Karnataka, India Good study and well-written manuscript. ○ But the discussion and conclusion is incomplete. Kindly mention about the importance of parameters taken for sexual dimporphism and its correlation with sex in the CBCT. ○ Highlight the important parameter having good co-relation co-efficient. ○ Kindly quote a few more studies using CBCT for sex dimorphism. ○ 1 Department of Oral Pathology, Forensic Odontology, Yenepoya Dental College, Yenepoya (Deemed to be University), Mangalore, Karnataka, India 2 Department of Oral Pathology, Forensic Odontology, Yenepoya Dental College, Yenepoya (Deemed to be University), Mangalore, Karnataka, India Good study and well-written manuscript. ○ But the discussion and conclusion is incomplete. Kindly mention about the importance of parameters taken for sexual dimporphism and its correlation with sex in the CBCT. ○ But the discussion and conclusion is incomplete. Kindly mention about the importance of parameters taken for sexual dimporphism and its correlation with sex in the CBCT. ○ Highlight the important parameter having good co-relation co-efficient. ○ Kindly quote a few more studies using CBCT for sex dimorphism. ○ Page 13 of 14 F1000Research 2023, 12:811 Last updated: 10 OCT 2024 This manuscript can be accepted for indexing with minor modifications. ○ Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? No Competing Interests: No competing interests were disclosed. Reviewer Expertise: oral pathology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com Page 14 of 14 This manuscript can be accepted for indexing with minor modifications. ○ Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? No Competing Interests: No competing interests were disclosed. Reviewer Expertise: oral pathology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Are all the source data underlying the results available to ensure full reproduc Yes The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com Page 14 of 14
https://openalex.org/W4361819829	https://figshare.com/articles/journal_contribution/Supplementary_Figure_1_from_Polycomb_Protein_EZH2_Regulates_Tumor_Invasion_via_the_Transcriptional_Repression_of_the_Metastasis_Suppressor_RKIP_in_Breast_and_Prostate_Cancer/22389609/1/files/39835125.pdf	Luxembourgish	null	Supplementary Figure 1 from Polycomb Protein EZH2 Regulates Tumor Invasion via the Transcriptional Repression of the Metastasis Suppressor RKIP in Breast and Prostate Cancer	null	2,023	cc-by	455	NAP BPH PCA MPC N = 62 0 1 -1 2 -2 white - data missing EZH2 RKIP E-Cadherin Snail1 N = 25 NAP BPH PCA MPC 0 1 -1 2 -0.5 0.5 EZH2 RKIP E-Cadherin Snail1 data set #2 data set #3 Ren et al. SUPP Fig 1 siLuc siEZH2 siRKIP, siEZH2 siRKIP 4 2 6 soft agar colonies X102 0 DU145 MDA-MB231 3 2 1 soft agar colonies X102 0 a) 0 2 4 6 8 siEZH2siRKIP siEZH2 siLuc 0 14 28 42 56 70 84 hours cell number X105 DU145 b) c) NAP BPH PCA MPC N = 62 0 1 -1 2 -2 white - data missing EZH2 RKIP E-Cadherin Snail1 N = 25 NAP BPH PCA MPC 0 1 -1 2 -0.5 0.5 EZH2 RKIP E-Cadherin Snail1 data set #2 data set #3 Ren et al. SUPP Fig 1 siLuc siEZH2 siRKIP, siEZH2 siRKIP 4 2 6 soft agar colonies X102 0 DU145 MDA-MB231 3 2 1 soft agar colonies X102 0 a) 0 2 4 6 8 siEZH2siRKIP siEZH2 siLuc 0 14 28 42 56 70 84 hours cell number X105 DU145 b) c) NAP BPH PCA MPC N = 62 0 1 -1 2 -2 white - data missing EZH2 RKIP E-Cadherin Snail1 N = 25 NAP BPH PCA MPC 0 1 -1 2 -0.5 0.5 EZH2 RKIP E-Cadherin Snail1 data set #2 data set #3 Ren et al. SUPP Fig 1 a) data set #2 a) 0 1 -1 2 -2 0 1 -1 2 -0.5 0.5 data set #2 data set #3 Ren et al. SUPP Fig 1 a) a) Ren et al. SUPP Fig 1 Ren et al. SUPP Fig 1 NAP BPH PCA MPC N = 62 0 1 -1 2 -2 white - data missing EZH2 RKIP E-Cadherin Snail1 N = 25 NAP BPH PCA MPC 0 1 -1 2 -0.5 0.5 EZH2 RKIP E-Cadherin Snail1 data set #2 data set #3 Ren et al. SUPP Fig 1 a) data set #2 EZH2 RKIP E-Cadherin Snail1 NAP BPH PCA MPC siLuc siEZH2 siRKIP, siEZH2 siRKIP 4 2 6 soft agar colonies X102 0 DU145 MDA-MB231 3 2 1 soft agar colonies X102 0 0 2 4 6 8 siEZH2siRKIP siEZH2 siLuc 0 14 28 42 56 70 84 hours cell number X105 DU145 b) c) siLuc siEZH2 siRKIP, siEZH2 siRKIP 4 2 6 soft agar colonies X102 0 DU145 MDA-MB231 3 2 1 soft agar colonies X102 0 0 2 4 6 8 siEZH2siRKIP siEZH2 siLuc 0 14 28 42 56 70 84 hours cell number X105 DU145 b) c) b) 0 2 4 6 8 siEZH2siRKIP siEZH2 siLuc 0 14 28 42 56 70 84 hours cell number X105 DU145 ) c)

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